Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Coronaviruses post-SARS: update on replication and pathogenesis

Coronaviruses post-SARS: update on replication and pathogenesis REVIEWS Coronaviruses post-SARS: update on replication and pathogenesis Stanley Perlman and Jason Netland Abstract | Although coronaviruses were first identified nearly 60 years ago, they only received notoriety in 2003 when one of their members was identified as the aetiological agent of severe acute respiratory syndrome. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis. It also highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers. Coronaviruses, a genus in the Coronaviridae family (order encode an additional haemagglutinin-esterase (HE) pro - Prothrombinase Nidovirales; fig. 1), are pleomorphic, enveloped viruses. tein (fig . 2a,b). The HE protein, which may be involved Molecule that cleaves Coronaviruses gained prominence during the severe acute in virus entry or egress, is not required for replica- thrombin, thereby initiating the coagulation cascade. respiratory syndrome (SARS) outbreaks of 2002–2003 tion, but appears to be important for infection of the (ref. 1) . The viral membrane contains the transmembrane natural host . (M) glycoprotein, the spike (S) glycoprotein and the enve - Receptors for several coronaviruses have been iden- lope (E) protein, and surrounds a disordered or flexible, tified (TABLe 1). The prototypical coronavirus, mouse 2,3 probably helical, nucleocapsid. The viral membrane is hepatitis virus (MHV), uses CEACAM1a, a member of unusually thick, probably because the carboxy-terminal the murine carcinoembryonic antigen family, to enter region of the M protein forms an extra internal layer, as cells. Deletion of this protein makes mice resistant to 2 6 revealed by cryo-electron tomography. Coronaviruses infection . Several group 1 coronaviruses use ami- are divided into three groups, and further subdivided nopeptidase N to adhere to host cells, consistent with into subgroups (TABLe 1), based initially on serologic, and their respiratory and enteric tract tropisms (reviewed in more recently on genetic, analyses. With the identification ref. 7 ). SARS-CoV, a group 2 coronavirus, enters host of more distantly related viruses, the taxonomy of these cells through an interaction of the S protein with human viruses is likely to undergo further changes. angiotensin converting enzyme 2 (ACE2) . Strikingly, Coronaviruses contain a single stranded, 5′-capped, human coronavirus-NL63 (HCoV-NL63), which causes positive strand RNA molecule that ranges from 26–32 kb mild disease, also uses ACE2, although it binds to a dif - and that contains at least 6 open reading frames (ORFs). ferent part of the protein than does SARS-coronavirus 9,10 The first ORF (ORF1a/b) comprises approximately two- (SARS-CoV) . ACE2 is postulated to have a protec- thirds of the genome and encodes replicase proteins tive role in the inflamed lung, and SARS-CoV S pr- o (fig . 2a). Translation begins in ORF1a and continues in tein binding to ACE2 is thought to contribute to disease 11,12 ORF1b after a –1 frameshift signal. The large ORF1a and severity . As infection with HCoV-NL63 produces ORF1ab polypeptides, commonly referred to as pp1a mild disease, however, binding to ACE2 by itself cannot Department of Microbiology and pp1ab, respectively, are processed primarily by the be sufficient for this process. and Interdisciplinary pro virally encoded chymotrypsin-like protease 3CL (also The N protein is important for encapsidation of viral Program in Immunology, pro called M or main protease) with additional cleavage RNA and acts as an interferon (IFN) antagonist (see University of Iowa, Iowa City, Iowa 52242, USA. performed by one or two viral papain-like proteases below). Additionally, it causes upregulation o FGL f 2, a Correspondence to S.P. 4 (PLPs), depending on the species of coronavirus . The prothrombinase that contributes to fatal hepatic disease e-mail: stanley-perlman@ majority of the remaining one-third of the genome in mice that are infected with MHV-3 (ref. 13) and that uiowa.edu encodes four structural proteins: S, E, M and nucleo - modifies transforming growth factor-β (TGFβ) signalling doi:10.1038/nrmicro2147 Published online 11 May 2009 capsid (N) proteins. A subset of group 2 coronaviruses in SARS-CoV-infected cells . NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 439 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS RNA polymerase, nsp8, may function as a primase . Order: Nidovirales The nsp3 protease has additional roles in the assembly Family: Coronaviridae of virus replication structures (see below) and possesses Genus: Coronavirus poly(ADP-ribose) binding capabilities, and deubiqui- t Torovirus\Bafinivirus ylating activity in its protease domain, although the role Family: Roniviridae of the latter in virus replication is not yet known . Nsp7, nsp8, nsp9 and nsp10 are postulated to have Genus: Okavirus a role in subgenomic and genomic RNA replication, Family: Arteriviridae and all four proteins are essential for viral replication . Nsp7 and nsp8 form a hexadecameric structure, with Genus: Arterivirus RNA binding activity . The structure of nsp9 also sug- Figure 1 | The Nidoviruses. Phylogenetic relationship of gests that it binds RNA . Mutations in nsp10 inhibit Nature Reviews | Microbiology viruses in the order Nidoviruses. minus strand RNA synthesis, but this effect may be indi- rect, as studies have showed that nsp10 is required for pro 46 The E proteins are small integral membrane proteins proper function of the main viral protease (M ) . with roles in virus morphogenesis, assembly and bud - Nsp14, a bifunctional protein, is a 3′→5′ exonuclease, ding. In the absence of E proteins, virus release is inhib- with a role in maintaining fidelity of RNA transcription , ited completely (in the case of transmissible gastroenteritis and a (guanine-N7)-methyl transferase (N7-MTase), virus (TGEV)) or partially (in the case of SARS-CoV and involved in RNA cap formation . Coronaviruses also 15–17 MHV) . The E protein also possesses ion channel activity , encode a novel uridylate-specific endoribonuclease 18,19 which is required for optimal virus replication . (Nendou ), nsp15, that distinguishes nidoviruses in gen- Interspersed between and in these structural genes eral from other RNA viruses and that is crucial for virus are one to eight genes that encode accessory proteins, replication . Cleavage of RNA by Nendou results in depending on the virus strain. These show no sequence 2′-3′ cyclic phosphate ends, but its function in the virus similarity with other viral or cellular proteins and are life cycle remains unknown. Nsp16 is an S-adenosyl-l- 20–22 not required for virus replication in cultured cells . methionine-dependent RNA (nucleoside-2′O)-methyl However, they are conserved in virus species isolated at transferase (2′O-MTase) and, like nsp14, is involved in cap 23 50 different times and locales (for example, for SARS-CoV), formation . Nsp15 has been postulated to function with which suggests that these proteins have an important role nsp14 and nsp16 in RNA processing or cap production, in replication in the natural host. Several accessory pr- o but this remains to be proven. 24–27 teins are virion-associated , although whether these RNA replication is thought to occur on double- 28 51 proteins are truly structural is controversial. membrane vesicles (DMVs) (fig . 4). Newly synthesized The genes that encode non-replicase proteins are genomic RNA is then incorporated into virions on expressed from a set of ‘nested’ subgenomic mRNAs membranes that are located between the endoplasmic that have common 3′ ends and a common leader that is reticulum (ER) and the Golgi apparatus (ER–Golgi inter - encoded at the 5′ end of genomic RNA. Proteins are pro- mediate compartment (ERGIC); reviewed in ref. 52 ). duced generally only from the first ORF of subgenomic Initial studies suggested that these DMVs assemble using mRNAs, which are produced during minus strand RNA components of the autophagy pathway , but other stud- synthesis. Transcription termination and subsequent ies showed replication proceeded normally and that acquisition of a leader RNA occurs at transcription reg- DMVs were produced in macrophages lacking ATG5, ulatory sequences (TRS), located between ORFs. These a key component of autophagosomes . Thus, whether minus strand subgenomic RNAs serve as templates for autophagy is involved at all or whether its involvement is the production of subgenomic mRNAs (fig . 3), an effi- cell-specific remains uncertain. In addition, the unfolded cient process that results in a high ratio of subgenomic protein response (u PR) is induced during coronavirus 29 55 mRNA to minus strand subgenomic RNA . infections and may contribute to DMV formation . Recent results show that DMVs are likely to Coronavirus replication originate from the ER. u sing electron tomography One consequence of the SARS epidemic was an increase of cryo-fixed SARS-CoV-infected Vero E6 cells and in efforts to understand coronavirus replication and three-dimensional reconstruction imaging, Knoops Primase identify additional possible targets for anti-viral therapy. et al. showed that DMVs are not isolated vesicles, in the case of nsp8, an ORF1 of most coronaviruses encodes 16 proteins that but rather are part of a reticulovesicular network of r NA-dependent r NA polymerase that produces are involved in viral replication (fig . 2a); ORF1 of group 3 modified ER membranes . At later times after infec- r NA primers that are required coronaviruses lacks nsp1 and thus encodes only 15 pro - tions, these networks appear to merge into large for initiation of r NA synthesis teins. The structure of many of these proteins has been single-membrane vesicles. Proteins involved in virus by the main viral r NA solved by X-ray crystallography or nuclear magnetic replication (nsp3, nsp5 and nsp8; TABLe 2) are located polymerase, nsp12. 30–40 resonance, facilitating structure–function studies . mainly outside of DMVs, in adjacent reticular struc - Double-membrane vesicle Functions were predicted and later confirmed for many tures. Double-stranded RNA, representing either rep- A structure that is observed in pro pro of these proteins (TABLe 2), including PL1 and PL2 licative intermediates or ‘dead end’ double-stranded electron micrographs of pro (papain-like proteases) contained in nsp3 and 3CL RNA, was detected primarily in DMVs and, surpris- infected cells and that is pro or M contained in nsp5, the RNA-dependent RNA ingly, no obvious connections between the interior of thought to be the site of virus replication. polymerase nsp12 and the helicase nsp13. A second these vesicles and the cytosol were detected . Thus, it 440 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Representative coronavirus species and their receptors g roup Host Virus c ellular receptor Group 1a Bat BtCoV Unknown Cat FCoV APN Cat FIPV APN Dog CCoV APN Pig TGEV APN Group 1b Human HCoV-229E APN Human HCoV-NL63 Angiotensin-converting enzyme 2 (ACE2) Pig PEDV Unknown Group 1* Rabbit RbCoV Unknown Group 2a Cattle, ruminants, BCoV and related viruses 9-O-acetylated sialic acid alpaca Dog CRCoV Unknown Human HCoV-HKU1 Unknown Human HCoV-OC43 9-O-acetylated sialic acid Mouse MHV Carcinoembryonic antigen adhesion molecule 1 Pig PHEV Unknown Group 2b Bat BtCoV (multiple species) Unknown Human SARS-CoV ACE2 Group 2* Manx shearwaters PCoV Unknown Rat RtCoV Unknown Rat SDAV Unknown Group 3a Chicken IBV Unknown Pheasant PhCoV Unknown Turkey TCoV Unknown Group 3b Beluga whale SW1 Unknown Group 3c Bulbul BuCoV-HKU11 Unknown Thrush ThCoV-HKU12 Unknown Munia MuCoV-HKU13 Unknown Asian leopard cat, ALCCoV Unknown Chinese ferret badger *Due to a lack of sequence data, subgroup has not been assigned. More than 60 bat coronavirus species have been identified and tentatively classified as members of group 1 or group 2 (ref. 91) . ALCCoV, Asian leopard cat coronavirus; APN, aminopeptidase N; BCoV, bovine coronavirus; BtCoV, bat coronavirus; BuCoV, bulbul coronavirus; CCoV, canine coronavirus; CRCoV, canine respiratory coronavirus; FCoV, feline coronavirus; FIPV, feline infectious peritonitis virus; HCoV, human coronavirus; IBV, infection bronchitis virus; MHV, mouse hepatitis virus; MuCoV, munia coronavirus; PCoV, puffinosis coronavirus; PEDV, porcine epidemic diarrhoea virus; PhCoV, pheasant coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; RbCoV, rabbit coronavirus; RtCoV, rat coronavirus; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; SDAV, sialodacryoadenitis virus; TCoV, turkey coronavirus; ThCoV, thrush coronavirus; TGEV, transmissible gastroenteritis virus. remains unknown how newly synthesized RNA might but both the amino and carboxyl termini of these proteins be transported to sites of virus assembly, assuming that are in the cytoplasm, suggesting that one hydrophobic RNA transcription occurs in DMVs. region does not span the membrane ; whether this region Formation of DMVs requires membrane curvature, contributes to membrane curvature or has another function and this may be initiated by insertion of specific viral pro - requires further investigation. teins into membranes. Based on studies of equine arteritis virus , a non-coronavirus member of the nidovirus order Coronavirus-mediated diseases (fig. 1) , nsp3 and nsp4 are probably sufficient for DMV for - Before the SARS epidemic of 2002–2003, two human mation. Mutations in nsp4 result in aberrant formation of coronaviruses, HCoV-OC43 and HCoV-229E, were DMVs, further supporting a role for this protein in estab - recognized as important causes of upper respira- lishing sites of virus replication . Nsp6, like nsp3 and nsp4, tory tract infections and were occasionally associated also contains multiple transmembrane regions and ma y with more severe pulmonary disease in the elderly, 59–61 62 be involved in membrane modification . Notably, nsp3 newborn and immunocompromised . SARS-CoV, and nsp6 encode an odd number of hydrophobic domains, unlike HCoV-OC43 and HCoV -229E, causes a severe NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 441 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS a b Group 1: TGEV SARS-CoV virion Leader ORF1a S b7 M 5′ ORF1b 3a E N Poly(A) Group 2: SARS-CoV 8a Leader ORF1a S 3b MN 7a 5′ ORF1b 3a E 6 Poly(A) 7b 8b 9b 12 34 56 78 91011 12 13 14 15 16 ADRP/ RDRP Hel ExoN 2′OMT pro pro PL2 3CL ssRBP NendoU Group 3: IBV 7a/b Poly(A) Leader ORF1a S E 5′ ORF1b MN ssRNA 3a/b Figure 2 | Structure of coronavirus genome and virion. a | Schematic diagram of representative genomes from each of the coronavirus groups. Approximately the first two-thirds of the 26–32 Kb, positive-sense RNA genome encodes a large Nature Reviews | Microbiology polyprotein (ORF1a/b; green) that is proteolytically cleaved to generate 15 or 16 non-structural proteins (nsps; nsps for severe acute respiratory syndrome coronavirus (SARS-CoV) are illustrated). The 3′-end third of the genome encodes four structural proteins — spike (S), membrane (M), envelope (E) and nucleocapsid (N) (all shown in blue) — along with a set of accessory proteins that are unique to each virus species (shown in red). Some group 2 coronaviruses express an additional structural protein, haemagglutinin-esterase (not shown). b | Schematic diagram of the coronavirus virion. 2′OMT, ribose-2′- O-methyltransferase; ExoN, 3′5′ exonuclease; Hel, helicase; IBV, infection bronchitis virus; NendoU, uridylate-specific endoribonuclease; RDRP, RNA-dependent RNA polymerase; ssRBP, single-stranded RNA binding protein; ssRNA, single-stranded RNA; TGEV, transmissible gastroenteritis virus. respiratory disease, and nearly 10% mortality was Although the severe disease forming capabilities of observed in 2002–2003 (ref. 1) . Notable features of the human coronaviruses were only recognized because disease were an apparent worsening of symptoms as the of the SARS epidemic, it was well known that animal virus was cleared (suggesting the disease had an immu- coronaviruses could cause life-threatening disease. nopathological basis), and a lack of contagion until lower TGEV, which causes diarrhoea in piglets, infectious respiratory tract symptoms were apparent. This latter bronchitis virus (IBV), a cause of severe upper respira- feature made control of the epidemic by quarantine fea- tory tract and kidney disease in chickens, and bovine sible, as it simplified identification of infected patients. coronavirus (BCoV), which causes respiratory tract u nlike HCoV-OC43 and HCoV -229E, SARS-CoV also disease and diarrhoea in cattle (‘winter dysentery’ caused systemic disease, with evidence of infection of and ‘shipping fever’), are all economically important the gastrointestinal tract, liver, kidney and brain, among pathogens. Feline infectious peritonitis virus (FIPV), other tissues . Although the virus spread primarily via a virulent feline coronavirus (FCoV), causes an respiratory droplets, infection of the gastrointestinal invariably fatal systemic disease in domestic cats and tract may have facilitated other routes of spread. other felines. u nlike most strains of FCoV, which are The recognition that SARS was caused by a corona- endemic causes of mild diarrhoea, FIPV arises spo- virus intensified the search for other pathogenic coro- radically, most likely by mutation or deletion in felines naviruses associated with human disease, which led to persistently infected with enteric strains of FCoV , the identification of HCoV-NL63 and HCoV-HKu 1. and is macrophage-tropic. These viruses were isolated from hospitalized patients, Perhaps the most convincing explanation for FIPV- either young children with severe respiratory disease mediated disease was suggested by the observation that 64,65 (HCoV-NL63) or elderly patients with underlying progressive waves of virus replication, lymphopenia and 65,66 medical problems (HCoV-HK u 1) . HCoV-NL63 ineffectual T cell responses occurred in feline infectious has infected human populations for centuries, as phylo - peritonitis (FIP) . In conjunction with previous stud- genetic studies show that it diverged from HCoV-229E ies, these results raised the possibility that FIPV infec - nearly 1,000 years ago . HCoV-NL63 and HCoV-HKu 1 tion of macrophages and dendritic cells caused aberrant have worldwide distributions and generally cause mild cytokine and/or chemokine expression and lymphocyte upper respiratory tract diseases, with the exception depletion, resulting in enhanced virus loads and, co- n that HCoV-NL63 is also an aetiological agent of croup. sequently, a fatal outcome. Although this explanation is HCoV-NL63 can be propagated in tissue culture cells, appealing, additional work is needed to prove its valid - and an infectious cDNA clone of this virus was recently ity. Notably, anti-FIPV antibody-mediated enhancement engineered, facilitating future studies . By contrast, has been implicated in pathogenesis, but this has been HCoV-HKu 1 cannot be grown in tissue culture cells, shown only after immunization with S protein express- which makes it imperative that an infectious cDNA ing vaccines ; it has not been shown to play a role in a clone be developed for future studies. natural feline infection. 442 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 23,80 Anti-genome from Chinese horseshoe bats (Rhinolophus spp.) , 3′ 5′ which were also present in the live animal markets, the Leader TRS Minus strand synthesis Body TRS a Replication virus may have recently spread from bats to other mam- mals, such as palm civets, and then to humans (fig . 5a). 5′ Genome 3′ Consistent with a recent spread, antibodies to SARS- CoV were detected at extremely low levels (0.008%) in Nascent minus strand population studies in Hong Kong . Bat SARS-like CoV transferred to leader TRS cannot replicate in cells that express bat ACE2, although productive infection of cells expressing human ACE2 occurs if the RBD of the bat S protein is replaced with 82,83 that of a human isolate . Collectively, these observa - tions suggest that virus spread from bats to other spe- cies. Host cell entry does not occur via ACE2 in bats, Subgenomic mRNA although it does in palm civets and humans. Besides SARS-CoV, there are other examples of coronavirus cross-species transmission. BCoV and Figure 3 | Mechanism of coronavirus replication and transcription. Following entry Nature Reviews | Microbiology into the cell and uncoating, the positive sense RNA genome is translated to generate HCoV-OC43 are similar and the virus may have crossed replicase proteins from open reading frame 1a/b (ORF1a/b). These proteins use the from bovine to human hosts approximately 100 years genome as a template to generate full-length negative sense RNAs, which subsequently ago . BCoV has continued to cross species, as a related serve as templates in generating additional full-length genomes (a). Coronavirus mRNAs virus (99.5% similarity) has been isolated from an alpaca all contain a common 5′ leader sequence fused to downstream gene sequences. These 85,86 with enteritis and from captive wild ruminants leaders are added by a discontinuous synthesis of minus sense subgenomic RNAs using (fig . 5b). Furthermore, canine coronavirus (CCoV), feline genome RNA as a template (reviewed in ref. 29 ). Subgenomic RNAs are initiated at the 3′ and porcine viruses show evidence they have recom- end of the genome and proceed until they encounter one of the transcriptional regulatory bined with each other, indicating that they were present sequences (TRS; red) that reside upstream of most open-reading frames (b). Through in the same host. Recombination events between early base-pairing interactions, the nascent transcript is transferred to the complementary CCoV and FCoV strains (CCoV-I and FCoV-I) and leader TRS (light red) (c) and transcription continues through the 5′ end of the genome (d). These subgenomic RNAs then serve as templates for viral mRNA production (e). an unknown coronavirus resulted in two sets of novel viruses — CCoV-II and FCoV-II. Sequence data suggest that TGEV resulted from a cross-species transmission of Cross-species transmission CCoV-II from an infected canine (fig . 5c). A striking feature of the 2002–2003 SARS epidemic Molecular surveillance studies have identified at least 88 50 was the ability of the SARS-CoV to cross species from 60 novel bat coronaviruses in China , North America , 89,90 91 Himalayan palm civets P ( aguma larvata), raccoon dogs Europe and Africa . These bat CoVs may have orig- (Nyctereutes procyonoides) and Chinese ferret badg- inated from a common source and then subsequently ers (Melogale moschata) to infect human populations diverged as they adapted to growth in different species of (fig . 5a). Transmission occurred in live animal retail (wet) bat; they are now only distantly related to other coron-a markets, where animal handlers became infected. In ret - viruses. These studies also identified several novel avian rospect, it seems that variants of SARS-CoV related to the group 3 coronaviruses that were related to a novel coro - epidemic strain infected human populations in the wet navirus isolated from Asian leopard cats P ( rionailurus markets fairly frequently, as is shown by the high sero - bengalensis) and Chinese ferret badgers sold in illegal positivity rate detected in animal handlers who did not wild animal markets in China , suggesting that this develop SARS-like illnesses . The epidemic began when virus, like SARS-CoV, can cross species. Another novel a physician who was treating personnel in the wet mar - group 3 coronavirus, isolated from a deceased beluga kets became infected and subsequently infected multiple whale (Delphinapterus leucas), is only distantly related contacts . to IBV-like and novel avian coronaviruses, suggesting Genetic analyses of virus isolates from infected palm that it comprises a third subgroup . Thus group 3 coro- civets and humans during the epidemic showed that naviruses, which formerly included only avian viruse s, 75,76 the virus underwent rapid adaptation in both hosts , now consist of at least 3 subgroups and include primarily in the receptor binding domain (RBD) of the viruses that infect mammalian hosts. S protein, to allow more efficient infection of human cells . In particular, mutations K479N and S487T in Immunopathology in coronavirus infections the RBD of the S protein were key to adaptation to the It is generally accepted that the host response is respon - human receptor (ACE2). These results were recently sible for many of the disease manifestations in infections 95,96 confirmed using cell lines expressing civet ACE2 or caused by coronaviruses . This was shown initially in human ACE2 (ref. 78) . mice infected with the neurotropic strains of mouse The obser vation that SARS-CoV could not be hepatitis virus (the j HMV and MHV-A59 strains). detected in either farmed or wild palm civets , together Many attenuated strains of j HMV cause a subacute with evidence of adaptive changes detected in virus iso- or persistent infection in the central nervous system, lated from infected animals, suggested that palm civets with persistence in glia, especially oligodendrocytes. A and other animals in wet markets were not the primary consequence of host efforts to clear the virus is myelin reservoir for the virus. As SARS-like CoV were isolated destruction (demyelination). However, j HMV infection NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 443 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS the onset of virus clearance . Although worsening clini - Table 2 | Coronavirus non-structural proteins and their functions cal disease occurring as a consequence of virus clea- r Protein Functions ance has not been duplicated in any animal model of Nsp1 Host mRNA degradation; translation inhibition; cell cycle arrest; inhibition SARS, the severe disease observed in older patients can of IFN signaling 102–104 be mimicked in SARS-CoV-infected aged mice . Nsp2 Unknown This has been attributed, in part, to a suboptimal T cell pro pro response resulting in delayed kinetics of virus clea -r Nsp3 Papain-like proteases (PL1 , PL2 ) (polyprotein processing); ance. A suboptimal T cell response, occurring as a con - poly(ADP-ribose) binding; DMV formation (?); IFN antagonist; nucleic acid binding; deubiquitinating activity sequence of infection of macrophages or dendritic cells, may also be critical for the immunopathological lethal Nsp4 DMV formation (?) disease that is observed in FIPV-infected felines . Thus, pro pro Nsp5 Main protease (M , 3CL ); polyprotein processing in many instances, host efforts to clear a coronavirus Nsp6 DMV formation (?) infection result in some tissue destruction. Nsp7 Single-stranded RNA binding Evasion of the innate immune response Nsp8 Primase Although anti-viral T cells and antibodies are crucial for Nsp9 Part of replicase complex virus clearance and for the prevention of recrudescence Nsp10 Part of replicase complex (reviewed in ref. 96 ), the efficacy of the innate immune Nsp11 Unknown response determines the extent of initial virus replica - tion and thus the load that the host must overcome to Nsp12 RNA-dependent RNA polymerase clear the infection (fig . 6a). Coronaviruses, like all other Nsp13 Helicase; nucleoside triphosphatase activity; RNA 5′-triphosphatase successful viruses, have developed strategies to counter activity the innate immune response (fig . 6b-d). IFN expres- Nsp14 3′→5′ exoribonuclease; RNA cap formation (guanine-N7)- sion is a crucial component of this initial response, and methyltransferase coronaviruses have developed ‘passive’ and ‘active’ tools Nsp15 Endonuclease to prevent IFN induction and signalling. Interferon is not induced in fibroblasts that are infected with either Nsp16 RNA cap formation (2′O-methytransferase) 105–107 SARS-CoV or MHV . However, in both instances, DMV, double-membrane vesicle; IFN, interferon. treatment of cells with polyinosinic:polycytidylic acid or with other IFN-inducing agents, results in activation of 105,106 of mice that lack T or B cells (sublethally irradiated IFN regulating factor 3 (IRF3) and IFN induction . mice or mice with severe combined immunodeficiency Thus, in these cells, viruses appear to be invisible to or genetically deficient in recombination activating intracellular viral sensors (such as RI- G I, MDA5 and –/– gene 1 (RAG1 )) results, eventually, in death in all TLR3), perhaps because double stranded RNA, a potent mice, but without demyelination. Adoptive transfer of stimulator of the innate immune system, is buried in a + + CD4 or CD8 T splenocytes 7 or 30 days after immu- DMV (fig . 6b). –/– nization with j HMV to infected RAG1 or SCID mice Additionally, viral proteins, in particular nsp1, nsp3, 95–97 results in virus clearance and demyelination . Myelin N protein and the SARS-CoV accessory proteins ORF6 108–113 destruction is also observed if anti-j HMV antibody is and ORF3b, also prevent IFN induction . The N pro- –/– transferred to infected RAG1 mice in the absence tein of MHV inhibits activator protein 1 (AP1) signal- of T cells , or if mice are infected with virus express- ling and protein kinase R (PKR) function, whereas the ing the macrophage chemoattractant CCL2 in the N protein of SARS-CoV also inhibits nuclear facto- rκB 99 108–110 absence of other interventions . In all cases, infiltratin g activation when expressed in transfection assays. macrophages appear to be crucial for virus clearance Whether these inhibitory functions of the N protein and subsequent demyelination; these results suggest are coronavirus or cell-type specific, and whether they that the process of macrophage infiltration can be initi- occur in infected cells, remains to be determined. The ated by T cells, antij-HMV antibody or overexpression ORF6 protein inhibits IFN signalling by binding to of a single macrophage chemoattractant. These results karyopherin-α2, thereby tethering karyopherin-β have been extended to mice with encephalitis caused to cytoplasmic membranes . This, in turn, pre- + + by virulent strains of j HMV. Although CD4 and CD8 vents nuclear translocation of proteins containing 100 115 T cells are both required for virus clearance , partial classical nuclear import signals, including STAT1, a abrogation of the CD4 T cell response (by mutating crucial component of IFNα, IFNβ and IFNγ signalling the immunodominant CD4 T cell epitope jr.M ) pathways. Of note, deletion of ORF6 does not increase Y135Q results in disease amelioration, and virulence is the IFN sensitivity of SARS-CoV , probably because regained if another CD4 T cell epitope is reintroduced mechanisms of IFN antagonism are redundant. into the rj.M genome . Thus acute encephali- SARS-CoV and MHV nsp1 function, at least in part, Y135Q tis, like chronic demyelination, is at least partial ly by degrading host cell mRNA and inhibiting transl-a 111–113,117 mediated by the immune system. tion . Nsp1 also inhibits IFN signalling in both Similar processes may occur in SARS-CoV-infected SARS-CoV- and MHV-infected cells, in part by inhib - 112,113 humans, as pulmonary disease often worsens at 1–2 weeks iting STAT1 phosphorylation . Mutation of nsp1 after onset of respiratory symptoms, concomitant with attenuates SARS-CoV and MHV growth in mice and 444 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS tissue culture cells in the presence of an intact IFN sys - nsp3 nsp5 nsp8 111–113 tem, but not when IFN function is deficient . Nsp3 RER is also an IFN antagonist, and it inhibits phosphorylation VP and nuclear importation of IRF3 (ref. 118) . dsRNA Both MHV and SARS-CoV inhibit IFNα and IFNβ induction and signalling. However, IFN α and/or IFNβ are 119,120 detected in infected mice and humans and mice deficient in IFNα and/or IFNβ receptor expression are 113,121 exquisitely sensitive to MHV infection , showing that IFNα and/or IFNβ has a major role in the anti- Ribosome virus immune response. Reconciling these disparate results, recent studies showed that IFNα is produced in large amounts in SARS-CoV- and MHV-infected plasmacytoid dendritic cells, via a T LR7-dependent mechanism . Furthermore, IFNβ is expressed by mac- rophages and microglia, but not by dendritic cells after MHV infection . Macrophages, and to a lesser extent dendritic cells, are the major targets for IFN α and/or CM IFNβ in MHV-infected mice . In addition to IFN, multiple chemokines and cytokines are also induced as part of the host response to coronaviruses such as MHV, SARS-CoV and FIPV. Cytokines such as interleukin 1 (IL -1), IL-6 and IL-12 and chemokines such as IL-8, CCL2 and CXCL10 are ele- vated in SARS patients. u sing genomics and proteomics, Cameron et al. found that IFNα and/or IFNβ and IFNγ, as well as chemokines such as CXCL10 and CCL2, are DMV elevated at early times post infection in all patients and diminished in those who recovered, accompanied by a Figure 4 | c oronavirus-induced membrane alterations robust anti-virus antibody response . However, levels Nature Reviews | Microbiology as platforms for viral replication. Coronavirus infection of CXCL10, CCL2 and other proinflammatory media- induces the formation of a reticulovesicular network of tors remained elevated and anti-SARS-CoV antibody modified membranes that are thought to be the sites titres were low in those patients who developed severe of virus replication. These modifications, which include disease. SARS-CoV-infected pulmonary epithelial cells double-membrane vesicles (DMVs), vesicle packets (VPs, were the source of at least some of the cytokines and/or single-membrane vesicles surrounded by a shared outer chemokines, such as CCL2, IL-6, IL-1β and tumour membrane) and convoluted membranes (CMs), are all necrosis factor (TNF) . Others have suggested that interconnected and contiguous with the rough a strong T 2 (IL4, IL-5 and IL-10) response correlated endoplasmic reticulum (RER). Viral double-stranded RNA is with a poor outcome . It has been postulated that an mostly localized to the interior of the DMVs and inner vesicles of the VPs, whereas replicase proteins (that is, over-exuberant cytokine response contributed to a poor nsp3, nsp5 and nsp8) are present on the surrounding CM. outcome in patients with SARS in 2002–2003 (reviewed Some nsp8 can be detected inside the DMVs. All in refs 95,127,128 ). Collectively, these results do not membranes are bound by ribosomes. (Figure based on data strongly prove or disprove a role for an exuberant cytokine from r efs. 56,141,142 .) and chemokine response in severe SARS, in part because virus titres could not be determined concomitantly and also because serum levels, but not pulmonary cytokine neuronal infection, accompanied by high cytokine and/ or chemokine levels were measured. or chemokine expression and minimal cellular infilt -ra tion in the brain . Although the severity of the brain Animal models for SARS infection observed in human ACE2 transgenic mice As human SARS has disappeared, the role of an exuber- is greater than that seen in human patients, infection ant (but perhaps appropriate for the titre of the virus) of this organ has been detected in some studies and immune response will need to be addressed using ani - patients who survived SARS had a greater incidence mal models of SARS. Mice, cats, ferrets, macaques and of neurological and psychiatric sequelae than antici - 63,133,134 civet cats are all susceptible to SARS-CoV, but none, pated . The high susceptibility of these mice to with the exception of aged mice, develop severe disease infection with SARS-CoV makes them useful for vac- (reviewed in ref. 129 ). In efforts to develop models that cine and therapeutic trials. Another approach to devel - closely mimic human disease, mice that are transgenic oping an animal model for SARS was to adapt the virus for the expression of human ACE2 were developed and by passage 10–15 times through the lungs of BALB/c 130,131 103,135,136 infected with SARS-CoV . Although these mice mice or rats . Three to six mutations were develop more severe pulmonary disease than non- detected in the adapted viruses, with changes most com- pro transgenic mice, they also develop an overwhelming monly observed in the S protein and in nsp5 (3CL ). NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 445 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Humans Bat Himalayan palm civet Human BtCoV SARS Alpacas Alpaca Cow Human Wild ruminants Wild ruminant (deer) HCoV-OC43BCoV Dog Unknown reservoir Dog Pig CCoV-I Unknown CCoV-II TGEV coronavirus Common ancestor Cat Cat Cat FCoV-I FCoV-II FCoV-II Figure 5 | c ross-species transmission of coronaviruses. a | Severe acute respiratory syndrome (SARS)-like bat coronavirus (BtCoV) spread and adapted to wild animals such as the Himalayan palm civet that was sold as food in Chinese wet markets. The virus frequently spread to animal handlers in these markets, but caused minimal or no disease. Further adaptation resulted in strains that replicated efficiently in the human host, caused disease and could spread from person to person. b | Human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV) are closely related and it is thought that the virus originated in one species and then crossed species. BCoV has also spread to numerous other animals, such as alpaca and wild ruminants. c | Feline coronavirus I (FCoV-I) and canine coronavirus I (CCoV-I) are thought to share a common ancestor. CCoV-I underwent recombination with an unknown coronavirus to give rise to canine coronavirus II Nature Reviews | Microbiology (CCoV-II). CCoV-II in turn underwent recombination with FCoV-I (in an unknown host) to give rise to feline coronavirus II (FCoV-II). CCoV-II probably also spread to pigs, resulting in transmissible gastroenteritis virus (TGEV). The adapted virus caused extensive pulmonary infec- mediators, such as IL-6, TNF, CXCL10 and CCL2, tion and disease was most severe in aged animals. accompanied by slower kinetics of virus clearance These viruses will be useful for studies of pathogenesis and worse outcomes in aged compared to young and for vaccine and therapeutic trials. animals , paralleling disease patterns in patients Some models have been tested on the genomic with SARS . These two studies also showed changes and proteomic level. Studies of SARS-CoV infected in expression of proteins that are involved in cell macaques showed that several chemokines and/or growth, cycling, cell-to-cell signalling and deve- lop cytokines, such as IL-6, IL-8, CXCL10 and CCL2, ment and death. It will be important to determine as well as IFNα, IFNβ and IFNγ, were upregu - whether these changes are useful as a ‘fingerprint’ lated . These animals recovered, showing that the for SARS or whether they represent generalized same inflammator y mediators that are associated responses to pulmonary stress. with severe human disease are also produced as part of the inflammatory response in animals that Future directions mount an appropriate response. Genomics studies Perhaps the most important insight made over the of mice infected with the u rbani strain of SARS- past several years is that coronaviruses have and will CoV showed continued expression of inflammatory likely continue to cross between species and cause 446 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Fibroblasts and dendritic cells a b IFNα and IFNβ Plasmacytoid dendritic cell Macrophage Virus STAT1, Viral RNA Viral RNA release STAT2 DMV dsRNA dsRNA MDA5 ssRNA RIG1 MDA5 Virus TLR7 IRF3 NF-κB production NF-κB AP1 AP1 IRF7 NF-κB IRF3 N, nsp1, nsp3, IFNα and IFNβ ORF6, IFNα and IFNβ ORF3b Nucleus Nucleus Nucleus cd Systemic disease due to cytokine storm B cell Infiltration of T cell infected tissue Macrophage Cytokines Microglial cell Figure 6 | inefficient activation of the type 1 interferon response, and immunopathological disease, in Nature Reviews | Microbiology coronavirus infections. a | Coronaviruses, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV), induce a type 1 interferon (IFN) response in plasmacytoid dendritic cells (pDC) and macrophages, via TLR7- and MDA5-dependent pathways, respectively. b | IFNα and/or IFNβ is not produced in either SARS-CoV fibroblasts or DCs, partly because coronavirus macromolecules appear to be invisible to immune sensors. Additionally, coronaviruses encode proteins that actively inhibit IFNα and/or IFNβ expression (such as nucleocapsid (N) protein, nsp3, ORF6 and ORF3b) or signalling through the type 1 IFN receptor (such as N, nsp1, ORF6 and ORF3b). c | Consequently, the kinetics of virus clearance is delayed, with subsequent robust T and B cell and cytokine and/or chemokine responses. d | This pro-inflammatory response results in immunopathological disease that occurs during the process of virus clearance. In MHV-infected mice, virus clearance involves recruitment of activated macrophages and microglia to sites of virus infection, leading to demyelination. Similar mechanisms with exuberant cytokine production may function in the lungs of SARS-CoV-infected humans, leading to severe pulmonary disease (adult respiratory distress syndrome, ARDS). AP1, activator protein 1; DMV, double-membrane vesicle; dsRNA, double-stranded RNA; NF-κB, nuclear factor-κB; ssRNA, single-stranded RNA. disease in unrelated hosts. This disease may be mild, species, the virus also needed to evolve strategies to like the disease caused by the SARS-like CoV that evade the innate immune response of the new hosts. was transmitted to animal handlers in wet markets in One future goal will be to further delineate how the China, but it may be severe, as illustrated by the tran- s virus evades the immune response and better under- mission that triggered the SARS epidemic. Further, stand its interaction with the T and B cell responses, SARS-CoV appeared to use an entirely new recep- both in the original host (bats), in which disease tor when it crossed species from bats to palm civets appears to be mild, and in humans and experimentall y and humans. As part of this transmission to a new infected animals. NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 447 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Although coronaviruses use host proteins as part roles of these proteins in virus replication still require Collaborative cross mice of their replication strategies, it has also become clear additional investigation. Progress in these fields will A panel of 1,000 recombinant inbred mouse strains derived that immune, metabolic, stress, cell cycling and other take advantage of new methodologies that allow from 8 genetically diverse pathways are activated by infection. Assessing the bio- detailed observations of both fixed and living cells at founder strains. The crosses logical function of these pathways in virus replication high resolution. were designed for complex and in disease outcome will be critical. Determining Finally, no effective treatments exist for any coro - trait analysis and will be useful the extent to which virus–host interactions are coro- navirus infections, including SARS ; vaccines, even for identifying and establishing the role of host genes in s Ars navirus-specific and organ-specific will be possible, for animal coronaviruses, are not effective; and live pathogenesis. using genomics and proteomics, as well as new rea - attenuated vaccines are prone to recombination with gents and collaborative cross mice. The collaborative circulating coronaviruses. One future goal will be to cross, a panel of approximately 1,000 recombinant translate new information about the structure and inbred mouse strains derived from 8 founder strains, function of coronavirus proteins into specific anti- will be useful for analyses of complex genetic traits. virus therapies. Also, development of live, attenuated, u sing sophisticated microscopy and biochemi- safe vaccines that do not recombine in the wild is cal approaches, details of coronavirus replication in another goal, made more feasible as more is learned infected cells have been revealed. However, these new about basic coronavirus biology. Over the past few results have led to a new set of questions about the years, the development of new technologies has sim - relationship between sites of viral RNA replication plified the identification of novel coronaviruses; the and virus assembly. Furthermore, although putative next major goals will be to understand viral path -o functions have been assigned to many of the proteins genesis and to design effective coronavirus vaccines encoded by the large ORF1 replicase gene, the precise and therapies. 1. Peiris, J. S., Guan, Y. & Yuen, K. Y. Severe acute 14. Zhao, X., Nicholls, J. M. & Chen, Y. G. Severe acute 28. Neuman, B. W. et al. Proteomics analysis unravels the respiratory syndrome. Nature Med. 10, S88–S97 respiratory syndrome-associated coronavirus functional repertoire of coronavirus nonstructural (2004). nucleocapsid protein interacts with Smad3 and protein 3. J. Virol. 82, 5279–5294 (2008). 2. Barcena, M. et al. Cryo-electron tomography of mouse modulates transforming growth factor-β signaling. 29. Sawicki, S. G., Sawicki, D. L. & Siddell, S. G. A hepatitis virus: insights into the structure of the J. Biol. Chem. 283, 3272–3280 (2008). contemporary view of coronavirus transcription. coronavirion. Proc. Natl Acad. Sci. USA 106, 15. DeDiego, M. L. et al. A severe acute respiratory J. Virol. 81, 20–29 (2007). 582–587 (2009). syndrome coronavirus that lacks the E gene is 30. Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. & This paper and Ref. 3 use novel methodologies to attenuated in vitro and in vivo. J. Virol. 81, Hilgenfeld, R. Coronavirus main proteinase (3CLpro) obtain detailed models of the structure of the 1701–1713 (2007). structure: basis for design of anti-SARS drugs. Science intact coronavirion. 16. Kuo, L. & Masters, P. S. The small envelope protein E 300, 1763–1767 (2003). 3. Neuman, B. W. et al. Supramolecular architecture of is not essential for murine coronavirus replication. 31. Zhai, Y. et al. Insights into SARS-CoV transcription and severe acute respiratory syndrome coronavirus J. Virol. 77, 4597–4608 (2003). replication from the structure of the nsp7-nsp8 revealed by electron cryomicroscopy. J. Virol. 80, 17. Ortego, J., Ceriani, J. E., Patino, C., Plana, J. & hexadecamer. Nature Struct. Mol. Biol. 12, 980–986 7918–7928 (2006). Enjuanes, L. Absence of E protein arrests transmissible (2005). 4. Masters, P. S. The molecular biology of coronaviruses. gastroenteritis coronavirus maturation in the secretory 32. Chatterjee, A. et al. Nuclear magnetic resonance Adv. Virus Res. 66, 193–292 (2006). pathway. Virology 368, 296–308 (2007). structure shows that the severe acute respiratory 5. Lissenberg, A. et al. Luxury at a cost? Recombinant 18. Madan, V., Garcia Mde, J., Sanz, M. A. & Carrasco, L. syndrome coronavirus-unique domain contains a mouse hepatitis viruses expressing the accessory Viroporin activity of murine hepatitis virus E protein. macrodomain fold. J. Virol. 83, 1823–1836 (2009). hemagglutinin esterase protein display reduced fitness FEBS Lett. 579, 3607–3612 (2005). 33. Joseph, J. S. et al. Crystal structure of nonstructural in vitro. J. Virol. 79, 15054–15063 (2005). 19. Wilson, L., McKinlay, C., Gage, P. & Ewart, G. SARS protein 10 from the severe acute respiratory syndrome –/– 6. Hemmila, E. et al. Ceacam1a mice are completely coronavirus E protein forms cation-selective ion coronavirus reveals a novel fold with two zinc-binding resistant to infection by murine coronavirus mouse channels. Virology 330, 322–331 (2004). motifs. J. Virol. 80, 7894–7901 (2006). hepatitis virus A59. J. Virol. 78, 10156–10165 20. Yount, B. et al. Severe acute respiratory syndrome 34. Joseph, J. S. et al. Crystal structure of a monomeric (2004). coronavirus group-specific open reading frames encode form of severe acute respiratory syndrome coronavirus 7. Weiss, S. R. & Navas-Martin, S. Coronavirus nonessential functions for replication in cell cultures endonuclease nsp15 suggests a role for pathogenesis and the emerging pathogen severe acute and mice. J. Virol. 79, 14909–14922 (2005). hexamerization as an allosteric switch. J. Virol. 81, respiratory syndrome coronavirus. Microbiol. Mol. 21. Ontiveros, E., Kuo, L., Masters, P. S. & Perlman, S. 6700–6708 (2007). Biol. Rev. 69, 635–664 (2005). Inactivation of expression of gene 4 of mouse hepatitis 35. Peti, W. et al. Structural genomics of the severe acute 8. Li, W. et al. Angiotensin-converting enzyme 2 is a virus strain JHM does not affect virulence in the respiratory syndrome coronavirus: nuclear magnetic functional receptor for the SARS coronavirus. Nature murine CNS. Virology 290, 230–238 (2001). resonance structure of the protein nsP7. J. Virol. 79, 426, 450–454 (2003). 22. de Haan, C. A., Masters, P. S., Shen, X., Weiss, S. & 12905–12913 (2005). This is the first report identifying the SARS-CoV Rottier, P. J. The group-specific murine coronavirus 36. Saikatendu, K. S. et al. Structural basis of severe acute receptor. genes are not essential, but their deletion, by reverse respiratory syndrome coronavirus ADP-ribose-1′′- 9. Li, W. et al. The S proteins of human coronavirus NL63 genetics, is attenuating in the natural host. Virology phosphate dephosphorylation by a conserved domain and severe acute respiratory syndrome coronavirus 296, 177–189 (2002). of nsP3. Structure 13, 1665–1675 (2005). bind overlapping regions of ACE2. Virology 367, 23. Li, W. et al. Bats are natural reservoirs of SARS-like 37. Almeida, M. S., Johnson, M. A., Herrmann, T., Geralt, 367–374 (2007). coronaviruses. Science 310, 676–679 (2005). M. & Wuthrich, K. Novel β -barrel fold in the nuclear 10. Hofmann, H. et al. Human coronavirus NL63 employs This paper and REF. 80 show that SARS-CoV magnetic resonance structure of the replicase the severe acute respiratory syndrome coronavirus probably originated in bats. nonstructural protein 1 from the severe acute receptor for cellular entry. Proc. Natl Acad. Sci. USA 24. Fischer, F., Peng, D., Hingley, S. T., Weiss, S. R. & respiratory syndrome coronavirus. J. Virol. 81, 102, 7988–7993 (2005). Masters, P. S. The internal open reading frame within 3151–3161 (2007). 11. Kuba, K. et al. A crucial role of angiotensin converting the nucleocapsid gene of mouse hepatitis virus 38. Serrano, P. et al. Nuclear magnetic resonance enzyme 2 (ACE2) in SARS coronavirus-induced lung encodes a structural protein that is not essential for structure of the N-terminal domain of nonstructural injury. Nature Med. 11, 875–879 (2005). viral replication. J. Virol. 71, 996–1003 (1997). protein 3 from the severe acute respiratory syndrome This report suggests that interactions between the 25. Huang, C., Peters, C. J. & Makino, S. Severe acute coronavirus. J. Virol. 81, 12049–12060 (2007). SARS-CoV S protein and its receptor may respiratory syndrome coronavirus accessory protein 6 is 39. Ricagno, S. et al. Crystal structure and mechanistic contribute to disease progression, in addition to a virion-associated protein and is released from 6 protein- determinants of SARS coronavirus nonstructural facilitating virus entry. expressing cells. J. Virol. 81, 5423–5426 (2007). protein 15 define an endoribonuclease family. Proc. 12. Imai, Y. et al. Angiotensin-converting enzyme 2 26. Ito, N. et al. Severe acute respiratory syndrome Natl Acad. Sci. USA 103, 11892–11897 (2006). protects from severe acute lung failure. Nature 436, coronavirus 3a protein is a viral structural protein. 40. Su, D. et al. Dodecamer structure of severe acute 112–116 (2005). J. Virol. 79, 3182–3186 (2005). respiratory syndrome coronavirus nonstructural 13. Ning, Q. et al. Induction of prothrombinase fgl2 by 27. Schaecher, S. R., Mackenzie, J. M. & Pekosz, A. The protein nsp10. J. Virol. 80, 7902–7908 (2006). the nucleocapsid protein of virulent mouse hepatitis ORF7b protein of severe acute respiratory syndrome 41. Snijder, E. J. et al. Unique and conserved features of virus is dependent on host hepatic nuclear coronavirus (SARS-CoV) is expressed in virus-infected genome and proteome of SARS-coronavirus, an early factor-4α. J. Biol. Chem. 278, 15541–15549 cells and incorporated into SARS-CoV particles. split-off from the coronavirus group 2 lineage. J. Mol. (2003). J. Virol. 81, 718–731 (2007). Biol. 331, 991–1004 (2003). 448 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 42. Imbert, I. et al. A second, non-canonical RNA- 65. van der Hoek, L. et al. Identification of a new human 90. Gloza-Rausch, F. et al. Detection and prevalence dependent RNA polymerase in SARS coronavirus. coronavirus. Nature Med. 10, 368–373 (2004). patterns of group I coronaviruses in bats, northern EMBO J. 25, 4933–4942 (2006). 66. Woo, P. C. et al. Characterization and complete Germany. Emerg. Infect. Dis. 14, 626–631 (2008). 43. Ratia, K. et al. Severe acute respiratory syndrome genome sequence of a novel coronavirus, coronavirus 91. Tong, S. et al. Detection of novel SARS-like and other coronavirus papain-like protease: structure of a viral HKU1, from patients with pneumonia. J. Virol. 79, coronaviruses in bats from Kenya. Emerg. Infect. Dis. deubiquitinating enzyme. Proc. Natl Acad. Sci. USA 884–895 (2005). 15, 482–485 (2009). 103, 5717–5722 (2006). 67. Pyrc, K. et al. Mosaic structure of human coronavirus 92. Woo, P. C. et al. Comparative analysis of complete 44. Deming, D. J., Graham, R. L., Denison, M. R. & Baric, NL63, one thousand years of evolution. J. Mol. Biol. genome sequences of three novel avian coronaviruses R. S. Processing of open reading frame 1a replicase 364, 964–973 (2006). reveals a novel group 3c coronavirus. J. Virol. 83, proteins nsp7 to nsp10 in murine hepatitis virus strain This manuscript delineates molecular evolution of 908–917 (2009). A59 replication. J. Virol. 81, 10280–10291 (2007). HCoV-229E and HCoV-NL63. 93. Dong, B. Q. et al. Detection of a novel and highly 45. Egloff, M. P. et al. The severe acute respiratory 68. van der Hoek, L. et al. Croup is associated with the divergent coronavirus from asian leopard cats and syndrome-coronavirus replicative protein nsp9 is a novel coronavirus NL63. PLoS Med. 2, e240 (2005). Chinese ferret badgers in Southern China. J. Virol. 81, single-stranded RNA-binding subunit unique in the RNA 69. Donaldson, E. F. et al. Systematic assembly of a full- 6920–6926 (2007). virus world. Proc. Natl Acad. Sci. USA 101, length infectious clone of human coronavirus NL63. 94. Mihindukulasuriya, K. A., Wu, G., St Leger, J., 3792–3796 (2004). J. Virol. 82, 11948–11957 (2008). Nordhausen, R. W. & Wang, D. Identification of a 46. Donaldson, E. F., Sims, A. C., Graham, R. L., Denison, 70. Rottier, P. J., Nakamura, K., Schellen, P., Volders, H. & novel coronavirus from a beluga whale by using a M. R. & Baric, R. S. Murine hepatitis virus replicase Haijema, B. J. Acquisition of macrophage tropism panviral microarray. J. Virol. 82, 5084–5088 protein nsp10 is a critical regulator of viral RNA during the pathogenesis of feline infectious peritonitis (2008). synthesis. J. Virol. 81, 6356–6368 (2007). is determined by mutations in the feline coronavirus 95. Perlman, S. & Dandekar, A. A. Immunopathogenesis 47. Eckerle, L. D., Lu, X., Sperry, S. M., Choi, L. & Denison, spike protein. J. Virol. 79, 14122–14130 (2005). of coronavirus infections: implications for SARS. M. R. High fidelity of murine hepatitis virus replication 71. de Groot-Mijnes, J. D., van Dun, J. M., van der Most, Nature Rev. Immunol. 5, 917–927 (2005). is decreased in nsp14 exoribonuclease mutants. R. G. & de Groot, R. J. Natural history of a recurrent 96. Bergmann, C. C., Lane, T. E. & Stohlman, S. A. J. Virol. 81, 12135–12144 (2007). feline coronavirus infection and the role of cellular Coronavirus infection of the central nervous system: 48. Chen, Y. et al. Functional screen reveals SARS immunity in survival and disease. J. Virol. 79, host–virus stand-off. Nature Rev. Microbiol. 4, coronavirus nonstructural protein nsp14 as a novel cap 1036–1044 (2005). 121–132 (2006). N7 methyltransferase. Proc. Natl Acad. Sci. USA 10 Feb 72. Vennema, H. et al. Early death after feline infectious 97. Savarin, C., Bergmann, C. C., Hinton, D. R., Ransohoff, 2009 (doi:10.1073/pnas.0808790106). peritonitis virus challenge due to recombinant vaccinia R. M. & Stohlman, S. A. Memory CD4 49. Ivanov, K. A. et al. Major genetic marker of nidoviruses virus immunization. J. Virol. 64, 1407–1409 (1990). T-cell-mediated protection from lethal coronavirus encodes a replicative endoribonuclease. Proc. Natl 73. Guan, Y. et al. Isolation and characterization of viruses encephalomyelitis. J. Virol. 82, 12432–12440 Acad. Sci. USA 101, 12694–12699 (2004). related to the SARS coronavirus from animals in (2008). 50. Decroly, E. et al. Coronavirus nonstructural protein southern China. Science 302, 276–278 (2003). 98. Kim, T. S. & Perlman, S. Virus-specific antibody, in the 16 is a cap-0 binding enzyme possessing 74. Riley, S. et al. Transmission dynamics of the etiological absence of T cells, mediates demyelination in mice (nucleoside-2′O)-methyltransferase activity. J. Virol. 82, agent of SARS in Hong Kong: impact of public health infected with a neurotropic coronavirus. Am. J. Pathol. 8071–8084 (2008). interventions. Science 300, 1961–1966 (2003). 166, 801–809 (2005). 51. Snijder, E. J. et al. Ultrastructure and origin of 75. Chinese, S. M. E. C. Molecular evolution of the SARS 99. Kim, T. S. & Perlman, S. Viral expression of CCL2 is –/– membrane vesicles associated with the severe acute coronavirus during the course of the SARS epidemic in sufficient to induce demyelination in RAG1 mice respiratory syndrome coronavirus replication complex. China. Science 303, 1666–1669 (2004). infected with a neurotropic coronavirus. J. Virol. 79, J. Virol. 80, 5927–5940 (2006). 76. Song, H. D. et al. Cross-host evolution of severe acute 7113–7120 (2005). 52. de Haan, C. A. & Rottier, P. J. Molecular interactions in respiratory syndrome coronavirus in palm civet and 100. Williamson, J. S. & Stohlman, S. A. Effective clearance the assembly of coronaviruses. Adv. Virus Res. 64, human. Proc. Natl Acad. Sci. USA 102, 2430–2435 of mouse hepatitis virus from the central nervous + + 165–230 (2005). (2005). system requires both CD4 and CD8 T cells. J. Virol. 53. Prentice, E., Jerome, W. G., Yoshimori, T., Mizushima, 77. Li, W. et al. Receptor and viral determinants of SARS- 64, 4589–4592 (1990). N. & Denison, M. R. Coronavirus replication complex coronavirus adaptation to human ACE2. EMBO J. 24, 101. Anghelina, D., Pewe, L. & Perlman, S. Pathogenic role formation utilizes components of cellular autophagy. 1634–1643 (2005). for virus-specific CD4 T cells in mice with coronavirus- J. Biol. Chem. 279, 10136–10141 (2004). 78. Sheahan, T., Rockx, B., Donaldson, E., Corti, D. & induced acute encephalitis. Am. J. Pathol. 169, 54. Zhao, Z. et al. Coronavirus replication does not require Baric, R. Pathways of cross-species transmission of 209–222 (2006). the autophagy gene ATG5. Autophagy 3, 581–585 synthetically reconstructed zoonotic severe acute 102. Deming, D. et al. Vaccine efficacy in senescent mice (2007). respiratory syndrome coronavirus. J. Virol. 82, challenged with recombinant SARS-CoV bearing 55. Bechill, J., Chen, Z., Brewer, J. W. & Baker, S. C. 8721–8732 (2008). epidemic and zoonotic spike variants. PLoS Med. 3, Coronavirus infection modulates the unfolded protein 79. Poon, L. L. et al. Identification of a novel coronavirus e525 (2006). response and mediates sustained translational in bats. J. Virol. 79, 2001–2009 (2005). 103. Roberts, A. et al. A mouse-adapted SARS-Coronavirus repression. J. Virol. 82, 4492–4501 (2008). 80. Lau, S. K. et al. Severe acute respiratory syndrome causes disease and mortality in BALB/c mice. PLoS 56. Knoops, K. et al. SARS-coronavirus replication is coronavirus-like virus in Chinese horseshoe bats. Proc. Pathog. 3, e5 (2007). supported by a reticulovesicular network of modified Natl Acad. Sci. USA 102, 14040–14045 (2005). This is the first report showing that a mouse- endoplasmic reticulum. PLoS Biol. 6, e226 (2008). 81. Leung, D. T. et al. Extremely low exposure of a adapted SARS-CoV causes a SARS-like illness in This is an elegant electron microscopic study that community to severe acute respiratory syndrome mice. uses 3D imaging of SARS-CoV-infected cells to coronavirus: false seropositivity due to use of 104. Roberts, A. et al. Aged BALB/c mice as a model for delineate the relationship between virus replication bacterially derived antigens. J. Virol. 80, 8920–8928 increased severity of severe acute respiratory and virus-induced membranous changes. (2006). syndrome in elderly humans. J. Virol. 79, 5833–5838 57. Snijder, E. J., van Tol, H., Roos, N. & Pedersen, K. W. 82. Becker, M. M. et al. Synthetic recombinant bat SARS-like (2005). Non-structural proteins 2 and 3 interact to modify host coronavirus is infectious in cultured cells and in mice. 105. Versteeg, G. A., Bredenbeek, P. J., van den Worm, cell membranes during the formation of the arterivirus Proc. Natl Acad. Sci. USA 105, 19944–19949 (2008). S. H. & Spaan, W. J. Group 2 coronaviruses prevent replication complex. J. Gen. Virol. 82, 985–994 (2001). This manuscript describes the engineering of a immediate early interferon induction by protection of 58. Clementz, M. A., Kanjanahaluethai, A., O’Brien, T. E. & non-cultivable bat SARS-like coronavirus using viral RNA from host cell recognition. Virology 361, Baker, S. C. Mutation in murine coronavirus replication synthetic DNA technology. 18–26 (2007). protein nsp4 alters assembly of double membrane 83. Ren, W. et al. Difference in receptor usage between This report and REF. 106 are the first to suggest vesicles. Virology 375, 118–129 (2008). severe acute respiratory syndrome (SARS) coronavirus that coronaviruses are invisible to the host innate 59. Kanjanahaluethai, A., Chen, Z., Jukneliene, D. & Baker, and SARS-like coronavirus of bat origin. J. Virol. 82, immune response in some cells. S. C. Membrane topology of murine coronavirus 1899–1907 (2008). 106. Zhou, H. & Perlman, S. Mouse hepatitis virus does not replicase nonstructural protein 3. Virology 361, 84. Vijgen, L. et al. Complete genomic sequence of human induce beta interferon synthesis and does not inhibit 391–401 (2007). coronavirus OC43: molecular clock analysis suggests a its induction by double-stranded RNA. J. Virol. 81, 60. Oostra, M. et al. Topology and membrane anchoring of relatively recent zoonotic coronavirus transmission 568–574 (2007). the coronavirus replication complex: Not all event. J. Virol. 79, 1595–1604 (2005). 107. Spiegel, M. et al. Inhibition of beta interferon hydrophobic domains of nsp3 and nsp6 are membrane 85. Alekseev, K. P. et al. Bovine-like coronaviruses isolated induction by severe acute respiratory syndrome spanning. J. Virol. 82, 12392–12405 (2008). from four species of captive wild ruminants are coronavirus suggests a two-step model for activation 61. Oostra, M. et al. Localization and membrane topology homologous to bovine coronaviruses based on of interferon regulatory factor 3. J. Virol. 79, of coronavirus nonstructural protein 4: involvement of complete genomic sequences. J. Virol. 82, 2079–2086 (2005). the early secretory pathway in replication. J. Virol. 81, 12422–12431 (2008). 108. He, R. et al. Activation of AP-1 signal transduction 12323–12336 (2007). 86. Jin, L. et al. Analysis of the genome sequence of an pathway by SARS coronavirus nucleocapsid protein. 62. Garbino, J. et al. A prospective hospital-based study of alpaca coronavirus. Virology 365, 198–203 (2007). Biochem. Biophys. Res. Commun. 311, 870–876 the clinical impact of non-severe acute respiratory 87. Lorusso, A. et al. Gain, preservation, and loss of a (2003). syndrome (Non-SARS)-related human coronavirus group 1a coronavirus accessory glycoprotein. J. Virol. 109. Kopecky-Bromberg, S. A., Martinez-Sobrido, L., infection. Clin. Infect. Dis. 43, 1009–1015 (2006). 82, 10312–10317 (2008). Frieman, M., Baric, R. A. & Palese, P. SARS 63. Gu, J. et al. Multiple organ infection and the 88. Woo, P. C. et al. Comparative analysis of twelve coronavirus proteins Orf 3b, Orf 6, and nucleocapsid pathogenesis of SARS. J. Exp. Med. 202, 415–424 genomes of three novel group 2c and group 2d function as interferon antagonists. J. Virol. 81, (2005). coronaviruses reveals unique group and subgroup 548–557 (2006). 64. Fouchier, R. A. et al. A previously undescribed features. J. Virol. 81, 1574–1585 (2007). 110. Ye, Y., Hauns, K., Langland, J. O., Jacobs, B. L. & coronavirus associated with respiratory disease in 89. Dominguez, S. R., O’Shea, T. J., Oko, L. M. & Holmes, Hogue, B. G. Mouse hepatitis coronavirus A59 humans. Proc. Natl Acad. Sci. USA 101, 6212–6216 K. V. Detection of group 1 coronaviruses in bats in North nucleocapsid protein is a type I interferon antagonist. (2004). America. Emerg. Infect. Dis. 13, 1295–1300 (2007). J. Virol. 81, 2554–2563 (2007). NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 449 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 111. Narayanan, K. et al. Severe acute respiratory essential in controlling neurotropic coronavirus the chemokine mig in pathogenesis. Clin. Infect. Dis. syndrome coronavirus nsp1 suppresses host gene infection irrespective of functional CD8 T cells. J. Virol. 41, 1089–1096 (2005). expression, including that of type I interferon, in 82, 300–310 (2008). 135. Nagata, N. et al. Mouse-passaged severe acute infected cells. J. Virol. 82, 4471–4479 (2008). 122. Cervantes-Barragan, L. et al. Control of coronavirus respiratory syndrome-associated coronavirus leads to 112. Wathelet, M. G., Orr, M., Frieman, M. B. & Baric, R. S. infection through plasmacytoid dendritic cell-derived lethal pulmonary edema and diffuse alveolar damage Severe acute respiratory syndrome coronavirus evades type I interferon. Blood 109, 1131–1137 (2006). in adult but not young mice. Am. J. Pathol. 172, antiviral signaling: role of nsp1 and rational design of 123. Roth-Cross, J. K., Bender, S. J. & Weiss, S. R. Murine 1625–1637 (2008). an attenuated strain. J. Virol. 81, 11620–11633 coronavirus mouse hepatitis virus is recognized by 136. Nagata, N. et al. Participation of both host and virus (2007). MDA5 and induces type I interferon in brain factors in induction of severe acute respiratory 113. Zust, R. et al. Coronavirus non-structural protein 1 is a macrophages/microglia. J. Virol. 82, 9829–9838 syndrome (SARS) in F344 rats infected with SARS major pathogenicity factor: implications for the (2008). coronavirus. J. Virol. 81, 1848–1857 (2007). rational design of coronavirus vaccines. PLoS Pathog. 124. Cervantes-Barragan, L. et al. Type I IFN-mediated 137. de Lang, A. et al. Functional genomics highlights 3, e109 (2007). protection of macrophages and dendritic cells secures differential induction of antiviral pathways in the lungs 114. Frieman, M. et al. Severe acute respiratory syndrome control of murine coronavirus infection. J. Immunol. of SARS-CoV-infected macaques. PLoS Pathog. 3, coronavirus ORF6 antagonizes STAT1 function by 182, 1099–1106 (2009). e112 (2007). sequestering nuclear import factors on the rough 125. He, L. et al. Expression of elevated levels of pro- 138. Baas, T. et al. Genomic analysis reveals age-dependent endoplasmic reticulum/Golgi membrane. J. Virol. 81, inflammatory cytokines in SARS-CoV-infected ACE2 innate immune responses to severe acute respiratory 9812–9824 (2007). cells in SARS patients: relation to the acute lung injury syndrome coronavirus. J. Virol. 82, 9465–9476 115. Hussain, S., Perlman, S. & Gallagher, T. M. Severe and pathogenesis of SARS. J. Pathol. 210, 288–297 (2008). acute respiratory syndrome coronavirus protein 6 (2006). 139. Morahan, G., Balmer, L. & Monley, D. Establishment accelerates murine hepatitis virus infections by more 126. Li, C. K. et al. T cell responses to whole SARS coronavirus of “The Gene Mine”: a resource for rapid identification than one mechanism. J. Virol. 82, 7212–7222 in humans. J. Immunol. 181, 5490–5500 (2008). of complex trait genes. Mamm. Genome 19, (2008). 127. Gu, J. & Korteweg, C. Pathology and pathogenesis of 390–393 (2008). 116. Zhao, J. et al. Severe acute respiratory syndrome-CoV severe acute respiratory syndrome. Am. J. Pathol. 140. Stockman, L. J., Bellamy, R. & Garner, P. SARS: protein 6 is required for optimal replication. J. Virol. 170, 1136–1147 (2007). systematic review of treatment effects. PLoS Med. 3, 83, 2368–2373 (2008). 128. Chen, J. & Subbarao, K. The immunobiology of SARS. e343 (2006). 117. Kamitani, W. et al. Severe acute respiratory syndrome Annu. Rev. Immunol. 25, 443–472 (2007). 141. Gosert, R., Kanjanahaluethai, A., Egger, D., Bienz, K. coronavirus nsp1 protein suppresses host gene 129. Subbarao, K. & Roberts, A. Is there an ideal animal & Baker, S. C. RNA replication of mouse hepatitis expression by promoting host mRNA degradation. model for SARS? Trends Microbiol. 14, 299–303 virus takes place at double-membrane vesicles. Proc. Natl Acad. Sci. USA 103, 12885–12890 (2006). (2006). J. Virol. 76, 3697–3708 (2002). Describes a novel mechanism of viral 130. McCray, P. B. Jr et al. Lethal infection in K18-hACE2 142. Sims, A. C., Ostermann, J. & Denison, M. R. Mouse protein-induced inhibition of host gene expression. mice infected with SARS-CoV. J. Virol. 81, 813–821 hepatitis virus replicase proteins associate with two 118. Devaraj, S. G. et al. Regulation of IRF-3-dependent (2006). distinct populations of intracellular membranes. innate immunity by the papain-like protease domain of This paper and REF. 131 show that mice transgenic J. Virol. 74, 5647–5654 (2000). the severe acute respiratory syndrome coronavirus. for the human SARS-CoV develop a lethal infection J. Biol. Chem. 282, 32208–32221 (2007). characterized by extensive infection of the brain. Acknowledgements 119. Cameron, M. J. et al. Interferon-mediated 131. Tseng, C. T. et al. SARS coronavirus infection of mice Supported in part by research (PO1 AI060699 and RO1 immunopathological events are associated with transgenic for the human angiotensin-converting NS36592) and training (T32 AI007533) grants from the atypical innate and adaptive immune responses in enzyme 2 (hACE2) virus receptor. J. Virol. 81, National Institutes of Health (USA). patients with severe acute respiratory syndrome. 1162–1173 (2006). J. Virol. 81, 8692–8706 (2007). 132. Netland, J., Meyerholz, D. K., Moore, S., Cassell, M. & This report provides a careful description of the Perlman, S. Severe acute respiratory syndrome DATABASES changes in cytokine and chemokine expression that coronavirus infection causes neuronal death in the UniProtKB: http://ca.expasy.org/sprot occurred in patients during the 2002–2003 absence of encephalitis in mice transgenic for human ACE2 | CCL2 | CXCL10 | FGL2 | IL-6 | IL-8 | IRF3 | MDA5 | epidemic. ACE2. J. Virol. 82, 7264–7275 (2008). RAG1 | STAT1 | TLR7 120. Rempel, J. D., Murray, S. J., Meisner, J. & Buchmeier, 133. Lee, D. T. et al. Factors associated with psychosis M. J. Differential regulation of innate and adaptive among patients with severe acute respiratory FURTHER INFORMATION immune responses in viral encephalitis. Virology 318, syndrome: a case-control study. Clin. Infect. Dis. 39, Stanley Perlman’s homepage: http://www.uiowa.edu/ 381–392 (2004). 1247–1249 (2004). microbiology/perlman.shtml 121. Ireland, D. D., Stohlman, S. A., Hinton, D. R., 134. Xu, J. et al. Detection of severe acute respiratory All li Nk S Are Ac TiVe iN THe o Nli Ne Pd F Atkinson, R. & Bergmann, C. C. Type I interferons are syndrome coronavirus in the brain: potential role of 450 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nature Reviews. Microbiology Pubmed Central

Coronaviruses post-SARS: update on replication and pathogenesis

Perlman, Stanley; Netland, Jason
Nature Reviews. Microbiology , Volume 7 (6) – May 11, 2009
Download PDF
12 pages

Loading next page...
 
/lp/pubmed-central/coronaviruses-post-sars-update-on-replication-and-pathogenesis-VQP04v6R04

References (159)

Publisher
Pubmed Central
Copyright
© Nature Publishing Group 2009
ISSN
1740-1526
eISSN
1740-1534
DOI
10.1038/nrmicro2147
Publisher site
See Article on Publisher Site

Abstract

REVIEWS Coronaviruses post-SARS: update on replication and pathogenesis Stanley Perlman and Jason Netland Abstract | Although coronaviruses were first identified nearly 60 years ago, they only received notoriety in 2003 when one of their members was identified as the aetiological agent of severe acute respiratory syndrome. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis. It also highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers. Coronaviruses, a genus in the Coronaviridae family (order encode an additional haemagglutinin-esterase (HE) pro - Prothrombinase Nidovirales; fig. 1), are pleomorphic, enveloped viruses. tein (fig . 2a,b). The HE protein, which may be involved Molecule that cleaves Coronaviruses gained prominence during the severe acute in virus entry or egress, is not required for replica- thrombin, thereby initiating the coagulation cascade. respiratory syndrome (SARS) outbreaks of 2002–2003 tion, but appears to be important for infection of the (ref. 1) . The viral membrane contains the transmembrane natural host . (M) glycoprotein, the spike (S) glycoprotein and the enve - Receptors for several coronaviruses have been iden- lope (E) protein, and surrounds a disordered or flexible, tified (TABLe 1). The prototypical coronavirus, mouse 2,3 probably helical, nucleocapsid. The viral membrane is hepatitis virus (MHV), uses CEACAM1a, a member of unusually thick, probably because the carboxy-terminal the murine carcinoembryonic antigen family, to enter region of the M protein forms an extra internal layer, as cells. Deletion of this protein makes mice resistant to 2 6 revealed by cryo-electron tomography. Coronaviruses infection . Several group 1 coronaviruses use ami- are divided into three groups, and further subdivided nopeptidase N to adhere to host cells, consistent with into subgroups (TABLe 1), based initially on serologic, and their respiratory and enteric tract tropisms (reviewed in more recently on genetic, analyses. With the identification ref. 7 ). SARS-CoV, a group 2 coronavirus, enters host of more distantly related viruses, the taxonomy of these cells through an interaction of the S protein with human viruses is likely to undergo further changes. angiotensin converting enzyme 2 (ACE2) . Strikingly, Coronaviruses contain a single stranded, 5′-capped, human coronavirus-NL63 (HCoV-NL63), which causes positive strand RNA molecule that ranges from 26–32 kb mild disease, also uses ACE2, although it binds to a dif - and that contains at least 6 open reading frames (ORFs). ferent part of the protein than does SARS-coronavirus 9,10 The first ORF (ORF1a/b) comprises approximately two- (SARS-CoV) . ACE2 is postulated to have a protec- thirds of the genome and encodes replicase proteins tive role in the inflamed lung, and SARS-CoV S pr- o (fig . 2a). Translation begins in ORF1a and continues in tein binding to ACE2 is thought to contribute to disease 11,12 ORF1b after a –1 frameshift signal. The large ORF1a and severity . As infection with HCoV-NL63 produces ORF1ab polypeptides, commonly referred to as pp1a mild disease, however, binding to ACE2 by itself cannot Department of Microbiology and pp1ab, respectively, are processed primarily by the be sufficient for this process. and Interdisciplinary pro virally encoded chymotrypsin-like protease 3CL (also The N protein is important for encapsidation of viral Program in Immunology, pro called M or main protease) with additional cleavage RNA and acts as an interferon (IFN) antagonist (see University of Iowa, Iowa City, Iowa 52242, USA. performed by one or two viral papain-like proteases below). Additionally, it causes upregulation o FGL f 2, a Correspondence to S.P. 4 (PLPs), depending on the species of coronavirus . The prothrombinase that contributes to fatal hepatic disease e-mail: stanley-perlman@ majority of the remaining one-third of the genome in mice that are infected with MHV-3 (ref. 13) and that uiowa.edu encodes four structural proteins: S, E, M and nucleo - modifies transforming growth factor-β (TGFβ) signalling doi:10.1038/nrmicro2147 Published online 11 May 2009 capsid (N) proteins. A subset of group 2 coronaviruses in SARS-CoV-infected cells . NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 439 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS RNA polymerase, nsp8, may function as a primase . Order: Nidovirales The nsp3 protease has additional roles in the assembly Family: Coronaviridae of virus replication structures (see below) and possesses Genus: Coronavirus poly(ADP-ribose) binding capabilities, and deubiqui- t Torovirus\Bafinivirus ylating activity in its protease domain, although the role Family: Roniviridae of the latter in virus replication is not yet known . Nsp7, nsp8, nsp9 and nsp10 are postulated to have Genus: Okavirus a role in subgenomic and genomic RNA replication, Family: Arteriviridae and all four proteins are essential for viral replication . Nsp7 and nsp8 form a hexadecameric structure, with Genus: Arterivirus RNA binding activity . The structure of nsp9 also sug- Figure 1 | The Nidoviruses. Phylogenetic relationship of gests that it binds RNA . Mutations in nsp10 inhibit Nature Reviews | Microbiology viruses in the order Nidoviruses. minus strand RNA synthesis, but this effect may be indi- rect, as studies have showed that nsp10 is required for pro 46 The E proteins are small integral membrane proteins proper function of the main viral protease (M ) . with roles in virus morphogenesis, assembly and bud - Nsp14, a bifunctional protein, is a 3′→5′ exonuclease, ding. In the absence of E proteins, virus release is inhib- with a role in maintaining fidelity of RNA transcription , ited completely (in the case of transmissible gastroenteritis and a (guanine-N7)-methyl transferase (N7-MTase), virus (TGEV)) or partially (in the case of SARS-CoV and involved in RNA cap formation . Coronaviruses also 15–17 MHV) . The E protein also possesses ion channel activity , encode a novel uridylate-specific endoribonuclease 18,19 which is required for optimal virus replication . (Nendou ), nsp15, that distinguishes nidoviruses in gen- Interspersed between and in these structural genes eral from other RNA viruses and that is crucial for virus are one to eight genes that encode accessory proteins, replication . Cleavage of RNA by Nendou results in depending on the virus strain. These show no sequence 2′-3′ cyclic phosphate ends, but its function in the virus similarity with other viral or cellular proteins and are life cycle remains unknown. Nsp16 is an S-adenosyl-l- 20–22 not required for virus replication in cultured cells . methionine-dependent RNA (nucleoside-2′O)-methyl However, they are conserved in virus species isolated at transferase (2′O-MTase) and, like nsp14, is involved in cap 23 50 different times and locales (for example, for SARS-CoV), formation . Nsp15 has been postulated to function with which suggests that these proteins have an important role nsp14 and nsp16 in RNA processing or cap production, in replication in the natural host. Several accessory pr- o but this remains to be proven. 24–27 teins are virion-associated , although whether these RNA replication is thought to occur on double- 28 51 proteins are truly structural is controversial. membrane vesicles (DMVs) (fig . 4). Newly synthesized The genes that encode non-replicase proteins are genomic RNA is then incorporated into virions on expressed from a set of ‘nested’ subgenomic mRNAs membranes that are located between the endoplasmic that have common 3′ ends and a common leader that is reticulum (ER) and the Golgi apparatus (ER–Golgi inter - encoded at the 5′ end of genomic RNA. Proteins are pro- mediate compartment (ERGIC); reviewed in ref. 52 ). duced generally only from the first ORF of subgenomic Initial studies suggested that these DMVs assemble using mRNAs, which are produced during minus strand RNA components of the autophagy pathway , but other stud- synthesis. Transcription termination and subsequent ies showed replication proceeded normally and that acquisition of a leader RNA occurs at transcription reg- DMVs were produced in macrophages lacking ATG5, ulatory sequences (TRS), located between ORFs. These a key component of autophagosomes . Thus, whether minus strand subgenomic RNAs serve as templates for autophagy is involved at all or whether its involvement is the production of subgenomic mRNAs (fig . 3), an effi- cell-specific remains uncertain. In addition, the unfolded cient process that results in a high ratio of subgenomic protein response (u PR) is induced during coronavirus 29 55 mRNA to minus strand subgenomic RNA . infections and may contribute to DMV formation . Recent results show that DMVs are likely to Coronavirus replication originate from the ER. u sing electron tomography One consequence of the SARS epidemic was an increase of cryo-fixed SARS-CoV-infected Vero E6 cells and in efforts to understand coronavirus replication and three-dimensional reconstruction imaging, Knoops Primase identify additional possible targets for anti-viral therapy. et al. showed that DMVs are not isolated vesicles, in the case of nsp8, an ORF1 of most coronaviruses encodes 16 proteins that but rather are part of a reticulovesicular network of r NA-dependent r NA polymerase that produces are involved in viral replication (fig . 2a); ORF1 of group 3 modified ER membranes . At later times after infec- r NA primers that are required coronaviruses lacks nsp1 and thus encodes only 15 pro - tions, these networks appear to merge into large for initiation of r NA synthesis teins. The structure of many of these proteins has been single-membrane vesicles. Proteins involved in virus by the main viral r NA solved by X-ray crystallography or nuclear magnetic replication (nsp3, nsp5 and nsp8; TABLe 2) are located polymerase, nsp12. 30–40 resonance, facilitating structure–function studies . mainly outside of DMVs, in adjacent reticular struc - Double-membrane vesicle Functions were predicted and later confirmed for many tures. Double-stranded RNA, representing either rep- A structure that is observed in pro pro of these proteins (TABLe 2), including PL1 and PL2 licative intermediates or ‘dead end’ double-stranded electron micrographs of pro (papain-like proteases) contained in nsp3 and 3CL RNA, was detected primarily in DMVs and, surpris- infected cells and that is pro or M contained in nsp5, the RNA-dependent RNA ingly, no obvious connections between the interior of thought to be the site of virus replication. polymerase nsp12 and the helicase nsp13. A second these vesicles and the cytosol were detected . Thus, it 440 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Table 1 | Representative coronavirus species and their receptors g roup Host Virus c ellular receptor Group 1a Bat BtCoV Unknown Cat FCoV APN Cat FIPV APN Dog CCoV APN Pig TGEV APN Group 1b Human HCoV-229E APN Human HCoV-NL63 Angiotensin-converting enzyme 2 (ACE2) Pig PEDV Unknown Group 1* Rabbit RbCoV Unknown Group 2a Cattle, ruminants, BCoV and related viruses 9-O-acetylated sialic acid alpaca Dog CRCoV Unknown Human HCoV-HKU1 Unknown Human HCoV-OC43 9-O-acetylated sialic acid Mouse MHV Carcinoembryonic antigen adhesion molecule 1 Pig PHEV Unknown Group 2b Bat BtCoV (multiple species) Unknown Human SARS-CoV ACE2 Group 2* Manx shearwaters PCoV Unknown Rat RtCoV Unknown Rat SDAV Unknown Group 3a Chicken IBV Unknown Pheasant PhCoV Unknown Turkey TCoV Unknown Group 3b Beluga whale SW1 Unknown Group 3c Bulbul BuCoV-HKU11 Unknown Thrush ThCoV-HKU12 Unknown Munia MuCoV-HKU13 Unknown Asian leopard cat, ALCCoV Unknown Chinese ferret badger *Due to a lack of sequence data, subgroup has not been assigned. More than 60 bat coronavirus species have been identified and tentatively classified as members of group 1 or group 2 (ref. 91) . ALCCoV, Asian leopard cat coronavirus; APN, aminopeptidase N; BCoV, bovine coronavirus; BtCoV, bat coronavirus; BuCoV, bulbul coronavirus; CCoV, canine coronavirus; CRCoV, canine respiratory coronavirus; FCoV, feline coronavirus; FIPV, feline infectious peritonitis virus; HCoV, human coronavirus; IBV, infection bronchitis virus; MHV, mouse hepatitis virus; MuCoV, munia coronavirus; PCoV, puffinosis coronavirus; PEDV, porcine epidemic diarrhoea virus; PhCoV, pheasant coronavirus; PHEV, porcine hemagglutinating encephalomyelitis virus; RbCoV, rabbit coronavirus; RtCoV, rat coronavirus; SARS-CoV, severe acute respiratory syndrome-associated coronavirus; SDAV, sialodacryoadenitis virus; TCoV, turkey coronavirus; ThCoV, thrush coronavirus; TGEV, transmissible gastroenteritis virus. remains unknown how newly synthesized RNA might but both the amino and carboxyl termini of these proteins be transported to sites of virus assembly, assuming that are in the cytoplasm, suggesting that one hydrophobic RNA transcription occurs in DMVs. region does not span the membrane ; whether this region Formation of DMVs requires membrane curvature, contributes to membrane curvature or has another function and this may be initiated by insertion of specific viral pro - requires further investigation. teins into membranes. Based on studies of equine arteritis virus , a non-coronavirus member of the nidovirus order Coronavirus-mediated diseases (fig. 1) , nsp3 and nsp4 are probably sufficient for DMV for - Before the SARS epidemic of 2002–2003, two human mation. Mutations in nsp4 result in aberrant formation of coronaviruses, HCoV-OC43 and HCoV-229E, were DMVs, further supporting a role for this protein in estab - recognized as important causes of upper respira- lishing sites of virus replication . Nsp6, like nsp3 and nsp4, tory tract infections and were occasionally associated also contains multiple transmembrane regions and ma y with more severe pulmonary disease in the elderly, 59–61 62 be involved in membrane modification . Notably, nsp3 newborn and immunocompromised . SARS-CoV, and nsp6 encode an odd number of hydrophobic domains, unlike HCoV-OC43 and HCoV -229E, causes a severe NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 441 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS a b Group 1: TGEV SARS-CoV virion Leader ORF1a S b7 M 5′ ORF1b 3a E N Poly(A) Group 2: SARS-CoV 8a Leader ORF1a S 3b MN 7a 5′ ORF1b 3a E 6 Poly(A) 7b 8b 9b 12 34 56 78 91011 12 13 14 15 16 ADRP/ RDRP Hel ExoN 2′OMT pro pro PL2 3CL ssRBP NendoU Group 3: IBV 7a/b Poly(A) Leader ORF1a S E 5′ ORF1b MN ssRNA 3a/b Figure 2 | Structure of coronavirus genome and virion. a | Schematic diagram of representative genomes from each of the coronavirus groups. Approximately the first two-thirds of the 26–32 Kb, positive-sense RNA genome encodes a large Nature Reviews | Microbiology polyprotein (ORF1a/b; green) that is proteolytically cleaved to generate 15 or 16 non-structural proteins (nsps; nsps for severe acute respiratory syndrome coronavirus (SARS-CoV) are illustrated). The 3′-end third of the genome encodes four structural proteins — spike (S), membrane (M), envelope (E) and nucleocapsid (N) (all shown in blue) — along with a set of accessory proteins that are unique to each virus species (shown in red). Some group 2 coronaviruses express an additional structural protein, haemagglutinin-esterase (not shown). b | Schematic diagram of the coronavirus virion. 2′OMT, ribose-2′- O-methyltransferase; ExoN, 3′5′ exonuclease; Hel, helicase; IBV, infection bronchitis virus; NendoU, uridylate-specific endoribonuclease; RDRP, RNA-dependent RNA polymerase; ssRBP, single-stranded RNA binding protein; ssRNA, single-stranded RNA; TGEV, transmissible gastroenteritis virus. respiratory disease, and nearly 10% mortality was Although the severe disease forming capabilities of observed in 2002–2003 (ref. 1) . Notable features of the human coronaviruses were only recognized because disease were an apparent worsening of symptoms as the of the SARS epidemic, it was well known that animal virus was cleared (suggesting the disease had an immu- coronaviruses could cause life-threatening disease. nopathological basis), and a lack of contagion until lower TGEV, which causes diarrhoea in piglets, infectious respiratory tract symptoms were apparent. This latter bronchitis virus (IBV), a cause of severe upper respira- feature made control of the epidemic by quarantine fea- tory tract and kidney disease in chickens, and bovine sible, as it simplified identification of infected patients. coronavirus (BCoV), which causes respiratory tract u nlike HCoV-OC43 and HCoV -229E, SARS-CoV also disease and diarrhoea in cattle (‘winter dysentery’ caused systemic disease, with evidence of infection of and ‘shipping fever’), are all economically important the gastrointestinal tract, liver, kidney and brain, among pathogens. Feline infectious peritonitis virus (FIPV), other tissues . Although the virus spread primarily via a virulent feline coronavirus (FCoV), causes an respiratory droplets, infection of the gastrointestinal invariably fatal systemic disease in domestic cats and tract may have facilitated other routes of spread. other felines. u nlike most strains of FCoV, which are The recognition that SARS was caused by a corona- endemic causes of mild diarrhoea, FIPV arises spo- virus intensified the search for other pathogenic coro- radically, most likely by mutation or deletion in felines naviruses associated with human disease, which led to persistently infected with enteric strains of FCoV , the identification of HCoV-NL63 and HCoV-HKu 1. and is macrophage-tropic. These viruses were isolated from hospitalized patients, Perhaps the most convincing explanation for FIPV- either young children with severe respiratory disease mediated disease was suggested by the observation that 64,65 (HCoV-NL63) or elderly patients with underlying progressive waves of virus replication, lymphopenia and 65,66 medical problems (HCoV-HK u 1) . HCoV-NL63 ineffectual T cell responses occurred in feline infectious has infected human populations for centuries, as phylo - peritonitis (FIP) . In conjunction with previous stud- genetic studies show that it diverged from HCoV-229E ies, these results raised the possibility that FIPV infec - nearly 1,000 years ago . HCoV-NL63 and HCoV-HKu 1 tion of macrophages and dendritic cells caused aberrant have worldwide distributions and generally cause mild cytokine and/or chemokine expression and lymphocyte upper respiratory tract diseases, with the exception depletion, resulting in enhanced virus loads and, co- n that HCoV-NL63 is also an aetiological agent of croup. sequently, a fatal outcome. Although this explanation is HCoV-NL63 can be propagated in tissue culture cells, appealing, additional work is needed to prove its valid - and an infectious cDNA clone of this virus was recently ity. Notably, anti-FIPV antibody-mediated enhancement engineered, facilitating future studies . By contrast, has been implicated in pathogenesis, but this has been HCoV-HKu 1 cannot be grown in tissue culture cells, shown only after immunization with S protein express- which makes it imperative that an infectious cDNA ing vaccines ; it has not been shown to play a role in a clone be developed for future studies. natural feline infection. 442 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 23,80 Anti-genome from Chinese horseshoe bats (Rhinolophus spp.) , 3′ 5′ which were also present in the live animal markets, the Leader TRS Minus strand synthesis Body TRS a Replication virus may have recently spread from bats to other mam- mals, such as palm civets, and then to humans (fig . 5a). 5′ Genome 3′ Consistent with a recent spread, antibodies to SARS- CoV were detected at extremely low levels (0.008%) in Nascent minus strand population studies in Hong Kong . Bat SARS-like CoV transferred to leader TRS cannot replicate in cells that express bat ACE2, although productive infection of cells expressing human ACE2 occurs if the RBD of the bat S protein is replaced with 82,83 that of a human isolate . Collectively, these observa - tions suggest that virus spread from bats to other spe- cies. Host cell entry does not occur via ACE2 in bats, Subgenomic mRNA although it does in palm civets and humans. Besides SARS-CoV, there are other examples of coronavirus cross-species transmission. BCoV and Figure 3 | Mechanism of coronavirus replication and transcription. Following entry Nature Reviews | Microbiology into the cell and uncoating, the positive sense RNA genome is translated to generate HCoV-OC43 are similar and the virus may have crossed replicase proteins from open reading frame 1a/b (ORF1a/b). These proteins use the from bovine to human hosts approximately 100 years genome as a template to generate full-length negative sense RNAs, which subsequently ago . BCoV has continued to cross species, as a related serve as templates in generating additional full-length genomes (a). Coronavirus mRNAs virus (99.5% similarity) has been isolated from an alpaca all contain a common 5′ leader sequence fused to downstream gene sequences. These 85,86 with enteritis and from captive wild ruminants leaders are added by a discontinuous synthesis of minus sense subgenomic RNAs using (fig . 5b). Furthermore, canine coronavirus (CCoV), feline genome RNA as a template (reviewed in ref. 29 ). Subgenomic RNAs are initiated at the 3′ and porcine viruses show evidence they have recom- end of the genome and proceed until they encounter one of the transcriptional regulatory bined with each other, indicating that they were present sequences (TRS; red) that reside upstream of most open-reading frames (b). Through in the same host. Recombination events between early base-pairing interactions, the nascent transcript is transferred to the complementary CCoV and FCoV strains (CCoV-I and FCoV-I) and leader TRS (light red) (c) and transcription continues through the 5′ end of the genome (d). These subgenomic RNAs then serve as templates for viral mRNA production (e). an unknown coronavirus resulted in two sets of novel viruses — CCoV-II and FCoV-II. Sequence data suggest that TGEV resulted from a cross-species transmission of Cross-species transmission CCoV-II from an infected canine (fig . 5c). A striking feature of the 2002–2003 SARS epidemic Molecular surveillance studies have identified at least 88 50 was the ability of the SARS-CoV to cross species from 60 novel bat coronaviruses in China , North America , 89,90 91 Himalayan palm civets P ( aguma larvata), raccoon dogs Europe and Africa . These bat CoVs may have orig- (Nyctereutes procyonoides) and Chinese ferret badg- inated from a common source and then subsequently ers (Melogale moschata) to infect human populations diverged as they adapted to growth in different species of (fig . 5a). Transmission occurred in live animal retail (wet) bat; they are now only distantly related to other coron-a markets, where animal handlers became infected. In ret - viruses. These studies also identified several novel avian rospect, it seems that variants of SARS-CoV related to the group 3 coronaviruses that were related to a novel coro - epidemic strain infected human populations in the wet navirus isolated from Asian leopard cats P ( rionailurus markets fairly frequently, as is shown by the high sero - bengalensis) and Chinese ferret badgers sold in illegal positivity rate detected in animal handlers who did not wild animal markets in China , suggesting that this develop SARS-like illnesses . The epidemic began when virus, like SARS-CoV, can cross species. Another novel a physician who was treating personnel in the wet mar - group 3 coronavirus, isolated from a deceased beluga kets became infected and subsequently infected multiple whale (Delphinapterus leucas), is only distantly related contacts . to IBV-like and novel avian coronaviruses, suggesting Genetic analyses of virus isolates from infected palm that it comprises a third subgroup . Thus group 3 coro- civets and humans during the epidemic showed that naviruses, which formerly included only avian viruse s, 75,76 the virus underwent rapid adaptation in both hosts , now consist of at least 3 subgroups and include primarily in the receptor binding domain (RBD) of the viruses that infect mammalian hosts. S protein, to allow more efficient infection of human cells . In particular, mutations K479N and S487T in Immunopathology in coronavirus infections the RBD of the S protein were key to adaptation to the It is generally accepted that the host response is respon - human receptor (ACE2). These results were recently sible for many of the disease manifestations in infections 95,96 confirmed using cell lines expressing civet ACE2 or caused by coronaviruses . This was shown initially in human ACE2 (ref. 78) . mice infected with the neurotropic strains of mouse The obser vation that SARS-CoV could not be hepatitis virus (the j HMV and MHV-A59 strains). detected in either farmed or wild palm civets , together Many attenuated strains of j HMV cause a subacute with evidence of adaptive changes detected in virus iso- or persistent infection in the central nervous system, lated from infected animals, suggested that palm civets with persistence in glia, especially oligodendrocytes. A and other animals in wet markets were not the primary consequence of host efforts to clear the virus is myelin reservoir for the virus. As SARS-like CoV were isolated destruction (demyelination). However, j HMV infection NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 443 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS the onset of virus clearance . Although worsening clini - Table 2 | Coronavirus non-structural proteins and their functions cal disease occurring as a consequence of virus clea- r Protein Functions ance has not been duplicated in any animal model of Nsp1 Host mRNA degradation; translation inhibition; cell cycle arrest; inhibition SARS, the severe disease observed in older patients can of IFN signaling 102–104 be mimicked in SARS-CoV-infected aged mice . Nsp2 Unknown This has been attributed, in part, to a suboptimal T cell pro pro response resulting in delayed kinetics of virus clea -r Nsp3 Papain-like proteases (PL1 , PL2 ) (polyprotein processing); ance. A suboptimal T cell response, occurring as a con - poly(ADP-ribose) binding; DMV formation (?); IFN antagonist; nucleic acid binding; deubiquitinating activity sequence of infection of macrophages or dendritic cells, may also be critical for the immunopathological lethal Nsp4 DMV formation (?) disease that is observed in FIPV-infected felines . Thus, pro pro Nsp5 Main protease (M , 3CL ); polyprotein processing in many instances, host efforts to clear a coronavirus Nsp6 DMV formation (?) infection result in some tissue destruction. Nsp7 Single-stranded RNA binding Evasion of the innate immune response Nsp8 Primase Although anti-viral T cells and antibodies are crucial for Nsp9 Part of replicase complex virus clearance and for the prevention of recrudescence Nsp10 Part of replicase complex (reviewed in ref. 96 ), the efficacy of the innate immune Nsp11 Unknown response determines the extent of initial virus replica - tion and thus the load that the host must overcome to Nsp12 RNA-dependent RNA polymerase clear the infection (fig . 6a). Coronaviruses, like all other Nsp13 Helicase; nucleoside triphosphatase activity; RNA 5′-triphosphatase successful viruses, have developed strategies to counter activity the innate immune response (fig . 6b-d). IFN expres- Nsp14 3′→5′ exoribonuclease; RNA cap formation (guanine-N7)- sion is a crucial component of this initial response, and methyltransferase coronaviruses have developed ‘passive’ and ‘active’ tools Nsp15 Endonuclease to prevent IFN induction and signalling. Interferon is not induced in fibroblasts that are infected with either Nsp16 RNA cap formation (2′O-methytransferase) 105–107 SARS-CoV or MHV . However, in both instances, DMV, double-membrane vesicle; IFN, interferon. treatment of cells with polyinosinic:polycytidylic acid or with other IFN-inducing agents, results in activation of 105,106 of mice that lack T or B cells (sublethally irradiated IFN regulating factor 3 (IRF3) and IFN induction . mice or mice with severe combined immunodeficiency Thus, in these cells, viruses appear to be invisible to or genetically deficient in recombination activating intracellular viral sensors (such as RI- G I, MDA5 and –/– gene 1 (RAG1 )) results, eventually, in death in all TLR3), perhaps because double stranded RNA, a potent mice, but without demyelination. Adoptive transfer of stimulator of the innate immune system, is buried in a + + CD4 or CD8 T splenocytes 7 or 30 days after immu- DMV (fig . 6b). –/– nization with j HMV to infected RAG1 or SCID mice Additionally, viral proteins, in particular nsp1, nsp3, 95–97 results in virus clearance and demyelination . Myelin N protein and the SARS-CoV accessory proteins ORF6 108–113 destruction is also observed if anti-j HMV antibody is and ORF3b, also prevent IFN induction . The N pro- –/– transferred to infected RAG1 mice in the absence tein of MHV inhibits activator protein 1 (AP1) signal- of T cells , or if mice are infected with virus express- ling and protein kinase R (PKR) function, whereas the ing the macrophage chemoattractant CCL2 in the N protein of SARS-CoV also inhibits nuclear facto- rκB 99 108–110 absence of other interventions . In all cases, infiltratin g activation when expressed in transfection assays. macrophages appear to be crucial for virus clearance Whether these inhibitory functions of the N protein and subsequent demyelination; these results suggest are coronavirus or cell-type specific, and whether they that the process of macrophage infiltration can be initi- occur in infected cells, remains to be determined. The ated by T cells, antij-HMV antibody or overexpression ORF6 protein inhibits IFN signalling by binding to of a single macrophage chemoattractant. These results karyopherin-α2, thereby tethering karyopherin-β have been extended to mice with encephalitis caused to cytoplasmic membranes . This, in turn, pre- + + by virulent strains of j HMV. Although CD4 and CD8 vents nuclear translocation of proteins containing 100 115 T cells are both required for virus clearance , partial classical nuclear import signals, including STAT1, a abrogation of the CD4 T cell response (by mutating crucial component of IFNα, IFNβ and IFNγ signalling the immunodominant CD4 T cell epitope jr.M ) pathways. Of note, deletion of ORF6 does not increase Y135Q results in disease amelioration, and virulence is the IFN sensitivity of SARS-CoV , probably because regained if another CD4 T cell epitope is reintroduced mechanisms of IFN antagonism are redundant. into the rj.M genome . Thus acute encephali- SARS-CoV and MHV nsp1 function, at least in part, Y135Q tis, like chronic demyelination, is at least partial ly by degrading host cell mRNA and inhibiting transl-a 111–113,117 mediated by the immune system. tion . Nsp1 also inhibits IFN signalling in both Similar processes may occur in SARS-CoV-infected SARS-CoV- and MHV-infected cells, in part by inhib - 112,113 humans, as pulmonary disease often worsens at 1–2 weeks iting STAT1 phosphorylation . Mutation of nsp1 after onset of respiratory symptoms, concomitant with attenuates SARS-CoV and MHV growth in mice and 444 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS tissue culture cells in the presence of an intact IFN sys - nsp3 nsp5 nsp8 111–113 tem, but not when IFN function is deficient . Nsp3 RER is also an IFN antagonist, and it inhibits phosphorylation VP and nuclear importation of IRF3 (ref. 118) . dsRNA Both MHV and SARS-CoV inhibit IFNα and IFNβ induction and signalling. However, IFN α and/or IFNβ are 119,120 detected in infected mice and humans and mice deficient in IFNα and/or IFNβ receptor expression are 113,121 exquisitely sensitive to MHV infection , showing that IFNα and/or IFNβ has a major role in the anti- Ribosome virus immune response. Reconciling these disparate results, recent studies showed that IFNα is produced in large amounts in SARS-CoV- and MHV-infected plasmacytoid dendritic cells, via a T LR7-dependent mechanism . Furthermore, IFNβ is expressed by mac- rophages and microglia, but not by dendritic cells after MHV infection . Macrophages, and to a lesser extent dendritic cells, are the major targets for IFN α and/or CM IFNβ in MHV-infected mice . In addition to IFN, multiple chemokines and cytokines are also induced as part of the host response to coronaviruses such as MHV, SARS-CoV and FIPV. Cytokines such as interleukin 1 (IL -1), IL-6 and IL-12 and chemokines such as IL-8, CCL2 and CXCL10 are ele- vated in SARS patients. u sing genomics and proteomics, Cameron et al. found that IFNα and/or IFNβ and IFNγ, as well as chemokines such as CXCL10 and CCL2, are DMV elevated at early times post infection in all patients and diminished in those who recovered, accompanied by a Figure 4 | c oronavirus-induced membrane alterations robust anti-virus antibody response . However, levels Nature Reviews | Microbiology as platforms for viral replication. Coronavirus infection of CXCL10, CCL2 and other proinflammatory media- induces the formation of a reticulovesicular network of tors remained elevated and anti-SARS-CoV antibody modified membranes that are thought to be the sites titres were low in those patients who developed severe of virus replication. These modifications, which include disease. SARS-CoV-infected pulmonary epithelial cells double-membrane vesicles (DMVs), vesicle packets (VPs, were the source of at least some of the cytokines and/or single-membrane vesicles surrounded by a shared outer chemokines, such as CCL2, IL-6, IL-1β and tumour membrane) and convoluted membranes (CMs), are all necrosis factor (TNF) . Others have suggested that interconnected and contiguous with the rough a strong T 2 (IL4, IL-5 and IL-10) response correlated endoplasmic reticulum (RER). Viral double-stranded RNA is with a poor outcome . It has been postulated that an mostly localized to the interior of the DMVs and inner vesicles of the VPs, whereas replicase proteins (that is, over-exuberant cytokine response contributed to a poor nsp3, nsp5 and nsp8) are present on the surrounding CM. outcome in patients with SARS in 2002–2003 (reviewed Some nsp8 can be detected inside the DMVs. All in refs 95,127,128 ). Collectively, these results do not membranes are bound by ribosomes. (Figure based on data strongly prove or disprove a role for an exuberant cytokine from r efs. 56,141,142 .) and chemokine response in severe SARS, in part because virus titres could not be determined concomitantly and also because serum levels, but not pulmonary cytokine neuronal infection, accompanied by high cytokine and/ or chemokine levels were measured. or chemokine expression and minimal cellular infilt -ra tion in the brain . Although the severity of the brain Animal models for SARS infection observed in human ACE2 transgenic mice As human SARS has disappeared, the role of an exuber- is greater than that seen in human patients, infection ant (but perhaps appropriate for the titre of the virus) of this organ has been detected in some studies and immune response will need to be addressed using ani - patients who survived SARS had a greater incidence mal models of SARS. Mice, cats, ferrets, macaques and of neurological and psychiatric sequelae than antici - 63,133,134 civet cats are all susceptible to SARS-CoV, but none, pated . The high susceptibility of these mice to with the exception of aged mice, develop severe disease infection with SARS-CoV makes them useful for vac- (reviewed in ref. 129 ). In efforts to develop models that cine and therapeutic trials. Another approach to devel - closely mimic human disease, mice that are transgenic oping an animal model for SARS was to adapt the virus for the expression of human ACE2 were developed and by passage 10–15 times through the lungs of BALB/c 130,131 103,135,136 infected with SARS-CoV . Although these mice mice or rats . Three to six mutations were develop more severe pulmonary disease than non- detected in the adapted viruses, with changes most com- pro transgenic mice, they also develop an overwhelming monly observed in the S protein and in nsp5 (3CL ). NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 445 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Humans Bat Himalayan palm civet Human BtCoV SARS Alpacas Alpaca Cow Human Wild ruminants Wild ruminant (deer) HCoV-OC43BCoV Dog Unknown reservoir Dog Pig CCoV-I Unknown CCoV-II TGEV coronavirus Common ancestor Cat Cat Cat FCoV-I FCoV-II FCoV-II Figure 5 | c ross-species transmission of coronaviruses. a | Severe acute respiratory syndrome (SARS)-like bat coronavirus (BtCoV) spread and adapted to wild animals such as the Himalayan palm civet that was sold as food in Chinese wet markets. The virus frequently spread to animal handlers in these markets, but caused minimal or no disease. Further adaptation resulted in strains that replicated efficiently in the human host, caused disease and could spread from person to person. b | Human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV) are closely related and it is thought that the virus originated in one species and then crossed species. BCoV has also spread to numerous other animals, such as alpaca and wild ruminants. c | Feline coronavirus I (FCoV-I) and canine coronavirus I (CCoV-I) are thought to share a common ancestor. CCoV-I underwent recombination with an unknown coronavirus to give rise to canine coronavirus II Nature Reviews | Microbiology (CCoV-II). CCoV-II in turn underwent recombination with FCoV-I (in an unknown host) to give rise to feline coronavirus II (FCoV-II). CCoV-II probably also spread to pigs, resulting in transmissible gastroenteritis virus (TGEV). The adapted virus caused extensive pulmonary infec- mediators, such as IL-6, TNF, CXCL10 and CCL2, tion and disease was most severe in aged animals. accompanied by slower kinetics of virus clearance These viruses will be useful for studies of pathogenesis and worse outcomes in aged compared to young and for vaccine and therapeutic trials. animals , paralleling disease patterns in patients Some models have been tested on the genomic with SARS . These two studies also showed changes and proteomic level. Studies of SARS-CoV infected in expression of proteins that are involved in cell macaques showed that several chemokines and/or growth, cycling, cell-to-cell signalling and deve- lop cytokines, such as IL-6, IL-8, CXCL10 and CCL2, ment and death. It will be important to determine as well as IFNα, IFNβ and IFNγ, were upregu - whether these changes are useful as a ‘fingerprint’ lated . These animals recovered, showing that the for SARS or whether they represent generalized same inflammator y mediators that are associated responses to pulmonary stress. with severe human disease are also produced as part of the inflammatory response in animals that Future directions mount an appropriate response. Genomics studies Perhaps the most important insight made over the of mice infected with the u rbani strain of SARS- past several years is that coronaviruses have and will CoV showed continued expression of inflammatory likely continue to cross between species and cause 446 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Fibroblasts and dendritic cells a b IFNα and IFNβ Plasmacytoid dendritic cell Macrophage Virus STAT1, Viral RNA Viral RNA release STAT2 DMV dsRNA dsRNA MDA5 ssRNA RIG1 MDA5 Virus TLR7 IRF3 NF-κB production NF-κB AP1 AP1 IRF7 NF-κB IRF3 N, nsp1, nsp3, IFNα and IFNβ ORF6, IFNα and IFNβ ORF3b Nucleus Nucleus Nucleus cd Systemic disease due to cytokine storm B cell Infiltration of T cell infected tissue Macrophage Cytokines Microglial cell Figure 6 | inefficient activation of the type 1 interferon response, and immunopathological disease, in Nature Reviews | Microbiology coronavirus infections. a | Coronaviruses, as exemplified by severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV), induce a type 1 interferon (IFN) response in plasmacytoid dendritic cells (pDC) and macrophages, via TLR7- and MDA5-dependent pathways, respectively. b | IFNα and/or IFNβ is not produced in either SARS-CoV fibroblasts or DCs, partly because coronavirus macromolecules appear to be invisible to immune sensors. Additionally, coronaviruses encode proteins that actively inhibit IFNα and/or IFNβ expression (such as nucleocapsid (N) protein, nsp3, ORF6 and ORF3b) or signalling through the type 1 IFN receptor (such as N, nsp1, ORF6 and ORF3b). c | Consequently, the kinetics of virus clearance is delayed, with subsequent robust T and B cell and cytokine and/or chemokine responses. d | This pro-inflammatory response results in immunopathological disease that occurs during the process of virus clearance. In MHV-infected mice, virus clearance involves recruitment of activated macrophages and microglia to sites of virus infection, leading to demyelination. Similar mechanisms with exuberant cytokine production may function in the lungs of SARS-CoV-infected humans, leading to severe pulmonary disease (adult respiratory distress syndrome, ARDS). AP1, activator protein 1; DMV, double-membrane vesicle; dsRNA, double-stranded RNA; NF-κB, nuclear factor-κB; ssRNA, single-stranded RNA. disease in unrelated hosts. This disease may be mild, species, the virus also needed to evolve strategies to like the disease caused by the SARS-like CoV that evade the innate immune response of the new hosts. was transmitted to animal handlers in wet markets in One future goal will be to further delineate how the China, but it may be severe, as illustrated by the tran- s virus evades the immune response and better under- mission that triggered the SARS epidemic. Further, stand its interaction with the T and B cell responses, SARS-CoV appeared to use an entirely new recep- both in the original host (bats), in which disease tor when it crossed species from bats to palm civets appears to be mild, and in humans and experimentall y and humans. As part of this transmission to a new infected animals. NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 447 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS Although coronaviruses use host proteins as part roles of these proteins in virus replication still require Collaborative cross mice of their replication strategies, it has also become clear additional investigation. Progress in these fields will A panel of 1,000 recombinant inbred mouse strains derived that immune, metabolic, stress, cell cycling and other take advantage of new methodologies that allow from 8 genetically diverse pathways are activated by infection. Assessing the bio- detailed observations of both fixed and living cells at founder strains. The crosses logical function of these pathways in virus replication high resolution. were designed for complex and in disease outcome will be critical. Determining Finally, no effective treatments exist for any coro - trait analysis and will be useful the extent to which virus–host interactions are coro- navirus infections, including SARS ; vaccines, even for identifying and establishing the role of host genes in s Ars navirus-specific and organ-specific will be possible, for animal coronaviruses, are not effective; and live pathogenesis. using genomics and proteomics, as well as new rea - attenuated vaccines are prone to recombination with gents and collaborative cross mice. The collaborative circulating coronaviruses. One future goal will be to cross, a panel of approximately 1,000 recombinant translate new information about the structure and inbred mouse strains derived from 8 founder strains, function of coronavirus proteins into specific anti- will be useful for analyses of complex genetic traits. virus therapies. Also, development of live, attenuated, u sing sophisticated microscopy and biochemi- safe vaccines that do not recombine in the wild is cal approaches, details of coronavirus replication in another goal, made more feasible as more is learned infected cells have been revealed. However, these new about basic coronavirus biology. Over the past few results have led to a new set of questions about the years, the development of new technologies has sim - relationship between sites of viral RNA replication plified the identification of novel coronaviruses; the and virus assembly. Furthermore, although putative next major goals will be to understand viral path -o functions have been assigned to many of the proteins genesis and to design effective coronavirus vaccines encoded by the large ORF1 replicase gene, the precise and therapies. 1. Peiris, J. S., Guan, Y. & Yuen, K. Y. Severe acute 14. Zhao, X., Nicholls, J. M. & Chen, Y. G. Severe acute 28. Neuman, B. W. et al. Proteomics analysis unravels the respiratory syndrome. Nature Med. 10, S88–S97 respiratory syndrome-associated coronavirus functional repertoire of coronavirus nonstructural (2004). nucleocapsid protein interacts with Smad3 and protein 3. J. Virol. 82, 5279–5294 (2008). 2. Barcena, M. et al. Cryo-electron tomography of mouse modulates transforming growth factor-β signaling. 29. Sawicki, S. G., Sawicki, D. L. & Siddell, S. G. A hepatitis virus: insights into the structure of the J. Biol. Chem. 283, 3272–3280 (2008). contemporary view of coronavirus transcription. coronavirion. Proc. Natl Acad. Sci. USA 106, 15. DeDiego, M. L. et al. A severe acute respiratory J. Virol. 81, 20–29 (2007). 582–587 (2009). syndrome coronavirus that lacks the E gene is 30. Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R. & This paper and Ref. 3 use novel methodologies to attenuated in vitro and in vivo. J. Virol. 81, Hilgenfeld, R. Coronavirus main proteinase (3CLpro) obtain detailed models of the structure of the 1701–1713 (2007). structure: basis for design of anti-SARS drugs. Science intact coronavirion. 16. Kuo, L. & Masters, P. S. The small envelope protein E 300, 1763–1767 (2003). 3. Neuman, B. W. et al. Supramolecular architecture of is not essential for murine coronavirus replication. 31. Zhai, Y. et al. Insights into SARS-CoV transcription and severe acute respiratory syndrome coronavirus J. Virol. 77, 4597–4608 (2003). replication from the structure of the nsp7-nsp8 revealed by electron cryomicroscopy. J. Virol. 80, 17. Ortego, J., Ceriani, J. E., Patino, C., Plana, J. & hexadecamer. Nature Struct. Mol. Biol. 12, 980–986 7918–7928 (2006). Enjuanes, L. Absence of E protein arrests transmissible (2005). 4. Masters, P. S. The molecular biology of coronaviruses. gastroenteritis coronavirus maturation in the secretory 32. Chatterjee, A. et al. Nuclear magnetic resonance Adv. Virus Res. 66, 193–292 (2006). pathway. Virology 368, 296–308 (2007). structure shows that the severe acute respiratory 5. Lissenberg, A. et al. Luxury at a cost? Recombinant 18. Madan, V., Garcia Mde, J., Sanz, M. A. & Carrasco, L. syndrome coronavirus-unique domain contains a mouse hepatitis viruses expressing the accessory Viroporin activity of murine hepatitis virus E protein. macrodomain fold. J. Virol. 83, 1823–1836 (2009). hemagglutinin esterase protein display reduced fitness FEBS Lett. 579, 3607–3612 (2005). 33. Joseph, J. S. et al. Crystal structure of nonstructural in vitro. J. Virol. 79, 15054–15063 (2005). 19. Wilson, L., McKinlay, C., Gage, P. & Ewart, G. SARS protein 10 from the severe acute respiratory syndrome –/– 6. Hemmila, E. et al. Ceacam1a mice are completely coronavirus E protein forms cation-selective ion coronavirus reveals a novel fold with two zinc-binding resistant to infection by murine coronavirus mouse channels. Virology 330, 322–331 (2004). motifs. J. Virol. 80, 7894–7901 (2006). hepatitis virus A59. J. Virol. 78, 10156–10165 20. Yount, B. et al. Severe acute respiratory syndrome 34. Joseph, J. S. et al. Crystal structure of a monomeric (2004). coronavirus group-specific open reading frames encode form of severe acute respiratory syndrome coronavirus 7. Weiss, S. R. & Navas-Martin, S. Coronavirus nonessential functions for replication in cell cultures endonuclease nsp15 suggests a role for pathogenesis and the emerging pathogen severe acute and mice. J. Virol. 79, 14909–14922 (2005). hexamerization as an allosteric switch. J. Virol. 81, respiratory syndrome coronavirus. Microbiol. Mol. 21. Ontiveros, E., Kuo, L., Masters, P. S. & Perlman, S. 6700–6708 (2007). Biol. Rev. 69, 635–664 (2005). Inactivation of expression of gene 4 of mouse hepatitis 35. Peti, W. et al. Structural genomics of the severe acute 8. Li, W. et al. Angiotensin-converting enzyme 2 is a virus strain JHM does not affect virulence in the respiratory syndrome coronavirus: nuclear magnetic functional receptor for the SARS coronavirus. Nature murine CNS. Virology 290, 230–238 (2001). resonance structure of the protein nsP7. J. Virol. 79, 426, 450–454 (2003). 22. de Haan, C. A., Masters, P. S., Shen, X., Weiss, S. & 12905–12913 (2005). This is the first report identifying the SARS-CoV Rottier, P. J. The group-specific murine coronavirus 36. Saikatendu, K. S. et al. Structural basis of severe acute receptor. genes are not essential, but their deletion, by reverse respiratory syndrome coronavirus ADP-ribose-1′′- 9. Li, W. et al. The S proteins of human coronavirus NL63 genetics, is attenuating in the natural host. Virology phosphate dephosphorylation by a conserved domain and severe acute respiratory syndrome coronavirus 296, 177–189 (2002). of nsP3. Structure 13, 1665–1675 (2005). bind overlapping regions of ACE2. Virology 367, 23. Li, W. et al. Bats are natural reservoirs of SARS-like 37. Almeida, M. S., Johnson, M. A., Herrmann, T., Geralt, 367–374 (2007). coronaviruses. Science 310, 676–679 (2005). M. & Wuthrich, K. Novel β -barrel fold in the nuclear 10. Hofmann, H. et al. Human coronavirus NL63 employs This paper and REF. 80 show that SARS-CoV magnetic resonance structure of the replicase the severe acute respiratory syndrome coronavirus probably originated in bats. nonstructural protein 1 from the severe acute receptor for cellular entry. Proc. Natl Acad. Sci. USA 24. Fischer, F., Peng, D., Hingley, S. T., Weiss, S. R. & respiratory syndrome coronavirus. J. Virol. 81, 102, 7988–7993 (2005). Masters, P. S. The internal open reading frame within 3151–3161 (2007). 11. Kuba, K. et al. A crucial role of angiotensin converting the nucleocapsid gene of mouse hepatitis virus 38. Serrano, P. et al. Nuclear magnetic resonance enzyme 2 (ACE2) in SARS coronavirus-induced lung encodes a structural protein that is not essential for structure of the N-terminal domain of nonstructural injury. Nature Med. 11, 875–879 (2005). viral replication. J. Virol. 71, 996–1003 (1997). protein 3 from the severe acute respiratory syndrome This report suggests that interactions between the 25. Huang, C., Peters, C. J. & Makino, S. Severe acute coronavirus. J. Virol. 81, 12049–12060 (2007). SARS-CoV S protein and its receptor may respiratory syndrome coronavirus accessory protein 6 is 39. Ricagno, S. et al. Crystal structure and mechanistic contribute to disease progression, in addition to a virion-associated protein and is released from 6 protein- determinants of SARS coronavirus nonstructural facilitating virus entry. expressing cells. J. Virol. 81, 5423–5426 (2007). protein 15 define an endoribonuclease family. Proc. 12. Imai, Y. et al. Angiotensin-converting enzyme 2 26. Ito, N. et al. Severe acute respiratory syndrome Natl Acad. Sci. USA 103, 11892–11897 (2006). protects from severe acute lung failure. Nature 436, coronavirus 3a protein is a viral structural protein. 40. Su, D. et al. Dodecamer structure of severe acute 112–116 (2005). J. Virol. 79, 3182–3186 (2005). respiratory syndrome coronavirus nonstructural 13. Ning, Q. et al. Induction of prothrombinase fgl2 by 27. Schaecher, S. R., Mackenzie, J. M. & Pekosz, A. The protein nsp10. J. Virol. 80, 7902–7908 (2006). the nucleocapsid protein of virulent mouse hepatitis ORF7b protein of severe acute respiratory syndrome 41. Snijder, E. J. et al. Unique and conserved features of virus is dependent on host hepatic nuclear coronavirus (SARS-CoV) is expressed in virus-infected genome and proteome of SARS-coronavirus, an early factor-4α. J. Biol. Chem. 278, 15541–15549 cells and incorporated into SARS-CoV particles. split-off from the coronavirus group 2 lineage. J. Mol. (2003). J. Virol. 81, 718–731 (2007). Biol. 331, 991–1004 (2003). 448 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 42. Imbert, I. et al. A second, non-canonical RNA- 65. van der Hoek, L. et al. Identification of a new human 90. Gloza-Rausch, F. et al. Detection and prevalence dependent RNA polymerase in SARS coronavirus. coronavirus. Nature Med. 10, 368–373 (2004). patterns of group I coronaviruses in bats, northern EMBO J. 25, 4933–4942 (2006). 66. Woo, P. C. et al. Characterization and complete Germany. Emerg. Infect. Dis. 14, 626–631 (2008). 43. Ratia, K. et al. Severe acute respiratory syndrome genome sequence of a novel coronavirus, coronavirus 91. Tong, S. et al. Detection of novel SARS-like and other coronavirus papain-like protease: structure of a viral HKU1, from patients with pneumonia. J. Virol. 79, coronaviruses in bats from Kenya. Emerg. Infect. Dis. deubiquitinating enzyme. Proc. Natl Acad. Sci. USA 884–895 (2005). 15, 482–485 (2009). 103, 5717–5722 (2006). 67. Pyrc, K. et al. Mosaic structure of human coronavirus 92. Woo, P. C. et al. Comparative analysis of complete 44. Deming, D. J., Graham, R. L., Denison, M. R. & Baric, NL63, one thousand years of evolution. J. Mol. Biol. genome sequences of three novel avian coronaviruses R. S. Processing of open reading frame 1a replicase 364, 964–973 (2006). reveals a novel group 3c coronavirus. J. Virol. 83, proteins nsp7 to nsp10 in murine hepatitis virus strain This manuscript delineates molecular evolution of 908–917 (2009). A59 replication. J. Virol. 81, 10280–10291 (2007). HCoV-229E and HCoV-NL63. 93. Dong, B. Q. et al. Detection of a novel and highly 45. Egloff, M. P. et al. The severe acute respiratory 68. van der Hoek, L. et al. Croup is associated with the divergent coronavirus from asian leopard cats and syndrome-coronavirus replicative protein nsp9 is a novel coronavirus NL63. PLoS Med. 2, e240 (2005). Chinese ferret badgers in Southern China. J. Virol. 81, single-stranded RNA-binding subunit unique in the RNA 69. Donaldson, E. F. et al. Systematic assembly of a full- 6920–6926 (2007). virus world. Proc. Natl Acad. Sci. USA 101, length infectious clone of human coronavirus NL63. 94. Mihindukulasuriya, K. A., Wu, G., St Leger, J., 3792–3796 (2004). J. Virol. 82, 11948–11957 (2008). Nordhausen, R. W. & Wang, D. Identification of a 46. Donaldson, E. F., Sims, A. C., Graham, R. L., Denison, 70. Rottier, P. J., Nakamura, K., Schellen, P., Volders, H. & novel coronavirus from a beluga whale by using a M. R. & Baric, R. S. Murine hepatitis virus replicase Haijema, B. J. Acquisition of macrophage tropism panviral microarray. J. Virol. 82, 5084–5088 protein nsp10 is a critical regulator of viral RNA during the pathogenesis of feline infectious peritonitis (2008). synthesis. J. Virol. 81, 6356–6368 (2007). is determined by mutations in the feline coronavirus 95. Perlman, S. & Dandekar, A. A. Immunopathogenesis 47. Eckerle, L. D., Lu, X., Sperry, S. M., Choi, L. & Denison, spike protein. J. Virol. 79, 14122–14130 (2005). of coronavirus infections: implications for SARS. M. R. High fidelity of murine hepatitis virus replication 71. de Groot-Mijnes, J. D., van Dun, J. M., van der Most, Nature Rev. Immunol. 5, 917–927 (2005). is decreased in nsp14 exoribonuclease mutants. R. G. & de Groot, R. J. Natural history of a recurrent 96. Bergmann, C. C., Lane, T. E. & Stohlman, S. A. J. Virol. 81, 12135–12144 (2007). feline coronavirus infection and the role of cellular Coronavirus infection of the central nervous system: 48. Chen, Y. et al. Functional screen reveals SARS immunity in survival and disease. J. Virol. 79, host–virus stand-off. Nature Rev. Microbiol. 4, coronavirus nonstructural protein nsp14 as a novel cap 1036–1044 (2005). 121–132 (2006). N7 methyltransferase. Proc. Natl Acad. Sci. USA 10 Feb 72. Vennema, H. et al. Early death after feline infectious 97. Savarin, C., Bergmann, C. C., Hinton, D. R., Ransohoff, 2009 (doi:10.1073/pnas.0808790106). peritonitis virus challenge due to recombinant vaccinia R. M. & Stohlman, S. A. Memory CD4 49. Ivanov, K. A. et al. Major genetic marker of nidoviruses virus immunization. J. Virol. 64, 1407–1409 (1990). T-cell-mediated protection from lethal coronavirus encodes a replicative endoribonuclease. Proc. Natl 73. Guan, Y. et al. Isolation and characterization of viruses encephalomyelitis. J. Virol. 82, 12432–12440 Acad. Sci. USA 101, 12694–12699 (2004). related to the SARS coronavirus from animals in (2008). 50. Decroly, E. et al. Coronavirus nonstructural protein southern China. Science 302, 276–278 (2003). 98. Kim, T. S. & Perlman, S. Virus-specific antibody, in the 16 is a cap-0 binding enzyme possessing 74. Riley, S. et al. Transmission dynamics of the etiological absence of T cells, mediates demyelination in mice (nucleoside-2′O)-methyltransferase activity. J. Virol. 82, agent of SARS in Hong Kong: impact of public health infected with a neurotropic coronavirus. Am. J. Pathol. 8071–8084 (2008). interventions. Science 300, 1961–1966 (2003). 166, 801–809 (2005). 51. Snijder, E. J. et al. Ultrastructure and origin of 75. Chinese, S. M. E. C. Molecular evolution of the SARS 99. Kim, T. S. & Perlman, S. Viral expression of CCL2 is –/– membrane vesicles associated with the severe acute coronavirus during the course of the SARS epidemic in sufficient to induce demyelination in RAG1 mice respiratory syndrome coronavirus replication complex. China. Science 303, 1666–1669 (2004). infected with a neurotropic coronavirus. J. Virol. 79, J. Virol. 80, 5927–5940 (2006). 76. Song, H. D. et al. Cross-host evolution of severe acute 7113–7120 (2005). 52. de Haan, C. A. & Rottier, P. J. Molecular interactions in respiratory syndrome coronavirus in palm civet and 100. Williamson, J. S. & Stohlman, S. A. Effective clearance the assembly of coronaviruses. Adv. Virus Res. 64, human. Proc. Natl Acad. Sci. USA 102, 2430–2435 of mouse hepatitis virus from the central nervous + + 165–230 (2005). (2005). system requires both CD4 and CD8 T cells. J. Virol. 53. Prentice, E., Jerome, W. G., Yoshimori, T., Mizushima, 77. Li, W. et al. Receptor and viral determinants of SARS- 64, 4589–4592 (1990). N. & Denison, M. R. Coronavirus replication complex coronavirus adaptation to human ACE2. EMBO J. 24, 101. Anghelina, D., Pewe, L. & Perlman, S. Pathogenic role formation utilizes components of cellular autophagy. 1634–1643 (2005). for virus-specific CD4 T cells in mice with coronavirus- J. Biol. Chem. 279, 10136–10141 (2004). 78. Sheahan, T., Rockx, B., Donaldson, E., Corti, D. & induced acute encephalitis. Am. J. Pathol. 169, 54. Zhao, Z. et al. Coronavirus replication does not require Baric, R. Pathways of cross-species transmission of 209–222 (2006). the autophagy gene ATG5. Autophagy 3, 581–585 synthetically reconstructed zoonotic severe acute 102. Deming, D. et al. Vaccine efficacy in senescent mice (2007). respiratory syndrome coronavirus. J. Virol. 82, challenged with recombinant SARS-CoV bearing 55. Bechill, J., Chen, Z., Brewer, J. W. & Baker, S. C. 8721–8732 (2008). epidemic and zoonotic spike variants. PLoS Med. 3, Coronavirus infection modulates the unfolded protein 79. Poon, L. L. et al. Identification of a novel coronavirus e525 (2006). response and mediates sustained translational in bats. J. Virol. 79, 2001–2009 (2005). 103. Roberts, A. et al. A mouse-adapted SARS-Coronavirus repression. J. Virol. 82, 4492–4501 (2008). 80. Lau, S. K. et al. Severe acute respiratory syndrome causes disease and mortality in BALB/c mice. PLoS 56. Knoops, K. et al. SARS-coronavirus replication is coronavirus-like virus in Chinese horseshoe bats. Proc. Pathog. 3, e5 (2007). supported by a reticulovesicular network of modified Natl Acad. Sci. USA 102, 14040–14045 (2005). This is the first report showing that a mouse- endoplasmic reticulum. PLoS Biol. 6, e226 (2008). 81. Leung, D. T. et al. Extremely low exposure of a adapted SARS-CoV causes a SARS-like illness in This is an elegant electron microscopic study that community to severe acute respiratory syndrome mice. uses 3D imaging of SARS-CoV-infected cells to coronavirus: false seropositivity due to use of 104. Roberts, A. et al. Aged BALB/c mice as a model for delineate the relationship between virus replication bacterially derived antigens. J. Virol. 80, 8920–8928 increased severity of severe acute respiratory and virus-induced membranous changes. (2006). syndrome in elderly humans. J. Virol. 79, 5833–5838 57. Snijder, E. J., van Tol, H., Roos, N. & Pedersen, K. W. 82. Becker, M. M. et al. Synthetic recombinant bat SARS-like (2005). Non-structural proteins 2 and 3 interact to modify host coronavirus is infectious in cultured cells and in mice. 105. Versteeg, G. A., Bredenbeek, P. J., van den Worm, cell membranes during the formation of the arterivirus Proc. Natl Acad. Sci. USA 105, 19944–19949 (2008). S. H. & Spaan, W. J. Group 2 coronaviruses prevent replication complex. J. Gen. Virol. 82, 985–994 (2001). This manuscript describes the engineering of a immediate early interferon induction by protection of 58. Clementz, M. A., Kanjanahaluethai, A., O’Brien, T. E. & non-cultivable bat SARS-like coronavirus using viral RNA from host cell recognition. Virology 361, Baker, S. C. Mutation in murine coronavirus replication synthetic DNA technology. 18–26 (2007). protein nsp4 alters assembly of double membrane 83. Ren, W. et al. Difference in receptor usage between This report and REF. 106 are the first to suggest vesicles. Virology 375, 118–129 (2008). severe acute respiratory syndrome (SARS) coronavirus that coronaviruses are invisible to the host innate 59. Kanjanahaluethai, A., Chen, Z., Jukneliene, D. & Baker, and SARS-like coronavirus of bat origin. J. Virol. 82, immune response in some cells. S. C. Membrane topology of murine coronavirus 1899–1907 (2008). 106. Zhou, H. & Perlman, S. Mouse hepatitis virus does not replicase nonstructural protein 3. Virology 361, 84. Vijgen, L. et al. Complete genomic sequence of human induce beta interferon synthesis and does not inhibit 391–401 (2007). coronavirus OC43: molecular clock analysis suggests a its induction by double-stranded RNA. J. Virol. 81, 60. Oostra, M. et al. Topology and membrane anchoring of relatively recent zoonotic coronavirus transmission 568–574 (2007). the coronavirus replication complex: Not all event. J. Virol. 79, 1595–1604 (2005). 107. Spiegel, M. et al. Inhibition of beta interferon hydrophobic domains of nsp3 and nsp6 are membrane 85. Alekseev, K. P. et al. Bovine-like coronaviruses isolated induction by severe acute respiratory syndrome spanning. J. Virol. 82, 12392–12405 (2008). from four species of captive wild ruminants are coronavirus suggests a two-step model for activation 61. Oostra, M. et al. Localization and membrane topology homologous to bovine coronaviruses based on of interferon regulatory factor 3. J. Virol. 79, of coronavirus nonstructural protein 4: involvement of complete genomic sequences. J. Virol. 82, 2079–2086 (2005). the early secretory pathway in replication. J. Virol. 81, 12422–12431 (2008). 108. He, R. et al. Activation of AP-1 signal transduction 12323–12336 (2007). 86. Jin, L. et al. Analysis of the genome sequence of an pathway by SARS coronavirus nucleocapsid protein. 62. Garbino, J. et al. A prospective hospital-based study of alpaca coronavirus. Virology 365, 198–203 (2007). Biochem. Biophys. Res. Commun. 311, 870–876 the clinical impact of non-severe acute respiratory 87. Lorusso, A. et al. Gain, preservation, and loss of a (2003). syndrome (Non-SARS)-related human coronavirus group 1a coronavirus accessory glycoprotein. J. Virol. 109. Kopecky-Bromberg, S. A., Martinez-Sobrido, L., infection. Clin. Infect. Dis. 43, 1009–1015 (2006). 82, 10312–10317 (2008). Frieman, M., Baric, R. A. & Palese, P. SARS 63. Gu, J. et al. Multiple organ infection and the 88. Woo, P. C. et al. Comparative analysis of twelve coronavirus proteins Orf 3b, Orf 6, and nucleocapsid pathogenesis of SARS. J. Exp. Med. 202, 415–424 genomes of three novel group 2c and group 2d function as interferon antagonists. J. Virol. 81, (2005). coronaviruses reveals unique group and subgroup 548–557 (2006). 64. Fouchier, R. A. et al. A previously undescribed features. J. Virol. 81, 1574–1585 (2007). 110. Ye, Y., Hauns, K., Langland, J. O., Jacobs, B. L. & coronavirus associated with respiratory disease in 89. Dominguez, S. R., O’Shea, T. J., Oko, L. M. & Holmes, Hogue, B. G. Mouse hepatitis coronavirus A59 humans. Proc. Natl Acad. Sci. USA 101, 6212–6216 K. V. Detection of group 1 coronaviruses in bats in North nucleocapsid protein is a type I interferon antagonist. (2004). America. Emerg. Infect. Dis. 13, 1295–1300 (2007). J. Virol. 81, 2554–2563 (2007). NATu RE REVIEWS | Microbiology VOLu M E 7 | ju NE 2009 | 449 © 2009 Macmillan Publishers Limited. All rights reserved REVIEWS 111. Narayanan, K. et al. Severe acute respiratory essential in controlling neurotropic coronavirus the chemokine mig in pathogenesis. Clin. Infect. Dis. syndrome coronavirus nsp1 suppresses host gene infection irrespective of functional CD8 T cells. J. Virol. 41, 1089–1096 (2005). expression, including that of type I interferon, in 82, 300–310 (2008). 135. Nagata, N. et al. Mouse-passaged severe acute infected cells. J. Virol. 82, 4471–4479 (2008). 122. Cervantes-Barragan, L. et al. Control of coronavirus respiratory syndrome-associated coronavirus leads to 112. Wathelet, M. G., Orr, M., Frieman, M. B. & Baric, R. S. infection through plasmacytoid dendritic cell-derived lethal pulmonary edema and diffuse alveolar damage Severe acute respiratory syndrome coronavirus evades type I interferon. Blood 109, 1131–1137 (2006). in adult but not young mice. Am. J. Pathol. 172, antiviral signaling: role of nsp1 and rational design of 123. Roth-Cross, J. K., Bender, S. J. & Weiss, S. R. Murine 1625–1637 (2008). an attenuated strain. J. Virol. 81, 11620–11633 coronavirus mouse hepatitis virus is recognized by 136. Nagata, N. et al. Participation of both host and virus (2007). MDA5 and induces type I interferon in brain factors in induction of severe acute respiratory 113. Zust, R. et al. Coronavirus non-structural protein 1 is a macrophages/microglia. J. Virol. 82, 9829–9838 syndrome (SARS) in F344 rats infected with SARS major pathogenicity factor: implications for the (2008). coronavirus. J. Virol. 81, 1848–1857 (2007). rational design of coronavirus vaccines. PLoS Pathog. 124. Cervantes-Barragan, L. et al. Type I IFN-mediated 137. de Lang, A. et al. Functional genomics highlights 3, e109 (2007). protection of macrophages and dendritic cells secures differential induction of antiviral pathways in the lungs 114. Frieman, M. et al. Severe acute respiratory syndrome control of murine coronavirus infection. J. Immunol. of SARS-CoV-infected macaques. PLoS Pathog. 3, coronavirus ORF6 antagonizes STAT1 function by 182, 1099–1106 (2009). e112 (2007). sequestering nuclear import factors on the rough 125. He, L. et al. Expression of elevated levels of pro- 138. Baas, T. et al. Genomic analysis reveals age-dependent endoplasmic reticulum/Golgi membrane. J. Virol. 81, inflammatory cytokines in SARS-CoV-infected ACE2 innate immune responses to severe acute respiratory 9812–9824 (2007). cells in SARS patients: relation to the acute lung injury syndrome coronavirus. J. Virol. 82, 9465–9476 115. Hussain, S., Perlman, S. & Gallagher, T. M. Severe and pathogenesis of SARS. J. Pathol. 210, 288–297 (2008). acute respiratory syndrome coronavirus protein 6 (2006). 139. Morahan, G., Balmer, L. & Monley, D. Establishment accelerates murine hepatitis virus infections by more 126. Li, C. K. et al. T cell responses to whole SARS coronavirus of “The Gene Mine”: a resource for rapid identification than one mechanism. J. Virol. 82, 7212–7222 in humans. J. Immunol. 181, 5490–5500 (2008). of complex trait genes. Mamm. Genome 19, (2008). 127. Gu, J. & Korteweg, C. Pathology and pathogenesis of 390–393 (2008). 116. Zhao, J. et al. Severe acute respiratory syndrome-CoV severe acute respiratory syndrome. Am. J. Pathol. 140. Stockman, L. J., Bellamy, R. & Garner, P. SARS: protein 6 is required for optimal replication. J. Virol. 170, 1136–1147 (2007). systematic review of treatment effects. PLoS Med. 3, 83, 2368–2373 (2008). 128. Chen, J. & Subbarao, K. The immunobiology of SARS. e343 (2006). 117. Kamitani, W. et al. Severe acute respiratory syndrome Annu. Rev. Immunol. 25, 443–472 (2007). 141. Gosert, R., Kanjanahaluethai, A., Egger, D., Bienz, K. coronavirus nsp1 protein suppresses host gene 129. Subbarao, K. & Roberts, A. Is there an ideal animal & Baker, S. C. RNA replication of mouse hepatitis expression by promoting host mRNA degradation. model for SARS? Trends Microbiol. 14, 299–303 virus takes place at double-membrane vesicles. Proc. Natl Acad. Sci. USA 103, 12885–12890 (2006). (2006). J. Virol. 76, 3697–3708 (2002). Describes a novel mechanism of viral 130. McCray, P. B. Jr et al. Lethal infection in K18-hACE2 142. Sims, A. C., Ostermann, J. & Denison, M. R. Mouse protein-induced inhibition of host gene expression. mice infected with SARS-CoV. J. Virol. 81, 813–821 hepatitis virus replicase proteins associate with two 118. Devaraj, S. G. et al. Regulation of IRF-3-dependent (2006). distinct populations of intracellular membranes. innate immunity by the papain-like protease domain of This paper and REF. 131 show that mice transgenic J. Virol. 74, 5647–5654 (2000). the severe acute respiratory syndrome coronavirus. for the human SARS-CoV develop a lethal infection J. Biol. Chem. 282, 32208–32221 (2007). characterized by extensive infection of the brain. Acknowledgements 119. Cameron, M. J. et al. Interferon-mediated 131. Tseng, C. T. et al. SARS coronavirus infection of mice Supported in part by research (PO1 AI060699 and RO1 immunopathological events are associated with transgenic for the human angiotensin-converting NS36592) and training (T32 AI007533) grants from the atypical innate and adaptive immune responses in enzyme 2 (hACE2) virus receptor. J. Virol. 81, National Institutes of Health (USA). patients with severe acute respiratory syndrome. 1162–1173 (2006). J. Virol. 81, 8692–8706 (2007). 132. Netland, J., Meyerholz, D. K., Moore, S., Cassell, M. & This report provides a careful description of the Perlman, S. Severe acute respiratory syndrome DATABASES changes in cytokine and chemokine expression that coronavirus infection causes neuronal death in the UniProtKB: http://ca.expasy.org/sprot occurred in patients during the 2002–2003 absence of encephalitis in mice transgenic for human ACE2 | CCL2 | CXCL10 | FGL2 | IL-6 | IL-8 | IRF3 | MDA5 | epidemic. ACE2. J. Virol. 82, 7264–7275 (2008). RAG1 | STAT1 | TLR7 120. Rempel, J. D., Murray, S. J., Meisner, J. & Buchmeier, 133. Lee, D. T. et al. Factors associated with psychosis M. J. Differential regulation of innate and adaptive among patients with severe acute respiratory FURTHER INFORMATION immune responses in viral encephalitis. Virology 318, syndrome: a case-control study. Clin. Infect. Dis. 39, Stanley Perlman’s homepage: http://www.uiowa.edu/ 381–392 (2004). 1247–1249 (2004). microbiology/perlman.shtml 121. Ireland, D. D., Stohlman, S. A., Hinton, D. R., 134. Xu, J. et al. Detection of severe acute respiratory All li Nk S Are Ac TiVe iN THe o Nli Ne Pd F Atkinson, R. & Bergmann, C. C. Type I interferons are syndrome coronavirus in the brain: potential role of 450 | ju NE 2009 | VOLu M E 7 w w w.nature.com/reviews/micro © 2009 Macmillan Publishers Limited. All rights reserved

Journal

Nature Reviews. Microbiology – Pubmed Central

Published: May 11, 2009

There are no references for this article.