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Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites... 5764–5777 Nucleic Acids Research, 2006, Vol. 34, No. 20 Published online 13 October 2006 doi:10.1093/nar/gkl722 Characterization of a nucleocapsid-like region Lys,2 and of two distinct primer tRNA binding sites in the endogenous retrovirus Gypsy 1 1 Caroline Gabus, Roland Ivanyi-Nagy, Julien Depollier , Alain Bucheton , Alain Pelisson and Jean-Luc Darlix* LaboRetro, Unite ´ de Virologie Humaine, INSERM, IFR 128, Ecole Normale Supe ´ rieure de Lyon, 46 Alle ´ e d’Italie, 69364 LYON Cedex 07, France and Institut de Ge ´ ne ´ tique Humaine, 141, rue de la Cardonille, 34396 MONTPELLIER Cedex 5, France Received August 28, 2006; Revised and Accepted September 18, 2006 with simple retroviruses, such as vertebrate gammaretro- ABSTRACT viruses and utilize similar basic mechanisms for their Mobile LTR-retroelements comprising retroviruses and replication. Genome replication proceeds via the conversion LTR-retrotransposons form a large part of eukaryotic of the single-stranded genomic RNA into a double-stranded genomes. Their mode of replication and abundance DNA copy with two LTRs by reverse transcriptase (RT), favour the notion that they are major actors in eukaryote followed by integration into the host genome by integrase evolution. The Gypsy retroelement can spread in the (1). By virtue of this copy-and-paste mechanism, retrotrans- posons are thought to have efficiently invaded eukaryotic germ line of the fruit fly Drosophila melanogaster via genomes. Being a large part of eukaryotic genomes, these both env-independent and env-dependent processes. LTR-retroelements are viewed as major players in eukaryote Thus, Gypsy is both an active retrotransposon and an evolution (2). infectious retrovirus resembling the gammaretrovirus A large number of studies on the early replication steps MuLV. However, unlike gammaretroviruses, the Gypsy of lentiviruses and gammaretroviruses, such as HIV-1 Gag structural precursor is not processed into Matrix, and MoMuLV, respectively, have been carried out in in Capsid and Nucleocapsid (NC) proteins. In contrast, it vitro reconstituted systems and in cell culture [reviewed in has features in common with Gag of the ancient yeast (3–5)]. Collectively the findings show that reverse transcrip- TY1 retroelement. These characteristics of Gypsy make tion occurs within the virion nucleocapsid (NC) structure it a very interesting model to study replication of a formed of the genomic RNA coated by molecules of NC retroelement at the frontier between ancient retrotrans- protein, and starts by NC-mediated annealing of a specific posons and retroviruses. We investigated Gypsy repli- cellular primer tRNA to a unique 5 genomic primer binding site (PBS), followed by RT-directed cDNA synthesis during cation using an in vitro model system and transfection which RT is assisted by NC (3–5). Much less studies have of insect cells. Results show that an unstructured been carried out on the early replication steps of ancient domain of Gypsy Gag has all the properties of a retrotransposons, such as TYs of yeast. Although a similar retroviral NC. This NC-like peptide forms ribonu- basic mechanism appears to operate, such as RT-directed cleoparticle-like complexes upon binding Gypsy RNA cDNA synthesis by extension of a specific cellular tRNA Lys,2 and directs the annealing of primer tRNA to two annealed to a genomic PBS within a ribonucleoprotein struc- distinct primer binding sites (PBS) at the genome 5 and ture, several important differences were discovered. The 0 0 3 ends. Only the 5 PBS is indispensable for cDNA genomic PBS is not unique but multipartite in TY1 and 0 0 synthesis in vitro and in Drosophila cells. TY3, and unique genomic 5 –3 interactions appear to exert a control over reverse transcription and consequently on the genetic amplification of these retrotransposons (6–8). As it is well documented for HIV-1, NC protein in its INTRODUCTION mature form plays critical roles in the conversion of the genomic RNA into proviral DNA by RT through Retrotransposons and retroviruses belong to a large family of specific and tight interactions with the genomic RNA, mobile genetic elements called long terminal repeat (LTR) Lys,3 primer tRNA , RT and the newly made cDNA [reviewed containing retroelements (1). Ancient retrotransposons, such in (3–5)]. Interestingly, retrotransposon NC proteins can as yeast transposons (TY) share common genetic features *To whom correspondence should be addressed. Tel: +33 4 72 72 81 69; Fax: +33 4 72 72 87 77; Email: [email protected] 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5765 either contain a canonical CCHC zinc finger RNA-binding of Gypsy, including the modified 3 LTR, was subcloned motif and be processed by protease cleavage of the Gag into pBluescript II KS(-) (Stratagene) to produce clone polyprotein, such as in TY3, or else Gag is not processed DNA #p18. The LTR+130UTR plasmid (18), kindly provided and does not contain a zinc finger motif, such as in TY1 by S. Jensen, was used as a template to PCR amplify the (9–11). Nevertheless, the retroviral NC functions appear to 130 bp long copia enhancer with the following KpnI and be conserved in these retrotransposons since the C-terminal XhoI primers: 5 -GGGGTACCCAGTCCATGCCTAATAA- 0 0 region of TY1 Gag chaperones the annealing of primer AC-3 and 5 -ACCGCTCGAGCTGAGAAGGAAATAATT- Met,i 0 0 tRNA to the 5 multipartite PBS, mediates TY1 RNA TC-3 . dimerization, and assists cDNA synthesis by the homologous The amplified DNA fragment was cut with KpnI and XhoI RT (12). and ligated to the 5 end of the 6.4 kb XhoI–EcoRI Gypsy Gypsy is a retroelement present in the germ line of the fruit fragment of pDm111 into the pBluescript II KS(-) vector, fly Drosophila melanogaster and can spread via cell-free yielding construct DNA #p19. The 800 bp EcoRI–BamHI viral infection. Thus, Gypsy can be considered both as an fragment from DNA #p18 was inserted into DNA #p19, active retrotransposon and an infectious retrovirus (13,14). giving rise to the full-length wild-type (WT) Gypsy construct. In agreement with this notion, the genetic structure of Mutant plasmid DNAs used for transfection. A frameshift Gypsy is similar to that of the murine gammaretrovirus starting at codon 40 of the Gag gene was obtained by filling MuLV with Gag, Pol and Env flanked by two LTRs in with Klenow polymerase the NcoI site of the WT plasmid. (15,16). However, the Gypsy Gag structural protein is not The 1.2 kb XhoI–KpnI fragment from WT was subcloned processed into Matrix, Capsid and NC proteins (B.V. Syomin into pBluescript II KS(-) to delete the putative 5 PBS and A. Pelisson, unpublished data), which is reminiscent of sequence (TGGCGCCCAAC) using the QuikChange Gag of the yeast TY1. system (Stratagene) with oligonucleotides used for pBSG8- These functional and genetic features of Gypsy make it a CG2 (Supplementary Table 1). The XhoI–NcoI 1 kb mutated very attractive model to study replication of a mobile genetic fragment was then excised and swapped with the correspond- element, which is at the frontier between ancient retrotrans- ing wild-type fragment of the WT plasmid, generating the D5 posons and retroviruses. To that end we set up an in vitro PBS Gypsy DNA. The same procedure was used to delete the replication system using Gypsy RNAs representing the 0 0 0 four 3 nt (CAAC) of the 5 PBS. The 3 PBS sequence 0 0 genomic RNA 5 and 3 regions, and cellular primer Lys,2 (CCACGACCCTG) was deleted from DNA #p18 using tRNA , and investigated their interactions with a putative the QuikChange system (Stratagene) with the oligonu- NC-like domain in Gag. Here we report that an unstructured cleotides used for pBSG8-CG4 (Supplementary Table 1). domain of Gypsy Gag has the hallmarks of an active retrovi- The modified EcoRI–BamHI fragment was then inserted ral NC. This NC-like domain forms ribonucleoparticle-like into DNA #p19 to yield the D3 PBS Gypsy DNA. All complexes upon binding Gypsy RNA in vitro. In addition, Lys,2 DNA constructs were verified by sequencing. two distinct primer tRNA binding sites of 11 nt were 0 0 identified at the 5 and 3 ends of the genome. We also Proteins and peptides found that the Gypsy NC-like peptide can direct the annealing Lys,2 0 of tRNA to these Gypsy PBSs but only the 5 PBS The TYA1-D NC-like peptide was obtained by opfp chemical appears to be indispensable for cDNA synthesis in vitro synthesis and purified by high-performance liquid chromatog- and in Drosophila cells. raphy (HPLC) as described before (12,19). The murine leukemia virus (MLV) and HIV-1 NC basic peptides were also obtained by opfp chemical synthesis and purified to homogeneity as described before (19–22). Using the one MATERIALS AND METHODS letter code representation, the MLV-NC peptide is: Plasmid construction RQGGERRRSQLDRDQCAYCKEKGHWAKDCPRKPRGP- PRAT where the unique zinc finger is underlined, and the Template DNA pBSG8. The XhoI fragment (39 to 6949) of HIV-1 NC peptide is: TVKCFNCGKEGHIAKNCRAPRKK- Gypsy DNA (GenBank accession no. M12927) was inserted 0 GCWKCGKEGHQMKDCTERQ where the two zinc fingers into the SalI site of the Bluescribe F . The clone was selected 0 are underlined. All peptides were dissolved at 1 mg/ml in a so that the EcoRI site of the polylinker was at the 5 end. 0 0 buffer containing 30 mM HEPES (pH 6.5), 30 mM NaCl Plasmid DNAs encoding Gypsy 5 and 3 RNAs were con- and 0.1 mM ZnCl . MLV RT was from Invitrogen. structed by cloning a high fidelity PCR generated fragment DNA encoding the NC-like region of Gypsy Gag was containing two restriction sites at its ends, using the pBSG8 obtained by PCR amplification using pBSG8 as template clone DNA as template and the oligonucleotide primers (see above) and inserted into pIVEX2.4d (Roche) using the described in Supplementary Table 1. Lys,2 SacII and BamHI sites. The sequence of the Gypsy NC-like The tRNA encoding DNA was constructed as peptide is shown in Figure 2B. In addition, a His-tag was pre- described previously (17). sent at its N-terminus (MSGSHHHHHHSSGIEGRG). The Wild-type plasmid DNA used for transfection. Wild-type con- peptide was expressed in Escherichia coli after overnight structs pDm111 (15) and 111xw (Nathalie, unpublished data) induction at 18 C since it proved to be highly toxic at were kindly provided by N. Lyubomirskaya. They both con- temperatures above 20 C. The peptide was purified in dena- tain the same functional Gypsy/mdg4 element except that, in turing conditions on Ni-NTA column (Qiagen) and eluted 111xw, a PvuI site replaced the XhoI restriction site of the with 100 mM NaH PO , 10 mM Tris–HCl, 8 M urea at 2 4 0 0 3 LTR. The EcoRI–PstI fragment containing the 816 3 nt pH 4.5. Fractions containing the peptide with the highest Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5766 Nucleic Acids Research, 2006, Vol. 34, No. 20 degree of purity were further diluted with 3 vol of 30 mM Synthesis of Gypsy cDNA in vitro HEPES (pH 6.5), 30 mM NaCl and 0.1 mM ZnCl and chro- 8 32 2 0 0 0 Gypsy 5 or 5 –3 RNA (5 · 10 M) and P-labelled matographed on an Amicon ultra-4 column. The peptide was Lys,2 7 tRNA (1 · 10 M) were incubated with nucleic acid stored at 1 mg/ml in the above buffer. Note that all buffers chaperone (at protein concentrations indicated in the figure were extensively degassed before use. legend) at room temperature for 10 min in 10 ml buffer con- taining 20 mM Tris–HCl (pH 7.0), 30 mM NaCl, 0.1 mM MgCl , 5 mM DTT, 0.01 mM ZnCl and 10 U RNasin DNA templates and RNA synthesis 2 2 (Promega) (20–22). Plasmid DNAs for 5 RNA synthesis were generated by The reaction volume was then increased to 25 ml by addi- digesting pBSG8-CG1 (wt) and pBSG8-CG2 (DPBS) with tion of 100 U of MLV RT (Invitrogen), 0.25 mM of each 0 0 0 XmnI (position 433). DNAs for 3 RNA and 5 –3 RNA syn- dNTP, 30 mM NaCl and 2.8 mM MgCl . Incubation was thesis were obtained by digesting pBSG8-CG3 to CG8 for 5 min at room temperature, and then 1 h at 30 Cto (wt and mutants) with NsiI (position 6941), treated by the allow synthesis of Gypsy minus strand cDNA. Reactions Klenow polymerase to remove the 3 strand overhang before were stopped by adding SDS/EDTA (0.5%/5 mM final con- in vitro transcription. centrations) and heated for 3 min at 60 C. Proteins were Lys,2 DNA template for tRNA synthesis was obtained by removed by phenol–chloroform extraction and nucleic acids Lys,2 digesting the tRNA plasmid with BstNI in order to gen- were precipitated by ethanol. Samples were resuspended in erate the primer tRNA ending at CCA. 10 ml formamide buffer (97% formamide, 1 mM EDTA, All RNAs were prepared by in vitro transcription with T7 0.02% bromophenol blue and 0.02% xylene cyanol), RNA polymerase according to the manufacturer’s instruc- denatured for 2 min at 95 C and resolved by denaturing tions (Promega). Gypsy RNAs were further purified by 8% PAGE-7 M urea in 0.5· TBE. Subsequently the gel spin-column chromatography (S-300 HR, Amersham Bio- was fixed, dried and autoradiographed. 5 P-labelled sciences) and dissolved at 1 mg/ml in sterile water. FX174 DNA HinfI markers (Promega) were used for size Lys,2 Primer tRNA was purified by PAGE in 7 M urea and determination. 0.5· TBE, recovered and dissolved at 0.05 mg/ml, and sub- sequently heat-denatured and slowly cooled down in the Inhibition of self-primed cDNA synthesis in vitro presence of 1 mM MgCl for proper folding. Primer Lys,2 32 Gypsy 5 RNA (5 · 10 M) was incubated with the NC-like tRNA was labelled by incorporation of [ P]UMP during peptide as described above but in the absence of the tRNA transcription. primer. For cDNA synthesis, the reaction volume was increased to 25 ml by addition of 100 U of MLV RT (Invitro- Binding of the Gypsy NC peptide to RNA gen), 30 mM NaCl and 2.8 mM MgCl , and nucleotides 32 8 0 0 (0.25 mM dATP, dGTP, dTTP, 25 mM dCTP and 20 mCi P-labelled Gypsy 5 or 3 RNA (5 · 10 M) was incubated [ P]dCTP). Incubation, product purification and gel analysis with Gypsy NC, TYA1-D, MLV-NC or HIV-1 NC at protein were the same as for initiation of cDNA synthesis by primer to nucleotide molar ratios as indicated in the figure legend, at tRNA (see section above). 30 C for 5 min in 10 ml assays containing 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.1 mM MgCl , 5 mM DTT, DNA transfection into Drosophila cells and 0.01 mM ZnCl and 8 U RNasin (Promega). Reactions Southern blotting were stopped by adding 5 mM EDTA and centrifuged for 30 min at 13 000 r.p.m. at 4 C. Supernatants were completely Exponentially growing Drosophila hydei Dh33 adherent cells removed by pipetting. Subsequently, P radioactivity in the were grown at 26 C in Schneider medium supplemented with supernatant and pellet fractions was monitored in a Packard 10% fetal calf serum (FCS). About 3 · 10 cells were seeded 1900 TR scintillation counter. in 9.6 cm wells containing 2 ml medium. They were trans- fected 24 h later with 0.8 mg of plasmid DNA, according to Lys,2 the protocol recommended by Qiagen: 100 ml DNA in EC Primer tRNA annealing to Gypsy RNA buffer, 6.4 ml enhancer solution, 20 ml Effectene and 600 ml 8 32 Lys,2 Gypsy RNA, wt or mutant (5 · 10 M), and P-tRNA culture supernatant were successively mixed at 10 min inter- (6 · 10 M) were incubated with nucleic acid chaperone (at vals and deposited on the remaining 1.4 ml of culture. Forty- concentrations indicated in figure legends) at 30 C for 5 min eight hours later, 2 ml of medium were added to the cells, in 10 ml assays containing 20 mM Tris–HCl (pH 7.5), 30 mM which were transferred into a 25 cm flask. Three days NaCl, 0.1 mM MgCl , 5 mM DTT, 0.01 mM ZnCl and 8 U after transfection with the LTR+130UTR control plasmid 2 2 RNasin (Promega). Reactions were stopped by addition of (18), up to 10% of the cells scored positive for X-Gal SDS/EDTA (0.5%/5 mM final concentrations) and proteins staining. were subsequently removed by proteinase K digestion (3 Transfected cells were collected at 72 h, then washed twice mg) at room temperature for 10 min and RNAs were phenol– with 8 ml PBS and the pellet was resuspended in 200 ml chloroform extracted. RNAs were analysed by 1.3% native [10 mM Tris–HCl (pH 8), 1 mM EDTA and 10 mg·ml agarose gel electrophoresis in 0.5· TBE. The gel was fixed RNase I). Lysis was achieved by adding 200 ml [200 mM with 10% trichloroacetic acid, dried and autoradiographed. Tris–HCl (pH 8), 200 mM EDTA and 2% SDS] and the 5 P-labelled FX174 DNA HinfI markers (Promega) were mixture was incubated for 30 min at 70 C. Proteins were used for size determination (data not shown). Quantifications precipitated by addition of 60 ml 3 M KoAc (pH 5.2) and were carried out by laser scanning as described before (8). incubating the mixture for 30 min at 0 C. The supernatant Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5767 was successively extracted with phenol/chloroform (1:1) and of well characterized RNA-binding domain(s) with CCHC chloroform/isoamyl alcohol (24:1) and adding 0.3 M sodium zinc fingers. acetate (pH 5.2) and 70% ethanol precipitated the DNA. Relatively long unstructured regions can be predicted from Half of each extract was digested with 60 U of the amino acid sequence with considerable accuracy. We used methylation-sensitive DpnI restriction enzyme (New England the DisProt VL3-H predictor [http://www.ist.temple.edu/ Biolabs), ethanol precipitated and then split into four parts, disprot/predictor.php, (35)] to assess the probability of disor- which were incubated with restriction enzymes as indicated der in Gypsy Gag. As shown in Figure 1A, the N-terminal in Figure 9. and central regions of Gag are predicted to be mostly ordered Identical amounts of nuclease restricted DNA were run on and do not show a distinctive basic character. However, an a 0.7% agarose gel in TBE 1·, partially depurinated, dena- approximately 100 amino acid region at the C-terminal end tured and capillary transferred in 10· SSC on a Nytran N of Gag (between 300 and 400 amino acid) contains predomi- membrane (Schleicher & Schuell). Hybridization with a nantly basic amino acid clusters in a putatively disordered randomly primed DNA probe—Gypsy coordinates 3097– environment, a pattern characteristic of retroviral NC proteins 5759 (16)—was performed in 50% formamide, 5· SSC, 5· [(34) and data not shown]. In agreement with this prediction, Denhardt’s solution, 0.1% SDS, at 42 C. After washing at an arginine-rich region (355–389 amino acid) has been previ- 55 C in (0.1· SSC and 0.5% SDS), the membrane was ously proposed to act as a possible RNA-binding domain of exposed in a Storm 820 phosphorimager (Molecular Gypsy Gag (36). Dynamics). The Gag region encompassing residues 289–404 was PCR-amplified and cloned into the pIVEX2.4d vector. The corresponding peptide was found to be toxic in E.coli and to aggregate (data not shown). To circumvent these difficul- RESULTS ties it was necessary to express the peptide at 18 C. Due to a Identification of a NC-like region in Gypsy Gag (ORF1) very low level of expression (Figure 1B, lane 1) we had to The NC proteins of retroviruses and retroelements are small insert a His-tag at the N-terminus in order to achieve an basic proteins with nucleic acid chaperone activities, gener- efficient purification and to obtain a peptide at about 95% ated upon processing of the Gag polyprotein by the viral pro- purity (Figure 1B, lane 2). This Gypsy Gag peptide was tease (3). Most NC proteins are characterized by the presence found to bind Gypsy RNA and tRNA with a high affinity of one or two Cx Cx Hx C motifs, called CCHC zinc fingers similar to the NC-like peptide derived from the yeast retro- 2 4 4 (23), constituting a specific RNA-binding motif (4,24). How- transposon TY1 Gag, termed TYA1-D (see Figure 2) (data ever, a number of LTR-containing retroelements—including not shown). This prompted us to investigate its RNA Gypsy—lack zinc fingers and their Gag is not processed by condensing and chaperoning properties using in vitro gener- 0 0 the protease into matrix, capsid and NC proteins ated RNAs mimicking the 5 and 3 regions of the Gypsy [(12,25,26), and B.V. Syomin and A. Pelisson, unpublished genome (Figure 3). data]. Nevertheless, NC functions may be conserved even in these cases, as evidenced by the Gag protein of the yeast TY1 retrotransposon. Indeed the Gag C-terminal domain of The Gypsy NC peptide has RNA-binding and TY1 possesses nucleic acid binding and chaperoning proper- condensing properties Met,i ties, promoting replication primer tRNA annealing to the RNA genome and initiation of cDNA synthesis by RT (12). One hallmark of viral RNA chaperone proteins, such as Since NC activity appears to be ubiquitous in functional retroviral NC proteins is that they cause RNA aggregation retroelements (27), we decided to search for an NC-like and condensation (12). Under in vitro conditions, large domain in Gypsy Gag (ORF1). Owing to the absence of a ribonucleoprotein complexes are formed and can be recov- consensus RNA-binding motif or considerable homology to ered by centrifugation. To analyse the ability of Gypsy NC known NC proteins [(26) and data not shown], we took to form large ribonucleoproteins, Gypsy NC– P-RNA advantage of physico-chemical features probably shared by complexes were formed at 30 C and recovered by centri- all NC and NC-like proteins. fugation at 13 000 r.p.m. (see Materials and Methods). The regions flanking the zinc fingers in NC proteins Radioactivity in the supernatant and in the nucleoprotein- contain low amino acid complexity regions and are rich in containing pellet was monitored. Results show that the proline and basic amino acid residues (26). Proline and Gypsy NC (G-NC) caused RNA condensation at a protein charged amino acids as well as low complexity were sug- to nucleotide molar ratio of 1:6, both with the Gypsy 5 gested to contribute to the flexibility of a polypeptide chain and 3 RNAs (Figure 4). At a protein to nt molar ratio (28). Indeed, the solution structures of NCp7 and NCp10 of 1:12, 50–60% of the RNA was found in nucleoprotein show that these proteins—with the exception of the short complexes (Figure 4) in agreement with the fact that about zinc finger region(s) (see Materials and Methods)—are half of the Gypsy P-RNA was bound to Gypsy NC as poorly structured when not bound to nucleic acids (29–31). indicated by gel retardation assays (data not shown). This flexibility (i.e. intrinsic disorder) might be an important The TYA1-D peptide (12) was used as a positive control prerequisite both for the multimerization of NC proteins (32) and, as expected, had a similar RNA condensing activity and for their RNA chaperone function (33,34). Based on (Figure 4). these considerations, we propose that the presence of a The observed RNA aggregation cannot be exclusively markedly basic, disordered region in retroviral/retroelement attributed to charge neutralization caused by the basic Gag may be a good indicator of NC function in the absence amino acids in Gypsy NC (pI ¼ 10.46) and TYA1-D Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5768 Nucleic Acids Research, 2006, Vol. 34, No. 20 Figure 1. Identification of the NC-like region in Gypsy Gag. (A) Putative NC-like region of Gypsy Gag. The bar chart illustrates the charge distribution of amino acids in Gypsy Gag (ORF1), as calculated by the charge function of the EMBOSS package, using default parameters and a sliding window of 5 amino acids. Computer prediction of disordered regions (solid line) was obtained using the DisProt VL3-H predictor (http://www.ist.temple.edu/disprot/predictor.php) (35). An amino acid with a disorder score above or equal to 0.5 is considered to be in a disordered environment, while below 0.5 in an ordered environment. (B) Expression and purification of the NC-like peptide. The C-terminal part of ORF1 contains several basic amino acid clusters and is predicted to be disordered in its unliganded state, a common feature of retroelement NC proteins (see text for explanation). This region encompassing 289–404 amino acids was amplified and cloned into the pIVEX2.4d vector. The peptide was amplified in E.coli and obtained in a purified form (see Materials and Methods). CL is the clear E.coli lysate (lane 1) and E the purified Gypsy NC-like peptide (lane 2). Note that the peptide was expressed only at a very low level in the CL. Purification steps allowed us to obtain a peptide more than 95% pure (lane 2). The minor protein band corresponds to an N-terminal cleavage product of the Gypsy NC-like peptide. Figure 2. The Gypsy NC and TYA1-D peptides. Amino acid sequences of the NC-like peptides of the yeast retrotransposon TY1 (TYA1-D) and Gypsy NC are shown according to the one letter code. Note the presence of a large number of basic residues and histidines, and the absence of a canonical ‘CCHC’ Zinc finger motif as in vertebrate retroviruses and in TY3 NCp9 (26). Both peptides were found to be basic and disordered according to computer predictions (see Figure 1A). Lys,2 The Gypsy NC peptide directs annealing of tRNA to (pI ¼ 10.24), since the similarly basic MLV-NC and HIV-1 0 0 two 5 and 3 PBSs NC peptides (with pI values of 10.2 and 9.42, respectively) failed to induce considerable RNA aggregation of the In order to set up an in vitro Gypsy replication system, we 0 0 0 Gypsy 5 and 3 RNAs (Figure 4; MLV and HIV-NC bars). needed to know whether the 5 PBS was unique, bipartite Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5769 0 0 Figure 3. Gypsy RNAs generated in vitro. Schematic representation of the Gypsy genomic RNA where the U3, R and U5 regions of the LTR, the 5 and 3 PBS and the Gag, Pol and Env open reading frames are depicted. PBS stands for primer tRNA-binding site and PPT for polypurine tract. The Gypsy 5 RNA (positions 0 0 0 0 0 1 to 433) includes the R repeat, the 3 region of the LTR (U5) and the 5 PBS. The Gypsy 3 RNA (positions 6416 to 6941) encompasses the 3 PBS and the 5 0 0 0 0 region of the LTR (U3). The recombinant 5 –3 Gypsy RNA contains the above 5 and 3 RNA sequences joined to form a single RNA. Numbering is with respect 0 0 to the genomic RNA positions. RNAs with the PBS mutated to a 6 nt SpeI site (D5 PBS) or to a 6 nt EcoRV site (D3 PBS) are depicted according to the same representation. See table for the cloning strategies and oligonucleotides used to that end (Supplementary Table 1). 5' RNA 3' RNA 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% 0% 0% CT 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 CT 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 TYA1-D G-NC MLV-NC HIV-NC TYA1-D G-NC MLV-NC HIV-NC 0 0 0 0 Figure 4. Binding of the Gypsy NC-like peptide to Gypsy 5 and 3 RNAs. Gypsy NC-like peptide and P-labelled 5 and 3 RNAs were synthesized as described in Materials and Methods. Binding of Gypsy NC (G-NC) was monitored by gel retardation (data not shown) and nucleoprotein complex formation. Complexes were recovered by centrifugation as reported in methods. The same experiments were carried out with the TYA1-D peptide and with MLV and HIV-NC peptides (see Materials and Methods for peptide sequences). Optimal complex formation with the G-NC and TYA1-D peptides was found to take place at a protein to nt molar ratio of 1 to 6, as previously observed for retroviral NC proteins, such as HIV-1 NCp7 and MuLV NCp10 (20,45). Note that the basic HIV-NC and MLV- NC peptides did not form large amounts of ribonucleoprotein complexes (HIV-NC and MLV-NC on both panels); although they bind RNA [see Refs (20–22)]. CT stands for RNA alone. Lys,2 or multipartite as in retroviruses and in the yeast retrotrans- Interestingly, another putative tRNA binding site was posons TY3 and TY1, respectively (3,6,7). Sequence found close to the genomic RNA 3 end at positions 6489 Lys,2 alignments using tRNA and the complete Gypsy to 6499, also 11 nt in length and rich in GC (Figure 5A). sequences were performed. As reported in Figure 5A, This second PBS is complementary to a different region Lys,2 a PBS of 11 nt in length and rich in GC is located immedi- of tRNA , namely part of the anticodon and TYC stems 0 0 0 0 ately 3 of the 5 LTR at positions 245–255, complementary (Figure 5B). To assess the role of these 5 and 3 PBSs in 0 Lys,2 Lys,2 to the 3 end of the tRNA acceptor stem (Figure 5B). primer tRNA annealing and initiation of reverse Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5770 Nucleic Acids Research, 2006, Vol. 34, No. 20 Lys,2 0 0 Figure 5. The primer tRNA binding sites on the Gypsy genomic RNA. (A) Gypsy RNA: the 5 and 3 regions of the Gypsy genomic RNA are shown. Lys,2 Lys,2 0 0 Sequence complementarities of 11 nt between 5 and 3 PBS and replication primer tRNA are indicated. (B) Secondary structure of cellular primer tRNA : 0 0 sequences possibly involved in tRNA annealing to the 5 and 3 PBSs of Gypsy RNA are indicated by lines. transcription, we generated several different RNAs mimick- they contain zinc fingers shown to specify direct NC/RNA 0 0 ing the 5 and 3 regions of Gypsy genomic RNA containing recognition (3,5,24). 0 0 0 the wild-type sequences, or a deleted PBS, and recombinant We used a recombinant 5 –3 RNA representing the 5 and 0 0 0 RNAs where the 5 and 3 regions have been fused in the 3 regions of the Gypsy genomic RNA to better understand wild-type sequence context or containing either one or what could be the interactions between the 5 PBS–tRNA Lys,2 both deleted PBSs (Figure 3; see Gypsy RNAs named and the 3 PBS–tRNA complexes since different tRNA BSG8-CG 1 to 9). sequences of 11 nt should be base paired to the Gypsy Lys,2 0 0 0 To examine primer tRNA annealing to the 5 and 5 and 3 PBSs (Figure 5B), thus possibly forming a link 0 0 0 0 0 3 PBSs we used the 5 ,3 and 5 –3 Gypsy RNAs generated between the ends of Gypsy genomic RNA in a manner similar Met,i in vitro (see Materials and Methods, and Figure 3). The to yeast TY3 RNA and primer tRNA (7). Overall, the Gypsy NC (G-NC) or the TYA1-D peptide was added Gypsy NC and TYA1-D peptides still directed tRNA anneal- to the assays together with P-labelled tRNA. At the end ing to the PBSs (Figure 6C, lanes 2–5 and 20–25). Deleting 0 0 of the incubation period, P-labelled nucleic acids were the 5 or 3 PBS did not result in levels of tRNA annealing 0 0 purified by proteinase K (PK) digestion and phenol extrac- to the 5 PBS or 3 PBS (Figure 6C, lanes 7–10 and 12–15, tion, and subsequently analysed by gel electrophoresis in respectively) similar to those obtained with the 3 RNA or native conditions followed by autoradiography. As reported 5 RNA alone (compare with wt RNAs in Figure 6A and in Figure 6A, the Gypsy NC and TYA1-D peptides promoted B). As before, the MLV and HIV-NC peptides were inactive Lys,2 extensive annealing of tRNA to the 5 PBS (lanes 2–7) (lanes 26–29). Taken together, these results indicate that and, as expected, not to the 5 RNA lacking the PBS (lanes complex interactions are probably taking place between 0 0 13–14). Both peptides also chaperoned the annealing of Gypsy 5 and 3 terminal regions, probably competing with Lys,2 Lys,2 tRNA to the 3 PBS, but somewhat less efficiently than to the binding to tRNA . Lys,2 the 5 PBS (Figure 6B, lanes 2–7). Despite many attempts, To investigate how tRNA could possibly establish a Lys,2 0 0 0 the level of tRNA annealing to the 3 PBS never reached bridge between the 5 and 3 ends of the Gypsy genomic more than 30–40% with TYA1-D and 40–60% with Gypsy RNA, we analysed the simultaneous binding of tRNA to NC as compared with the 5 PBS. As expected, in the absence the PBSs situated on two RNA molecules. To this end, 0 0 0 of 3 PBS no tRNA annealing took place (lanes 13–14). At Gypsy 5 and 3 RNAs were co-incubated with tRNA and the same time, the MLV and HIV-NC peptides were found the TYA1-D or Gypsy NC peptide. After peptide removal to be poorly active (Figure 6A and B, lanes 8–11) although by SDS-proteinase K digestion and phenol extraction, Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5771 Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5772 Nucleic Acids Research, 2006, Vol. 34, No. 20 RNAs were subjected to electrophoresis in native conditions. the specific role of NC protein in proviral DNA synthesis As shown in Figure 7, an RNA complex formed of the 5 and in most, if not all retroviruses (3). 3 RNAs, and tRNA was generated upon addition of TYA1-D or G-NC in a dose dependent manner (lanes 2–5) when both Analysis of Gypsy cDNA synthesis in D.hydei cells PBSs were present. However, this complex did not form upon deleting the PBS from the 3 RNA (lanes 7–10). Similar Lyubomirskaya et al. (37) have developed a simple ex vivo results were obtained with the Gypsy 5 RNA with a deleted assay to look for Gypsy cDNA synthesis, which allows the PBS (data not shown). These results indicate that the Gypsy direct detection of Gypsy reverse transcription by Southern primer tRNA can establish a bridge between the 5 and blotting in stably transformed Dh14 cells. These D.hydei 3 ends of the genomic RNA (see Discussion). cells were chosen because their genome does not cross- hybridize with the D.melanogaster Gypsy sequences. The rationale of this assay is that a small 5 end deletion in a Influence of the Gypsy NC peptide on LTR retroelement still allows the production of a full-length cDNA synthesis in vitro transcript, which is subsequently used as a template to gener- To examine the influence of the Gypsy NC peptide and the ate a complete cDNA with a restriction map different from 0 0 role of the 5 and 3 PBSs on Gypsy cDNA synthesis, we that of the deleted donor DNA. This assay benefited here 0 0 0 0 used the 5 ,3 and 5 –3 RNAs as well as the PBS-mutated from a slight modification of the donor plasmid where a RNAs (Figure 3). Reverse transcription by the related MLV strong enhancer sequence from the copia retrotransposon RT proceeded at 30 C for 1 h, after which cDNAs were (18) was inserted upstream of the Gypsy promoter, replacing recovered by SDS-PK treatment and phenol extraction, and the U3 sequence from the 5 LTR (see Materials and Meth- analysed by gel electrophoresis in denaturing conditions ods). To improve the signal-to-noise ratio, we took advantage (see Materials and Methods). The Gypsy NC and TYA1-D of the fact that the donor plasmid DNA was of prokaryotic Lys,2 peptides directed tRNA hybridization to the 5 PBS origin and could be selectively degraded into small DNA (Figures 6 and 7) that allowed strong stop cDNA (ss-cDNA) fragments by the DpnI enzyme. The newly made cDNA synthesis by RT using the 5 RNA template (Figure 8, and a minority of the parental DNA, which happened to lanes 2–7). In the absence of NC peptide no ss-cDNA was have replicated in these Dh cells, are devoid of the methyla- made, in agreement with the fact that little or no primer tion tags and are therefore resistant to DpnI. tRNA was annealed to the 5 PBS (lane 1). Since tRNA The results reported in Figure 9 (lanes 5–8, WT) show that 0 0 annealing to the 3 PBS did not involve its 3 terminal the unintegrated cDNA products, i.e. the linear and both the nucleotides (Figure 5), no cDNA was synthesized using the 1-LTR and 2-LTR circles, were found by Southern blotting as 3 RNA (lanes 9–10). early as three days after transfection. To investigate the role 0 0 0 We next examined the influence of the 3 PBS on ss-cDNA of the 5 PBS we constructed a D5 PBS mutant producing a 0 0 0 synthesis initiated at the 5 PBS using the 5 –3 RNA genomic RNA missing the 11 nt, which hybridize to the Lys,2 template. Results were very similar to the previous ones 3 terminus of primer tRNA . Using Gypsy anti-Gag and since only the 5 PBS was capable of directing ss-cDNA syn- anti-Env antibodies, we showed by western immuno-blotting thesis by RT (compare lanes 15–17 and 20) with only a little of cellular protein extracts that this 5 PBS deletion did not influence of the 3 PBS (compare lanes 15–17 and 25–27). affect Gypsy protein expression (data not shown). Results However, only the Gypsy NC peptide was effective at show that only the mutant donor plasmid DNA was detected 0 0 0 stimulating cDNA synthesis using the 5 –3 RNA template by Southern blotting, clearly indicating that the 5 PBS is (compare lanes 12–14 and 15–17). Similar results were required for Gypsy cDNA synthesis. This is also in complete 0 0 obtained upon mixing the 5 and 3 RNA templates (data agreement with the in vitro data (Figure 8, lanes 17 and 20). not shown). Both the TYA1-D and Gypsy NC peptides A smaller 5 PBS deletion has been made (see Materials and were found to strongly inhibit cDNA synthesis by self- Methods) and results confirmed that the 5 PBS is essential priming (Supplementary Figure 1), which further support for Gypsy cDNA synthesis ex vivo (data not shown). Lys,2 32 Lys,2 0 0 Figure 6. Hybridization of primer tRNA to the Gypsy 5 and 3 PBSs. Gypsy RNA and P-labelled primer tRNA were incubated at 30 C for 5 min. 0 0 0 0 RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5 wt, D5 PBS, 3 wt, D3 PBS, 0 0 0 0 0 0 0 0 0 recombinant 5 –3 (either wt, D5 PBS, D3 PBS or D5 –D3 PBS) is indicated at the top of each panel. Panel (A) stands for 5 RNA, (B) for 3 RNA and (C) for 5 – 32 Lys,2 3 RNA. Control was with Gypsy RNA and P-labelled tRNA but without NC protein as shown in lanes 1 and 12 (A and B), and 1 and 19 (C). (A and B): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:30, 1:15 and 1:7 (lanes 2–4 and 5–7, respectively). MLV and 0 0 HIV-NC peptides were at peptide to nt molar ratios of 1:15 and 1:7 (lanes 8–9 and 10–11, respectively). When the D5 PBS RNA or the D3 PBS RNA was used, Lys,2 0 the peptide to nt ratio was 1:7 (lanes 13–14). Note that primer tRNA annealed at a low level to the 5 RNA without the chaperone [(A) lane 1] and annealing was optimal at protein to nt molar ratios of 1:15 to 1:7 [lanes 3–4 and 6–7 in (A)]. Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5 PBS increased from 15–20 to 55–65% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV Lys,2 0 peptides (average values of three independent assays). Primer tRNA did not anneal to the 3 RNA without chaperone [(B), lane 1] and annealing was optimal at protein to nt molar ratio 1:7 (lanes 4 and 7). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 3 PBS increased from 0 to 40–50% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). (C): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 (lanes 2–3, 7–8 and 12–13, and 4–5, 0 0 9–10, and 14–15, respectively). For D 5 –D3 PBS RNA, ratio was 1:7 (lanes 17–18). Additional experiments with TYA1-D and G-NC were at molar NC to nt ratios of 1:30, 1:15 and 1:7 (lanes 20–22 and 23–25, respectively) and with MLV and HIV peptides ratios were 1:15 and 1:7 (lanes 26–27 and 28–29, 0 0 respectively). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5 and 3 PBSs increased from 8–12 to 40–85% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays; see also Lys,2 0 0 0 0 Figure 7). Sizes of tRNA and Gypsy RNAs (in nt) are indicated on the right for 5 RNA (433 nt), 3 RNA (525 nt), 5 –3 RNA (959 nt) and tRNA (76 nt). Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5773 Lys,2 32 Lys,2 0 0 0 0 Figure 7. Primer tRNA bridges the 5 and 3 RNA ends. Gypsy 5 and 3 RNAs and P-labelled primer tRNA were incubated together at 30 C for 5 min. 0 0 0 RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5 wt, 3 wt and D3 PBS RNA 32 Lys,2 is indicated at the top. Control Gypsy RNAs with P-tRNA but without protein are shown in lanes 1 and 6. TYA1-D or Gypsy NC (G-NC) peptide was added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 as shown in lanes 2–3, 4–5, 7–8 and 9–10, respectively. Arrow is direction of Lys,2 electrophoresis. Primer tRNA annealed at a low level to the RNA without chaperone (lanes 1 and 6) and this was optimal at a molar ratio of 1:7 (lane 5). Note Lys,2 0 0 0 0 that annealing of tRNA to both 5 and 3 PBS caused the formation of a complex made of 5 and 3 Gypsy RNAs and tRNA (lanes 4–5) but not when tRNA could not anneal to the 3 RNA (lanes 9–10). Next, we constructed the D3 PBS mutant by deleting nt extensively invade the genome is conditioned by the cell 6727–6737 (Materials and Methods). This D3 PBS mutant- capacity to repress this retroelement. This control results in displayed a wild-type level of cDNA made (compare lanes reduced steady state levels of Gypsy transcripts (42,43). 1–2 with 5–6, and 11–12 with 7–8) despite the fact that it Moreover, it has recently been shown that synthesis of does not produce any envelope protein due to the frameshift Gypsy Gag is strictly controlled at the translational level (44). generated by this 11 nt deletion at the Env 3 end (data not To understand the dual nature of the D.melanogaster shown). To further address the role of Gag in the synthesis Gypsy and the fact that it appears to be at the frontier between of Gypsy cDNA, we inserted a frameshift mutation at the retrotransposons of yeast and simple vertebrate retroviruses, 5 end of Gag. This Gypsy mutant that cannot express the we investigated Gypsy replication in vitro and in cell culture. Gag protein was totally unable to direct cDNA synthesis First, we searched for a NC-like region in Gypsy Gag with all (data not shown). In conclusion, these results confirm the the properties of a bona fide retroviral RNA chaperone in vitro findings (Figure 8; lanes 17 and 27) and show that protein (45) [reviewed in (3,4)]. To this end, we used a the 3 PBS is not required for cDNA synthesis, at least at computer-assisted search to look for a peptide domain of this stage of Gypsy replication. low complexity, rich in proline and basic residues, and with a predicted disordered state (33–35). Such a peptide domain, named G-NC, was found at the C-terminal end of the Gypsy Gag, showing notable similarities with the NC-like domain of DISCUSSION TY1 Gag (Figures 1 and 2). In fact, the Gypsy NC-like The D.melanogaster Gypsy retroelement has a genetic orga- peptide was active in the formation of high molecular weight nization similar to that of the widespread vertebrate nucleoprotein complexes and in replication primer annealing gammaretroviruses, such as the MLV. In fact the provirus to the genomic PBS in a manner very similar to that of the form of Gypsy contains the three canonical open reading C-terminal Gag peptide of yeast TY1 (TYA1-D; Figures 4 frames gag, pol and env flanked by two LTRs. As an infec- and 6). Like other retroviral NC proteins, the G-NC peptide tious enveloped retrovirus, Gypsy can efficiently invade the was found to possess general RNA chaperoning properties in vitro, which can promote profound rearrangements of germ line of permissive flies, upon infection of naive females (38,39). These new Gypsy proviruses can then increase their nucleic acid conformation [reviewed in (27,34)]. Along this number by expression in the somatic cells of permissive line, the optimal chaperoning activity of G-NC takes place flamenco females followed by recurrent invasion of the at a peptide to nucleic acid molar ratio of one peptide per germ line by a process, which, in contrast to virus infection, 6 to 10 nt (see Figure 6) as holds true for retroviral NC pro- does not need env (40). Thus, Gypsy can behave both as an teins and NC of the yeast TY3 retrotransposon (27,34,45). infectious retrovirus and as an active retrotransposon (13,14). Interestingly, NCp9 of TY3 was found to be active in the A high level of Gypsy transposition might be deleterious Gypsy system (data not shown) whereas the basic MLV for the host genome due to insertional mutagenesis, espe- and HIV-1 NC peptides were poorly active (Figures 4 and cially because Gypsy carries a strong DNA insulator (41), 6) although they contain zinc fingers shown to specify which can disrupt the activity of the genes, into which it is direct NC–RNA interactions in HIV and MLV (3,24,29). integrated by blocking the interactions of distal enhancers The primary structure of the Gypsy Gag and the presence with the target promoter. Thus, the inability of Gypsy to of an active RNA chaperoning domain at its C-terminus Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5774 Nucleic Acids Research, 2006, Vol. 34, No. 20 Figure 8. Role of the Gypsy NC peptide in cDNA synthesis in vitro.(A) Schematic representation of the initiation of reverse transcription on Gypsy 5 RNA. Reverse transcription of Gypsy 5 U5 and R RNA sequences leads to the synthesis of the so-called minus strand strong stop cDNA, ss-cDNA(), by RT extension Lys,2 32 Lys,2 0 0 0 0 of primer tRNA .(B) Gypsy 5 RNA, 3 RNA or recombinant 5 –3 RNA and P-tRNA were incubated with or without TYA1-D or Gypsy NC peptide. MLV RT was added together with dNTPs to allow reverse transcription. Assays were processed as described in Materials and Methods and ss-cDNA() was denatured and analysed by 10% PAGE in 7 M urea. Controls without protein are shown in lanes 1, 8, 11, 18 and 21. Protein to RNA nucleotide molar ratios were 1:48 (lanes 2, 5, 12, 15, 22 and 25), 1:24 (lanes 3, 6, 13, 16, 23 and 26) and 1:12 (lanes 4, 7, 9, 10, 14, 17, 19, 20, 24 and 27), corresponding to 1.25 · 10 , 2.5 · 7 7 7 7 6 Lys,2 0 0 0 0 10 and 5 · 10 M for 5 RNA and 2.5 · 10 ,5 · 10 and 10 M for 5 –3 RNA. Note that hybridization of tRNA to the 3 PBS caused an inhibition of the initiation of Gypsy reverse transcription which was severe with the TYA1-D peptide (compare lanes 2–4 with 12–14) and moderate with the homologous peptide (compare lanes 5–7 with 15–17). suggest that the Gypsy Gag resembles more that of an ancient from TY3 since the 3 PBS is not implicated in the process retrotransposon than the canonical Gag of the gammaretro- of cDNA synthesis in vitro and ex vivo (Figures 8 and 9). virus MLV, formed of the well-defined matrix, capsid Yet the exact role of the Gypsy 3 PBS could be at the and NC domains. On the opposite the Gypsy genomic level of genome maintenance or translation via formation RNA has long structured untranslated regions, the 5 - and of a circular RNA. This is presently under investigation. 3 -untranslated regions (UTRs) (Figure 3) similar to the In conclusion, these findings on the NC-like domain of MLV genomic RNA. In addition, the Gypsy 5 -UTR directs Gypsy Gag and on the existence of two PBSs indicate that translation of Gag and Env via specific internal ribosome Gypsy resembles active retrotransposons and is distinct from entry sites (IRES) that resemble the MLV Gag and Env canonical simple retroviruses, such as the MLVs and avian IRESes (44,46,47). leukosis viruses. In agreement with this conclusion, the env- Second, we characterized two distinct PBSs located at independent reverse transcription of Gypsy in D.hydei cells 0 0 the 5 and 3 ends of the Gypsy genomic RNA (Figure 5), (Figure 9) is reminiscent of the fact that the efficiency of which are reminiscent of those found in the yeast TY3 Gypsy replication in D.melanogaster females was not affected retrotransposon (7). The G-NC peptide promoted the by the frameshift disruption of the env gene (40). On the other Lys,2 0 0 0 annealing of primer tRNA to both the 5 and 3 PBSs hand, the presence of a single 5 PBS necessary for cDNA syn- (Figure 6). Furthermore, the simultaneous annealing of thesis (Figures 8 and 9), of long structured UTRs, of functional Lys,2 primer tRNA to these two PBSs (Figure 7) resulted in IRESes and Env, and the fact that cell-free Gypsy particles are 0 0 the formation of a 5 –3 RNA complex, indicating that a infectious (13,14) indicate that Gypsy should be considered as tRNA bridge can possibly generate a circular Gypsy RNA an ancient simple retrovirus, similar to the gammaretrovirus as proposed for the TY3 RNA (7). However, Gypsy differs MLV. To investigate how much the NC functions are Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5775 Figure 9. The 5 PBS is critical for Gypsy cDNA synthesis ex vivo. D.hydei cells were transfected with WT or PBS-mutated Gypsy DNA as reported in materials and methods. The 10.3 kb transfected DNA is shown at the top. The dotted line represents the vector DNA that was used to clone the Gypsy element with a 0 0 complete 3 LTR (rectangle with P, a PvuI site) and a shortened 5 LTR (square) the U3 sequence of which was replaced by a Copia enhancer (black box). Reverse transcription is expected to regenerate the missing sequence, giving rise to the full-length 7.5 kb Gypsy DNA shown at the bottom. The 2-LTR and 1-LTR circular DNA products of 7.5 and 7.0 kb, respectively, are shown. A total of 1 mg of total DNA were digested with either EcoRI (R) or NdeI (N), blotted and probed with the fragment schematized at the bottom. A unique band is expected to correspond to each category of molecules. The only exception corresponds to the integrated proviruses (not represented here), which if randomly inserted at various distances from the EcoRI or NdeI genomic restriction sites, are expected to produce a smear corresponding to a heterogeneous population of molecules larger than 6.6 and 5.7 kb, respectively. In contrast, in double digests involving PvuI (P) and either NdeI (N) or EcoRI (R), all types of cDNA products should merge into a single band, as shown in lanes 1, 5, 8 and 12). Most of the transfected plasmids have run out of the gel after being degraded into small DNA fragments by the methylation-dependent DpnI enzyme; therefore, the upper band only 0 0 corresponds to the minority of plasmids, which have lost these prokaryotic methylation tags. D5 and D3 PBS are mutants which only differ from the wild-type Gypsy sequence (WT) by the DPBS mutations described in Figure 3, at positions (proviral DNA sequence) 482–492 and 6727–6737, respectively (16). Note that 0 0 the D5 PBS, but not the D3 PBS deletion resulted in the absence of cDNA synthesis (lanes 3–4 and 9–10). conserved from Drosophila Gypsy to the MLV, substituting ACKNOWLEDGEMENTS MLV-NC by the Gypsy NC-like is now in progress using The authors thank Marc Ruff (Strasbourg) for helpful advice MLV-based vectors. on peptide expression and purification procedures. Thanks are due to Damien Ficheux (IBCP CNRS) for TYA1-D, MLV- NC and HIV-1 NC peptide synthesis and purification. Gag SUPPLEMENTARY DATA mutant plasmids were generated by Emeline Sarot with the ` ˆ Supplementary Data are available at NAR Online. technical assistance of Genevieve Payen-Groschene. Work Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5776 Nucleic Acids Research, 2006, Vol. 34, No. 20 supported by CNRS, ANRS and INSERM to J.L.D., by ARC melanogaster cell lines. Biochem. Biophys. Res. Commun., 203, 392–399. to A.P. and by CNRS to A.B. R.I.N. is a recipient of an ANRS 19. de Rocquigny,H., Ficheux,D., Gabus,C., Fournie-Zaluski,M.C., pre-doctoral fellowship. Funding to pay the Open Access Darlix,J.L. and Roques,B.P. (1991) First large scale chemical synthesis publication charges for this article was provided by ANRS of the 72 amino acid HIV-1 nucleocapsid protein NCp7 in an active (Agence Nationale de recherches sur le SIDA). form. Biochem. Biophys. Res. Commun., 180, 1010–1018. 20. Barat,C., Lullien,V., Schatz,O., Keith,G., Nugeyre,M.T., Conflict of interest statement. None declared. Gruninger-Leitch,F., Barre-Sinoussi,F., Le Grice,S.F. and Darlix,J.L. (1989) HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. EMBO J., 8, 3279–3285. REFERENCES 21. Darlix,J.L., Vincent,A., Gabus,C., de Rocquigny,H. and Roques,B. 0 0 (1993) Trans-activation of the 5 to 3 viral DNA strand transfer by 1. Boeke,J.D. and Stoye,J.P. 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(1994) Conformational behaviour of the active and retrotransposon is apparently an infectious retrovirus of Drosophila inactive forms of the nucleocapsid NCp7 of HIV-1 studied by melanogaster. Proc. Natl Acad. Sci. USA., 91, 1285–1289. 1H-NMR. J. Mol. Biol., 235, 287–301. 14. Song,S.U., Gerasimova,T., Kurkulos,M., Boeke,J.D. and Corces,V.G. 32. Namba,K. (2001) Roles of partly unfolded conformations in (1994) An env-like protein encoded by a Drosophila retroelement: macromolecular self-assembly. Genes Cells, 6, 1–12. evidence that gypsy is an infectious retrovirus. Genes Dev., 8, 33. Tompa,P. and Csermely,P. (2004) The role of structural disorder in 2046–2057. the function of RNA and protein chaperones. FASEB J., 18, 15. Bayev,A.A., Jr, Lyubomirskaya,N.V., Dzhumagaliev,E.B., 1169–1175. Ananiev,E.V., Amiantova,I.G. and Ilyin,Y.V. (1984) Structural 34. Ivanyi-Nagy,R., Davidovic,L., Khandjian,E.W. and Darlix,J.L. (2005) organization of transposable element mdg4 from Drosophila Disordered RNA chaperone proteins: from functions to disease. Cell. melanogaster and a nucleotide sequence of its long terminal repeats. Mol. Life Sci., 62, 1409–1417. Nucleic Acids Res., 12, 3707–3723. 35. Obradovic,Z., Peng,K., Vucetic,S., Radivojac,P., Brown,C.J. and 16. Marlor,R.L., Parkhurst,S.M. and Corces,V.G. (1986) The Drosophila Dunker,A.K. (2003) Predicting intrinsic disorder from amino acid melanogaster gypsy transposable element encodes putative gene sequence. Proteins, 53, 566–572. products homologous to retroviral proteins. Mol. Cell. Biol., 6, 36. Alberola,T.M. and de Frutos,R. (1996) Molecular structure of a gypsy 1129–1134. element of Drosophila subobscura (gypsyDs) constituting a degenerate 17. Barat,C., Le Grice,S.F.J. and Darlix,J.L. (1991) Interaction of HIV-1 form of insect retroviruses. 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Gypsy endogenous retrovirus of Drosophila melanogaster involves (1988) Small finger protein of avian and murine retroviruses env-independent invasion of the female germline. EMBO J., 18, has nucleic acid annealing activity and positions the replication 2659–2669. primer tRNA onto genomic RNA. EMBO J., 7, 41. Gdula,D.A., Gerasimova,T.I. and Corces,V.G. (1996) Genetic and 1777–1783. molecular analysis of the gypsy chromatin insulator of Drosophila. 46. Berlioz,C. and Darlix,J.-L. (1995) An internal ribosomal entry Proc. Natl Acad. Sci. USA, 93, 9378–9383. mechanism promotes translation of MuLV gag polyprotein precursors. 42. Pelisson,A., Song,S.U., Prudhomme,N., Smith,P.A., Bucheton,A. and J. Virol., 69, 2214–2222. Corces,V.G. (1994) Gypsy transposition correlates with the production 47. Deffaud,C. and Darlix,J.-L. (2000) Characterization of an internal of a retroviral envelope-like protein under the tissue-specific control of ribosomal entry segment in the 5 leader of murine leukemia virus env the Drosophila flamenco gene. EMBO J., 13, 4401–4411. RNA. J. Virol., 74, 846–850. Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

Characterization of a nucleocapsid-like region and of two distinct primer tRNALys,2 binding sites in the endogenous retrovirus Gypsy

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10.1093/nar/gkl722
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Abstract

5764–5777 Nucleic Acids Research, 2006, Vol. 34, No. 20 Published online 13 October 2006 doi:10.1093/nar/gkl722 Characterization of a nucleocapsid-like region Lys,2 and of two distinct primer tRNA binding sites in the endogenous retrovirus Gypsy 1 1 Caroline Gabus, Roland Ivanyi-Nagy, Julien Depollier , Alain Bucheton , Alain Pelisson and Jean-Luc Darlix* LaboRetro, Unite ´ de Virologie Humaine, INSERM, IFR 128, Ecole Normale Supe ´ rieure de Lyon, 46 Alle ´ e d’Italie, 69364 LYON Cedex 07, France and Institut de Ge ´ ne ´ tique Humaine, 141, rue de la Cardonille, 34396 MONTPELLIER Cedex 5, France Received August 28, 2006; Revised and Accepted September 18, 2006 with simple retroviruses, such as vertebrate gammaretro- ABSTRACT viruses and utilize similar basic mechanisms for their Mobile LTR-retroelements comprising retroviruses and replication. Genome replication proceeds via the conversion LTR-retrotransposons form a large part of eukaryotic of the single-stranded genomic RNA into a double-stranded genomes. Their mode of replication and abundance DNA copy with two LTRs by reverse transcriptase (RT), favour the notion that they are major actors in eukaryote followed by integration into the host genome by integrase evolution. The Gypsy retroelement can spread in the (1). By virtue of this copy-and-paste mechanism, retrotrans- posons are thought to have efficiently invaded eukaryotic germ line of the fruit fly Drosophila melanogaster via genomes. Being a large part of eukaryotic genomes, these both env-independent and env-dependent processes. LTR-retroelements are viewed as major players in eukaryote Thus, Gypsy is both an active retrotransposon and an evolution (2). infectious retrovirus resembling the gammaretrovirus A large number of studies on the early replication steps MuLV. However, unlike gammaretroviruses, the Gypsy of lentiviruses and gammaretroviruses, such as HIV-1 Gag structural precursor is not processed into Matrix, and MoMuLV, respectively, have been carried out in in Capsid and Nucleocapsid (NC) proteins. In contrast, it vitro reconstituted systems and in cell culture [reviewed in has features in common with Gag of the ancient yeast (3–5)]. Collectively the findings show that reverse transcrip- TY1 retroelement. These characteristics of Gypsy make tion occurs within the virion nucleocapsid (NC) structure it a very interesting model to study replication of a formed of the genomic RNA coated by molecules of NC retroelement at the frontier between ancient retrotrans- protein, and starts by NC-mediated annealing of a specific posons and retroviruses. We investigated Gypsy repli- cellular primer tRNA to a unique 5 genomic primer binding site (PBS), followed by RT-directed cDNA synthesis during cation using an in vitro model system and transfection which RT is assisted by NC (3–5). Much less studies have of insect cells. Results show that an unstructured been carried out on the early replication steps of ancient domain of Gypsy Gag has all the properties of a retrotransposons, such as TYs of yeast. Although a similar retroviral NC. This NC-like peptide forms ribonu- basic mechanism appears to operate, such as RT-directed cleoparticle-like complexes upon binding Gypsy RNA cDNA synthesis by extension of a specific cellular tRNA Lys,2 and directs the annealing of primer tRNA to two annealed to a genomic PBS within a ribonucleoprotein struc- distinct primer binding sites (PBS) at the genome 5 and ture, several important differences were discovered. The 0 0 3 ends. Only the 5 PBS is indispensable for cDNA genomic PBS is not unique but multipartite in TY1 and 0 0 synthesis in vitro and in Drosophila cells. TY3, and unique genomic 5 –3 interactions appear to exert a control over reverse transcription and consequently on the genetic amplification of these retrotransposons (6–8). As it is well documented for HIV-1, NC protein in its INTRODUCTION mature form plays critical roles in the conversion of the genomic RNA into proviral DNA by RT through Retrotransposons and retroviruses belong to a large family of specific and tight interactions with the genomic RNA, mobile genetic elements called long terminal repeat (LTR) Lys,3 primer tRNA , RT and the newly made cDNA [reviewed containing retroelements (1). Ancient retrotransposons, such in (3–5)]. Interestingly, retrotransposon NC proteins can as yeast transposons (TY) share common genetic features *To whom correspondence should be addressed. Tel: +33 4 72 72 81 69; Fax: +33 4 72 72 87 77; Email: [email protected] 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5765 either contain a canonical CCHC zinc finger RNA-binding of Gypsy, including the modified 3 LTR, was subcloned motif and be processed by protease cleavage of the Gag into pBluescript II KS(-) (Stratagene) to produce clone polyprotein, such as in TY3, or else Gag is not processed DNA #p18. The LTR+130UTR plasmid (18), kindly provided and does not contain a zinc finger motif, such as in TY1 by S. Jensen, was used as a template to PCR amplify the (9–11). Nevertheless, the retroviral NC functions appear to 130 bp long copia enhancer with the following KpnI and be conserved in these retrotransposons since the C-terminal XhoI primers: 5 -GGGGTACCCAGTCCATGCCTAATAA- 0 0 region of TY1 Gag chaperones the annealing of primer AC-3 and 5 -ACCGCTCGAGCTGAGAAGGAAATAATT- Met,i 0 0 tRNA to the 5 multipartite PBS, mediates TY1 RNA TC-3 . dimerization, and assists cDNA synthesis by the homologous The amplified DNA fragment was cut with KpnI and XhoI RT (12). and ligated to the 5 end of the 6.4 kb XhoI–EcoRI Gypsy Gypsy is a retroelement present in the germ line of the fruit fragment of pDm111 into the pBluescript II KS(-) vector, fly Drosophila melanogaster and can spread via cell-free yielding construct DNA #p19. The 800 bp EcoRI–BamHI viral infection. Thus, Gypsy can be considered both as an fragment from DNA #p18 was inserted into DNA #p19, active retrotransposon and an infectious retrovirus (13,14). giving rise to the full-length wild-type (WT) Gypsy construct. In agreement with this notion, the genetic structure of Mutant plasmid DNAs used for transfection. A frameshift Gypsy is similar to that of the murine gammaretrovirus starting at codon 40 of the Gag gene was obtained by filling MuLV with Gag, Pol and Env flanked by two LTRs in with Klenow polymerase the NcoI site of the WT plasmid. (15,16). However, the Gypsy Gag structural protein is not The 1.2 kb XhoI–KpnI fragment from WT was subcloned processed into Matrix, Capsid and NC proteins (B.V. Syomin into pBluescript II KS(-) to delete the putative 5 PBS and A. Pelisson, unpublished data), which is reminiscent of sequence (TGGCGCCCAAC) using the QuikChange Gag of the yeast TY1. system (Stratagene) with oligonucleotides used for pBSG8- These functional and genetic features of Gypsy make it a CG2 (Supplementary Table 1). The XhoI–NcoI 1 kb mutated very attractive model to study replication of a mobile genetic fragment was then excised and swapped with the correspond- element, which is at the frontier between ancient retrotrans- ing wild-type fragment of the WT plasmid, generating the D5 posons and retroviruses. To that end we set up an in vitro PBS Gypsy DNA. The same procedure was used to delete the replication system using Gypsy RNAs representing the 0 0 0 four 3 nt (CAAC) of the 5 PBS. The 3 PBS sequence 0 0 genomic RNA 5 and 3 regions, and cellular primer Lys,2 (CCACGACCCTG) was deleted from DNA #p18 using tRNA , and investigated their interactions with a putative the QuikChange system (Stratagene) with the oligonu- NC-like domain in Gag. Here we report that an unstructured cleotides used for pBSG8-CG4 (Supplementary Table 1). domain of Gypsy Gag has the hallmarks of an active retrovi- The modified EcoRI–BamHI fragment was then inserted ral NC. This NC-like domain forms ribonucleoparticle-like into DNA #p19 to yield the D3 PBS Gypsy DNA. All complexes upon binding Gypsy RNA in vitro. In addition, Lys,2 DNA constructs were verified by sequencing. two distinct primer tRNA binding sites of 11 nt were 0 0 identified at the 5 and 3 ends of the genome. We also Proteins and peptides found that the Gypsy NC-like peptide can direct the annealing Lys,2 0 of tRNA to these Gypsy PBSs but only the 5 PBS The TYA1-D NC-like peptide was obtained by opfp chemical appears to be indispensable for cDNA synthesis in vitro synthesis and purified by high-performance liquid chromatog- and in Drosophila cells. raphy (HPLC) as described before (12,19). The murine leukemia virus (MLV) and HIV-1 NC basic peptides were also obtained by opfp chemical synthesis and purified to homogeneity as described before (19–22). Using the one MATERIALS AND METHODS letter code representation, the MLV-NC peptide is: Plasmid construction RQGGERRRSQLDRDQCAYCKEKGHWAKDCPRKPRGP- PRAT where the unique zinc finger is underlined, and the Template DNA pBSG8. The XhoI fragment (39 to 6949) of HIV-1 NC peptide is: TVKCFNCGKEGHIAKNCRAPRKK- Gypsy DNA (GenBank accession no. M12927) was inserted 0 GCWKCGKEGHQMKDCTERQ where the two zinc fingers into the SalI site of the Bluescribe F . The clone was selected 0 are underlined. All peptides were dissolved at 1 mg/ml in a so that the EcoRI site of the polylinker was at the 5 end. 0 0 buffer containing 30 mM HEPES (pH 6.5), 30 mM NaCl Plasmid DNAs encoding Gypsy 5 and 3 RNAs were con- and 0.1 mM ZnCl . MLV RT was from Invitrogen. structed by cloning a high fidelity PCR generated fragment DNA encoding the NC-like region of Gypsy Gag was containing two restriction sites at its ends, using the pBSG8 obtained by PCR amplification using pBSG8 as template clone DNA as template and the oligonucleotide primers (see above) and inserted into pIVEX2.4d (Roche) using the described in Supplementary Table 1. Lys,2 SacII and BamHI sites. The sequence of the Gypsy NC-like The tRNA encoding DNA was constructed as peptide is shown in Figure 2B. In addition, a His-tag was pre- described previously (17). sent at its N-terminus (MSGSHHHHHHSSGIEGRG). The Wild-type plasmid DNA used for transfection. Wild-type con- peptide was expressed in Escherichia coli after overnight structs pDm111 (15) and 111xw (Nathalie, unpublished data) induction at 18 C since it proved to be highly toxic at were kindly provided by N. Lyubomirskaya. They both con- temperatures above 20 C. The peptide was purified in dena- tain the same functional Gypsy/mdg4 element except that, in turing conditions on Ni-NTA column (Qiagen) and eluted 111xw, a PvuI site replaced the XhoI restriction site of the with 100 mM NaH PO , 10 mM Tris–HCl, 8 M urea at 2 4 0 0 3 LTR. The EcoRI–PstI fragment containing the 816 3 nt pH 4.5. Fractions containing the peptide with the highest Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5766 Nucleic Acids Research, 2006, Vol. 34, No. 20 degree of purity were further diluted with 3 vol of 30 mM Synthesis of Gypsy cDNA in vitro HEPES (pH 6.5), 30 mM NaCl and 0.1 mM ZnCl and chro- 8 32 2 0 0 0 Gypsy 5 or 5 –3 RNA (5 · 10 M) and P-labelled matographed on an Amicon ultra-4 column. The peptide was Lys,2 7 tRNA (1 · 10 M) were incubated with nucleic acid stored at 1 mg/ml in the above buffer. Note that all buffers chaperone (at protein concentrations indicated in the figure were extensively degassed before use. legend) at room temperature for 10 min in 10 ml buffer con- taining 20 mM Tris–HCl (pH 7.0), 30 mM NaCl, 0.1 mM MgCl , 5 mM DTT, 0.01 mM ZnCl and 10 U RNasin DNA templates and RNA synthesis 2 2 (Promega) (20–22). Plasmid DNAs for 5 RNA synthesis were generated by The reaction volume was then increased to 25 ml by addi- digesting pBSG8-CG1 (wt) and pBSG8-CG2 (DPBS) with tion of 100 U of MLV RT (Invitrogen), 0.25 mM of each 0 0 0 XmnI (position 433). DNAs for 3 RNA and 5 –3 RNA syn- dNTP, 30 mM NaCl and 2.8 mM MgCl . Incubation was thesis were obtained by digesting pBSG8-CG3 to CG8 for 5 min at room temperature, and then 1 h at 30 Cto (wt and mutants) with NsiI (position 6941), treated by the allow synthesis of Gypsy minus strand cDNA. Reactions Klenow polymerase to remove the 3 strand overhang before were stopped by adding SDS/EDTA (0.5%/5 mM final con- in vitro transcription. centrations) and heated for 3 min at 60 C. Proteins were Lys,2 DNA template for tRNA synthesis was obtained by removed by phenol–chloroform extraction and nucleic acids Lys,2 digesting the tRNA plasmid with BstNI in order to gen- were precipitated by ethanol. Samples were resuspended in erate the primer tRNA ending at CCA. 10 ml formamide buffer (97% formamide, 1 mM EDTA, All RNAs were prepared by in vitro transcription with T7 0.02% bromophenol blue and 0.02% xylene cyanol), RNA polymerase according to the manufacturer’s instruc- denatured for 2 min at 95 C and resolved by denaturing tions (Promega). Gypsy RNAs were further purified by 8% PAGE-7 M urea in 0.5· TBE. Subsequently the gel spin-column chromatography (S-300 HR, Amersham Bio- was fixed, dried and autoradiographed. 5 P-labelled sciences) and dissolved at 1 mg/ml in sterile water. FX174 DNA HinfI markers (Promega) were used for size Lys,2 Primer tRNA was purified by PAGE in 7 M urea and determination. 0.5· TBE, recovered and dissolved at 0.05 mg/ml, and sub- sequently heat-denatured and slowly cooled down in the Inhibition of self-primed cDNA synthesis in vitro presence of 1 mM MgCl for proper folding. Primer Lys,2 32 Gypsy 5 RNA (5 · 10 M) was incubated with the NC-like tRNA was labelled by incorporation of [ P]UMP during peptide as described above but in the absence of the tRNA transcription. primer. For cDNA synthesis, the reaction volume was increased to 25 ml by addition of 100 U of MLV RT (Invitro- Binding of the Gypsy NC peptide to RNA gen), 30 mM NaCl and 2.8 mM MgCl , and nucleotides 32 8 0 0 (0.25 mM dATP, dGTP, dTTP, 25 mM dCTP and 20 mCi P-labelled Gypsy 5 or 3 RNA (5 · 10 M) was incubated [ P]dCTP). Incubation, product purification and gel analysis with Gypsy NC, TYA1-D, MLV-NC or HIV-1 NC at protein were the same as for initiation of cDNA synthesis by primer to nucleotide molar ratios as indicated in the figure legend, at tRNA (see section above). 30 C for 5 min in 10 ml assays containing 20 mM Tris–HCl (pH 7.5), 30 mM NaCl, 0.1 mM MgCl , 5 mM DTT, DNA transfection into Drosophila cells and 0.01 mM ZnCl and 8 U RNasin (Promega). Reactions Southern blotting were stopped by adding 5 mM EDTA and centrifuged for 30 min at 13 000 r.p.m. at 4 C. Supernatants were completely Exponentially growing Drosophila hydei Dh33 adherent cells removed by pipetting. Subsequently, P radioactivity in the were grown at 26 C in Schneider medium supplemented with supernatant and pellet fractions was monitored in a Packard 10% fetal calf serum (FCS). About 3 · 10 cells were seeded 1900 TR scintillation counter. in 9.6 cm wells containing 2 ml medium. They were trans- fected 24 h later with 0.8 mg of plasmid DNA, according to Lys,2 the protocol recommended by Qiagen: 100 ml DNA in EC Primer tRNA annealing to Gypsy RNA buffer, 6.4 ml enhancer solution, 20 ml Effectene and 600 ml 8 32 Lys,2 Gypsy RNA, wt or mutant (5 · 10 M), and P-tRNA culture supernatant were successively mixed at 10 min inter- (6 · 10 M) were incubated with nucleic acid chaperone (at vals and deposited on the remaining 1.4 ml of culture. Forty- concentrations indicated in figure legends) at 30 C for 5 min eight hours later, 2 ml of medium were added to the cells, in 10 ml assays containing 20 mM Tris–HCl (pH 7.5), 30 mM which were transferred into a 25 cm flask. Three days NaCl, 0.1 mM MgCl , 5 mM DTT, 0.01 mM ZnCl and 8 U after transfection with the LTR+130UTR control plasmid 2 2 RNasin (Promega). Reactions were stopped by addition of (18), up to 10% of the cells scored positive for X-Gal SDS/EDTA (0.5%/5 mM final concentrations) and proteins staining. were subsequently removed by proteinase K digestion (3 Transfected cells were collected at 72 h, then washed twice mg) at room temperature for 10 min and RNAs were phenol– with 8 ml PBS and the pellet was resuspended in 200 ml chloroform extracted. RNAs were analysed by 1.3% native [10 mM Tris–HCl (pH 8), 1 mM EDTA and 10 mg·ml agarose gel electrophoresis in 0.5· TBE. The gel was fixed RNase I). Lysis was achieved by adding 200 ml [200 mM with 10% trichloroacetic acid, dried and autoradiographed. Tris–HCl (pH 8), 200 mM EDTA and 2% SDS] and the 5 P-labelled FX174 DNA HinfI markers (Promega) were mixture was incubated for 30 min at 70 C. Proteins were used for size determination (data not shown). Quantifications precipitated by addition of 60 ml 3 M KoAc (pH 5.2) and were carried out by laser scanning as described before (8). incubating the mixture for 30 min at 0 C. The supernatant Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5767 was successively extracted with phenol/chloroform (1:1) and of well characterized RNA-binding domain(s) with CCHC chloroform/isoamyl alcohol (24:1) and adding 0.3 M sodium zinc fingers. acetate (pH 5.2) and 70% ethanol precipitated the DNA. Relatively long unstructured regions can be predicted from Half of each extract was digested with 60 U of the amino acid sequence with considerable accuracy. We used methylation-sensitive DpnI restriction enzyme (New England the DisProt VL3-H predictor [http://www.ist.temple.edu/ Biolabs), ethanol precipitated and then split into four parts, disprot/predictor.php, (35)] to assess the probability of disor- which were incubated with restriction enzymes as indicated der in Gypsy Gag. As shown in Figure 1A, the N-terminal in Figure 9. and central regions of Gag are predicted to be mostly ordered Identical amounts of nuclease restricted DNA were run on and do not show a distinctive basic character. However, an a 0.7% agarose gel in TBE 1·, partially depurinated, dena- approximately 100 amino acid region at the C-terminal end tured and capillary transferred in 10· SSC on a Nytran N of Gag (between 300 and 400 amino acid) contains predomi- membrane (Schleicher & Schuell). Hybridization with a nantly basic amino acid clusters in a putatively disordered randomly primed DNA probe—Gypsy coordinates 3097– environment, a pattern characteristic of retroviral NC proteins 5759 (16)—was performed in 50% formamide, 5· SSC, 5· [(34) and data not shown]. In agreement with this prediction, Denhardt’s solution, 0.1% SDS, at 42 C. After washing at an arginine-rich region (355–389 amino acid) has been previ- 55 C in (0.1· SSC and 0.5% SDS), the membrane was ously proposed to act as a possible RNA-binding domain of exposed in a Storm 820 phosphorimager (Molecular Gypsy Gag (36). Dynamics). The Gag region encompassing residues 289–404 was PCR-amplified and cloned into the pIVEX2.4d vector. The corresponding peptide was found to be toxic in E.coli and to aggregate (data not shown). To circumvent these difficul- RESULTS ties it was necessary to express the peptide at 18 C. Due to a Identification of a NC-like region in Gypsy Gag (ORF1) very low level of expression (Figure 1B, lane 1) we had to The NC proteins of retroviruses and retroelements are small insert a His-tag at the N-terminus in order to achieve an basic proteins with nucleic acid chaperone activities, gener- efficient purification and to obtain a peptide at about 95% ated upon processing of the Gag polyprotein by the viral pro- purity (Figure 1B, lane 2). This Gypsy Gag peptide was tease (3). Most NC proteins are characterized by the presence found to bind Gypsy RNA and tRNA with a high affinity of one or two Cx Cx Hx C motifs, called CCHC zinc fingers similar to the NC-like peptide derived from the yeast retro- 2 4 4 (23), constituting a specific RNA-binding motif (4,24). How- transposon TY1 Gag, termed TYA1-D (see Figure 2) (data ever, a number of LTR-containing retroelements—including not shown). This prompted us to investigate its RNA Gypsy—lack zinc fingers and their Gag is not processed by condensing and chaperoning properties using in vitro gener- 0 0 the protease into matrix, capsid and NC proteins ated RNAs mimicking the 5 and 3 regions of the Gypsy [(12,25,26), and B.V. Syomin and A. Pelisson, unpublished genome (Figure 3). data]. Nevertheless, NC functions may be conserved even in these cases, as evidenced by the Gag protein of the yeast TY1 retrotransposon. Indeed the Gag C-terminal domain of The Gypsy NC peptide has RNA-binding and TY1 possesses nucleic acid binding and chaperoning proper- condensing properties Met,i ties, promoting replication primer tRNA annealing to the RNA genome and initiation of cDNA synthesis by RT (12). One hallmark of viral RNA chaperone proteins, such as Since NC activity appears to be ubiquitous in functional retroviral NC proteins is that they cause RNA aggregation retroelements (27), we decided to search for an NC-like and condensation (12). Under in vitro conditions, large domain in Gypsy Gag (ORF1). Owing to the absence of a ribonucleoprotein complexes are formed and can be recov- consensus RNA-binding motif or considerable homology to ered by centrifugation. To analyse the ability of Gypsy NC known NC proteins [(26) and data not shown], we took to form large ribonucleoproteins, Gypsy NC– P-RNA advantage of physico-chemical features probably shared by complexes were formed at 30 C and recovered by centri- all NC and NC-like proteins. fugation at 13 000 r.p.m. (see Materials and Methods). The regions flanking the zinc fingers in NC proteins Radioactivity in the supernatant and in the nucleoprotein- contain low amino acid complexity regions and are rich in containing pellet was monitored. Results show that the proline and basic amino acid residues (26). Proline and Gypsy NC (G-NC) caused RNA condensation at a protein charged amino acids as well as low complexity were sug- to nucleotide molar ratio of 1:6, both with the Gypsy 5 gested to contribute to the flexibility of a polypeptide chain and 3 RNAs (Figure 4). At a protein to nt molar ratio (28). Indeed, the solution structures of NCp7 and NCp10 of 1:12, 50–60% of the RNA was found in nucleoprotein show that these proteins—with the exception of the short complexes (Figure 4) in agreement with the fact that about zinc finger region(s) (see Materials and Methods)—are half of the Gypsy P-RNA was bound to Gypsy NC as poorly structured when not bound to nucleic acids (29–31). indicated by gel retardation assays (data not shown). This flexibility (i.e. intrinsic disorder) might be an important The TYA1-D peptide (12) was used as a positive control prerequisite both for the multimerization of NC proteins (32) and, as expected, had a similar RNA condensing activity and for their RNA chaperone function (33,34). Based on (Figure 4). these considerations, we propose that the presence of a The observed RNA aggregation cannot be exclusively markedly basic, disordered region in retroviral/retroelement attributed to charge neutralization caused by the basic Gag may be a good indicator of NC function in the absence amino acids in Gypsy NC (pI ¼ 10.46) and TYA1-D Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5768 Nucleic Acids Research, 2006, Vol. 34, No. 20 Figure 1. Identification of the NC-like region in Gypsy Gag. (A) Putative NC-like region of Gypsy Gag. The bar chart illustrates the charge distribution of amino acids in Gypsy Gag (ORF1), as calculated by the charge function of the EMBOSS package, using default parameters and a sliding window of 5 amino acids. Computer prediction of disordered regions (solid line) was obtained using the DisProt VL3-H predictor (http://www.ist.temple.edu/disprot/predictor.php) (35). An amino acid with a disorder score above or equal to 0.5 is considered to be in a disordered environment, while below 0.5 in an ordered environment. (B) Expression and purification of the NC-like peptide. The C-terminal part of ORF1 contains several basic amino acid clusters and is predicted to be disordered in its unliganded state, a common feature of retroelement NC proteins (see text for explanation). This region encompassing 289–404 amino acids was amplified and cloned into the pIVEX2.4d vector. The peptide was amplified in E.coli and obtained in a purified form (see Materials and Methods). CL is the clear E.coli lysate (lane 1) and E the purified Gypsy NC-like peptide (lane 2). Note that the peptide was expressed only at a very low level in the CL. Purification steps allowed us to obtain a peptide more than 95% pure (lane 2). The minor protein band corresponds to an N-terminal cleavage product of the Gypsy NC-like peptide. Figure 2. The Gypsy NC and TYA1-D peptides. Amino acid sequences of the NC-like peptides of the yeast retrotransposon TY1 (TYA1-D) and Gypsy NC are shown according to the one letter code. Note the presence of a large number of basic residues and histidines, and the absence of a canonical ‘CCHC’ Zinc finger motif as in vertebrate retroviruses and in TY3 NCp9 (26). Both peptides were found to be basic and disordered according to computer predictions (see Figure 1A). Lys,2 The Gypsy NC peptide directs annealing of tRNA to (pI ¼ 10.24), since the similarly basic MLV-NC and HIV-1 0 0 two 5 and 3 PBSs NC peptides (with pI values of 10.2 and 9.42, respectively) failed to induce considerable RNA aggregation of the In order to set up an in vitro Gypsy replication system, we 0 0 0 Gypsy 5 and 3 RNAs (Figure 4; MLV and HIV-NC bars). needed to know whether the 5 PBS was unique, bipartite Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5769 0 0 Figure 3. Gypsy RNAs generated in vitro. Schematic representation of the Gypsy genomic RNA where the U3, R and U5 regions of the LTR, the 5 and 3 PBS and the Gag, Pol and Env open reading frames are depicted. PBS stands for primer tRNA-binding site and PPT for polypurine tract. The Gypsy 5 RNA (positions 0 0 0 0 0 1 to 433) includes the R repeat, the 3 region of the LTR (U5) and the 5 PBS. The Gypsy 3 RNA (positions 6416 to 6941) encompasses the 3 PBS and the 5 0 0 0 0 region of the LTR (U3). The recombinant 5 –3 Gypsy RNA contains the above 5 and 3 RNA sequences joined to form a single RNA. Numbering is with respect 0 0 to the genomic RNA positions. RNAs with the PBS mutated to a 6 nt SpeI site (D5 PBS) or to a 6 nt EcoRV site (D3 PBS) are depicted according to the same representation. See table for the cloning strategies and oligonucleotides used to that end (Supplementary Table 1). 5' RNA 3' RNA 100% 100% 80% 80% 60% 60% 40% 40% 20% 20% 0% 0% CT 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 CT 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 1:24 1:12 1:6 TYA1-D G-NC MLV-NC HIV-NC TYA1-D G-NC MLV-NC HIV-NC 0 0 0 0 Figure 4. Binding of the Gypsy NC-like peptide to Gypsy 5 and 3 RNAs. Gypsy NC-like peptide and P-labelled 5 and 3 RNAs were synthesized as described in Materials and Methods. Binding of Gypsy NC (G-NC) was monitored by gel retardation (data not shown) and nucleoprotein complex formation. Complexes were recovered by centrifugation as reported in methods. The same experiments were carried out with the TYA1-D peptide and with MLV and HIV-NC peptides (see Materials and Methods for peptide sequences). Optimal complex formation with the G-NC and TYA1-D peptides was found to take place at a protein to nt molar ratio of 1 to 6, as previously observed for retroviral NC proteins, such as HIV-1 NCp7 and MuLV NCp10 (20,45). Note that the basic HIV-NC and MLV- NC peptides did not form large amounts of ribonucleoprotein complexes (HIV-NC and MLV-NC on both panels); although they bind RNA [see Refs (20–22)]. CT stands for RNA alone. Lys,2 or multipartite as in retroviruses and in the yeast retrotrans- Interestingly, another putative tRNA binding site was posons TY3 and TY1, respectively (3,6,7). Sequence found close to the genomic RNA 3 end at positions 6489 Lys,2 alignments using tRNA and the complete Gypsy to 6499, also 11 nt in length and rich in GC (Figure 5A). sequences were performed. As reported in Figure 5A, This second PBS is complementary to a different region Lys,2 a PBS of 11 nt in length and rich in GC is located immedi- of tRNA , namely part of the anticodon and TYC stems 0 0 0 0 ately 3 of the 5 LTR at positions 245–255, complementary (Figure 5B). To assess the role of these 5 and 3 PBSs in 0 Lys,2 Lys,2 to the 3 end of the tRNA acceptor stem (Figure 5B). primer tRNA annealing and initiation of reverse Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5770 Nucleic Acids Research, 2006, Vol. 34, No. 20 Lys,2 0 0 Figure 5. The primer tRNA binding sites on the Gypsy genomic RNA. (A) Gypsy RNA: the 5 and 3 regions of the Gypsy genomic RNA are shown. Lys,2 Lys,2 0 0 Sequence complementarities of 11 nt between 5 and 3 PBS and replication primer tRNA are indicated. (B) Secondary structure of cellular primer tRNA : 0 0 sequences possibly involved in tRNA annealing to the 5 and 3 PBSs of Gypsy RNA are indicated by lines. transcription, we generated several different RNAs mimick- they contain zinc fingers shown to specify direct NC/RNA 0 0 ing the 5 and 3 regions of Gypsy genomic RNA containing recognition (3,5,24). 0 0 0 the wild-type sequences, or a deleted PBS, and recombinant We used a recombinant 5 –3 RNA representing the 5 and 0 0 0 RNAs where the 5 and 3 regions have been fused in the 3 regions of the Gypsy genomic RNA to better understand wild-type sequence context or containing either one or what could be the interactions between the 5 PBS–tRNA Lys,2 both deleted PBSs (Figure 3; see Gypsy RNAs named and the 3 PBS–tRNA complexes since different tRNA BSG8-CG 1 to 9). sequences of 11 nt should be base paired to the Gypsy Lys,2 0 0 0 To examine primer tRNA annealing to the 5 and 5 and 3 PBSs (Figure 5B), thus possibly forming a link 0 0 0 0 0 3 PBSs we used the 5 ,3 and 5 –3 Gypsy RNAs generated between the ends of Gypsy genomic RNA in a manner similar Met,i in vitro (see Materials and Methods, and Figure 3). The to yeast TY3 RNA and primer tRNA (7). Overall, the Gypsy NC (G-NC) or the TYA1-D peptide was added Gypsy NC and TYA1-D peptides still directed tRNA anneal- to the assays together with P-labelled tRNA. At the end ing to the PBSs (Figure 6C, lanes 2–5 and 20–25). Deleting 0 0 of the incubation period, P-labelled nucleic acids were the 5 or 3 PBS did not result in levels of tRNA annealing 0 0 purified by proteinase K (PK) digestion and phenol extrac- to the 5 PBS or 3 PBS (Figure 6C, lanes 7–10 and 12–15, tion, and subsequently analysed by gel electrophoresis in respectively) similar to those obtained with the 3 RNA or native conditions followed by autoradiography. As reported 5 RNA alone (compare with wt RNAs in Figure 6A and in Figure 6A, the Gypsy NC and TYA1-D peptides promoted B). As before, the MLV and HIV-NC peptides were inactive Lys,2 extensive annealing of tRNA to the 5 PBS (lanes 2–7) (lanes 26–29). Taken together, these results indicate that and, as expected, not to the 5 RNA lacking the PBS (lanes complex interactions are probably taking place between 0 0 13–14). Both peptides also chaperoned the annealing of Gypsy 5 and 3 terminal regions, probably competing with Lys,2 Lys,2 tRNA to the 3 PBS, but somewhat less efficiently than to the binding to tRNA . Lys,2 the 5 PBS (Figure 6B, lanes 2–7). Despite many attempts, To investigate how tRNA could possibly establish a Lys,2 0 0 0 the level of tRNA annealing to the 3 PBS never reached bridge between the 5 and 3 ends of the Gypsy genomic more than 30–40% with TYA1-D and 40–60% with Gypsy RNA, we analysed the simultaneous binding of tRNA to NC as compared with the 5 PBS. As expected, in the absence the PBSs situated on two RNA molecules. To this end, 0 0 0 of 3 PBS no tRNA annealing took place (lanes 13–14). At Gypsy 5 and 3 RNAs were co-incubated with tRNA and the same time, the MLV and HIV-NC peptides were found the TYA1-D or Gypsy NC peptide. After peptide removal to be poorly active (Figure 6A and B, lanes 8–11) although by SDS-proteinase K digestion and phenol extraction, Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5771 Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5772 Nucleic Acids Research, 2006, Vol. 34, No. 20 RNAs were subjected to electrophoresis in native conditions. the specific role of NC protein in proviral DNA synthesis As shown in Figure 7, an RNA complex formed of the 5 and in most, if not all retroviruses (3). 3 RNAs, and tRNA was generated upon addition of TYA1-D or G-NC in a dose dependent manner (lanes 2–5) when both Analysis of Gypsy cDNA synthesis in D.hydei cells PBSs were present. However, this complex did not form upon deleting the PBS from the 3 RNA (lanes 7–10). Similar Lyubomirskaya et al. (37) have developed a simple ex vivo results were obtained with the Gypsy 5 RNA with a deleted assay to look for Gypsy cDNA synthesis, which allows the PBS (data not shown). These results indicate that the Gypsy direct detection of Gypsy reverse transcription by Southern primer tRNA can establish a bridge between the 5 and blotting in stably transformed Dh14 cells. These D.hydei 3 ends of the genomic RNA (see Discussion). cells were chosen because their genome does not cross- hybridize with the D.melanogaster Gypsy sequences. The rationale of this assay is that a small 5 end deletion in a Influence of the Gypsy NC peptide on LTR retroelement still allows the production of a full-length cDNA synthesis in vitro transcript, which is subsequently used as a template to gener- To examine the influence of the Gypsy NC peptide and the ate a complete cDNA with a restriction map different from 0 0 role of the 5 and 3 PBSs on Gypsy cDNA synthesis, we that of the deleted donor DNA. This assay benefited here 0 0 0 0 used the 5 ,3 and 5 –3 RNAs as well as the PBS-mutated from a slight modification of the donor plasmid where a RNAs (Figure 3). Reverse transcription by the related MLV strong enhancer sequence from the copia retrotransposon RT proceeded at 30 C for 1 h, after which cDNAs were (18) was inserted upstream of the Gypsy promoter, replacing recovered by SDS-PK treatment and phenol extraction, and the U3 sequence from the 5 LTR (see Materials and Meth- analysed by gel electrophoresis in denaturing conditions ods). To improve the signal-to-noise ratio, we took advantage (see Materials and Methods). The Gypsy NC and TYA1-D of the fact that the donor plasmid DNA was of prokaryotic Lys,2 peptides directed tRNA hybridization to the 5 PBS origin and could be selectively degraded into small DNA (Figures 6 and 7) that allowed strong stop cDNA (ss-cDNA) fragments by the DpnI enzyme. The newly made cDNA synthesis by RT using the 5 RNA template (Figure 8, and a minority of the parental DNA, which happened to lanes 2–7). In the absence of NC peptide no ss-cDNA was have replicated in these Dh cells, are devoid of the methyla- made, in agreement with the fact that little or no primer tion tags and are therefore resistant to DpnI. tRNA was annealed to the 5 PBS (lane 1). Since tRNA The results reported in Figure 9 (lanes 5–8, WT) show that 0 0 annealing to the 3 PBS did not involve its 3 terminal the unintegrated cDNA products, i.e. the linear and both the nucleotides (Figure 5), no cDNA was synthesized using the 1-LTR and 2-LTR circles, were found by Southern blotting as 3 RNA (lanes 9–10). early as three days after transfection. To investigate the role 0 0 0 We next examined the influence of the 3 PBS on ss-cDNA of the 5 PBS we constructed a D5 PBS mutant producing a 0 0 0 synthesis initiated at the 5 PBS using the 5 –3 RNA genomic RNA missing the 11 nt, which hybridize to the Lys,2 template. Results were very similar to the previous ones 3 terminus of primer tRNA . Using Gypsy anti-Gag and since only the 5 PBS was capable of directing ss-cDNA syn- anti-Env antibodies, we showed by western immuno-blotting thesis by RT (compare lanes 15–17 and 20) with only a little of cellular protein extracts that this 5 PBS deletion did not influence of the 3 PBS (compare lanes 15–17 and 25–27). affect Gypsy protein expression (data not shown). Results However, only the Gypsy NC peptide was effective at show that only the mutant donor plasmid DNA was detected 0 0 0 stimulating cDNA synthesis using the 5 –3 RNA template by Southern blotting, clearly indicating that the 5 PBS is (compare lanes 12–14 and 15–17). Similar results were required for Gypsy cDNA synthesis. This is also in complete 0 0 obtained upon mixing the 5 and 3 RNA templates (data agreement with the in vitro data (Figure 8, lanes 17 and 20). not shown). Both the TYA1-D and Gypsy NC peptides A smaller 5 PBS deletion has been made (see Materials and were found to strongly inhibit cDNA synthesis by self- Methods) and results confirmed that the 5 PBS is essential priming (Supplementary Figure 1), which further support for Gypsy cDNA synthesis ex vivo (data not shown). Lys,2 32 Lys,2 0 0 Figure 6. Hybridization of primer tRNA to the Gypsy 5 and 3 PBSs. Gypsy RNA and P-labelled primer tRNA were incubated at 30 C for 5 min. 0 0 0 0 RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5 wt, D5 PBS, 3 wt, D3 PBS, 0 0 0 0 0 0 0 0 0 recombinant 5 –3 (either wt, D5 PBS, D3 PBS or D5 –D3 PBS) is indicated at the top of each panel. Panel (A) stands for 5 RNA, (B) for 3 RNA and (C) for 5 – 32 Lys,2 3 RNA. Control was with Gypsy RNA and P-labelled tRNA but without NC protein as shown in lanes 1 and 12 (A and B), and 1 and 19 (C). (A and B): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:30, 1:15 and 1:7 (lanes 2–4 and 5–7, respectively). MLV and 0 0 HIV-NC peptides were at peptide to nt molar ratios of 1:15 and 1:7 (lanes 8–9 and 10–11, respectively). When the D5 PBS RNA or the D3 PBS RNA was used, Lys,2 0 the peptide to nt ratio was 1:7 (lanes 13–14). Note that primer tRNA annealed at a low level to the 5 RNA without the chaperone [(A) lane 1] and annealing was optimal at protein to nt molar ratios of 1:15 to 1:7 [lanes 3–4 and 6–7 in (A)]. Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5 PBS increased from 15–20 to 55–65% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV Lys,2 0 peptides (average values of three independent assays). Primer tRNA did not anneal to the 3 RNA without chaperone [(B), lane 1] and annealing was optimal at protein to nt molar ratio 1:7 (lanes 4 and 7). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 3 PBS increased from 0 to 40–50% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays). (C): TYA1-D and Gypsy NC (G-NC) were added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 (lanes 2–3, 7–8 and 12–13, and 4–5, 0 0 9–10, and 14–15, respectively). For D 5 –D3 PBS RNA, ratio was 1:7 (lanes 17–18). Additional experiments with TYA1-D and G-NC were at molar NC to nt ratios of 1:30, 1:15 and 1:7 (lanes 20–22 and 23–25, respectively) and with MLV and HIV peptides ratios were 1:15 and 1:7 (lanes 26–27 and 28–29, 0 0 respectively). Quantifications made by laser scanning indicated that total percentages of tRNA annealed to the 5 and 3 PBSs increased from 8–12 to 40–85% upon addition of TYA1-D and G-NC, but did not change after addition of the MLV and HIV peptides (average values of three independent assays; see also Lys,2 0 0 0 0 Figure 7). Sizes of tRNA and Gypsy RNAs (in nt) are indicated on the right for 5 RNA (433 nt), 3 RNA (525 nt), 5 –3 RNA (959 nt) and tRNA (76 nt). Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5773 Lys,2 32 Lys,2 0 0 0 0 Figure 7. Primer tRNA bridges the 5 and 3 RNA ends. Gypsy 5 and 3 RNAs and P-labelled primer tRNA were incubated together at 30 C for 5 min. 0 0 0 RNA complexes were purified by SDS-PK treatment and phenol extraction (see Materials and Methods). The nature of the RNA, 5 wt, 3 wt and D3 PBS RNA 32 Lys,2 is indicated at the top. Control Gypsy RNAs with P-tRNA but without protein are shown in lanes 1 and 6. TYA1-D or Gypsy NC (G-NC) peptide was added to the assays at protein to nucleotide molar ratios of 1:15 and 1:7 as shown in lanes 2–3, 4–5, 7–8 and 9–10, respectively. Arrow is direction of Lys,2 electrophoresis. Primer tRNA annealed at a low level to the RNA without chaperone (lanes 1 and 6) and this was optimal at a molar ratio of 1:7 (lane 5). Note Lys,2 0 0 0 0 that annealing of tRNA to both 5 and 3 PBS caused the formation of a complex made of 5 and 3 Gypsy RNAs and tRNA (lanes 4–5) but not when tRNA could not anneal to the 3 RNA (lanes 9–10). Next, we constructed the D3 PBS mutant by deleting nt extensively invade the genome is conditioned by the cell 6727–6737 (Materials and Methods). This D3 PBS mutant- capacity to repress this retroelement. This control results in displayed a wild-type level of cDNA made (compare lanes reduced steady state levels of Gypsy transcripts (42,43). 1–2 with 5–6, and 11–12 with 7–8) despite the fact that it Moreover, it has recently been shown that synthesis of does not produce any envelope protein due to the frameshift Gypsy Gag is strictly controlled at the translational level (44). generated by this 11 nt deletion at the Env 3 end (data not To understand the dual nature of the D.melanogaster shown). To further address the role of Gag in the synthesis Gypsy and the fact that it appears to be at the frontier between of Gypsy cDNA, we inserted a frameshift mutation at the retrotransposons of yeast and simple vertebrate retroviruses, 5 end of Gag. This Gypsy mutant that cannot express the we investigated Gypsy replication in vitro and in cell culture. Gag protein was totally unable to direct cDNA synthesis First, we searched for a NC-like region in Gypsy Gag with all (data not shown). In conclusion, these results confirm the the properties of a bona fide retroviral RNA chaperone in vitro findings (Figure 8; lanes 17 and 27) and show that protein (45) [reviewed in (3,4)]. To this end, we used a the 3 PBS is not required for cDNA synthesis, at least at computer-assisted search to look for a peptide domain of this stage of Gypsy replication. low complexity, rich in proline and basic residues, and with a predicted disordered state (33–35). Such a peptide domain, named G-NC, was found at the C-terminal end of the Gypsy Gag, showing notable similarities with the NC-like domain of DISCUSSION TY1 Gag (Figures 1 and 2). In fact, the Gypsy NC-like The D.melanogaster Gypsy retroelement has a genetic orga- peptide was active in the formation of high molecular weight nization similar to that of the widespread vertebrate nucleoprotein complexes and in replication primer annealing gammaretroviruses, such as the MLV. In fact the provirus to the genomic PBS in a manner very similar to that of the form of Gypsy contains the three canonical open reading C-terminal Gag peptide of yeast TY1 (TYA1-D; Figures 4 frames gag, pol and env flanked by two LTRs. As an infec- and 6). Like other retroviral NC proteins, the G-NC peptide tious enveloped retrovirus, Gypsy can efficiently invade the was found to possess general RNA chaperoning properties in vitro, which can promote profound rearrangements of germ line of permissive flies, upon infection of naive females (38,39). These new Gypsy proviruses can then increase their nucleic acid conformation [reviewed in (27,34)]. Along this number by expression in the somatic cells of permissive line, the optimal chaperoning activity of G-NC takes place flamenco females followed by recurrent invasion of the at a peptide to nucleic acid molar ratio of one peptide per germ line by a process, which, in contrast to virus infection, 6 to 10 nt (see Figure 6) as holds true for retroviral NC pro- does not need env (40). Thus, Gypsy can behave both as an teins and NC of the yeast TY3 retrotransposon (27,34,45). infectious retrovirus and as an active retrotransposon (13,14). Interestingly, NCp9 of TY3 was found to be active in the A high level of Gypsy transposition might be deleterious Gypsy system (data not shown) whereas the basic MLV for the host genome due to insertional mutagenesis, espe- and HIV-1 NC peptides were poorly active (Figures 4 and cially because Gypsy carries a strong DNA insulator (41), 6) although they contain zinc fingers shown to specify which can disrupt the activity of the genes, into which it is direct NC–RNA interactions in HIV and MLV (3,24,29). integrated by blocking the interactions of distal enhancers The primary structure of the Gypsy Gag and the presence with the target promoter. Thus, the inability of Gypsy to of an active RNA chaperoning domain at its C-terminus Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5774 Nucleic Acids Research, 2006, Vol. 34, No. 20 Figure 8. Role of the Gypsy NC peptide in cDNA synthesis in vitro.(A) Schematic representation of the initiation of reverse transcription on Gypsy 5 RNA. Reverse transcription of Gypsy 5 U5 and R RNA sequences leads to the synthesis of the so-called minus strand strong stop cDNA, ss-cDNA(), by RT extension Lys,2 32 Lys,2 0 0 0 0 of primer tRNA .(B) Gypsy 5 RNA, 3 RNA or recombinant 5 –3 RNA and P-tRNA were incubated with or without TYA1-D or Gypsy NC peptide. MLV RT was added together with dNTPs to allow reverse transcription. Assays were processed as described in Materials and Methods and ss-cDNA() was denatured and analysed by 10% PAGE in 7 M urea. Controls without protein are shown in lanes 1, 8, 11, 18 and 21. Protein to RNA nucleotide molar ratios were 1:48 (lanes 2, 5, 12, 15, 22 and 25), 1:24 (lanes 3, 6, 13, 16, 23 and 26) and 1:12 (lanes 4, 7, 9, 10, 14, 17, 19, 20, 24 and 27), corresponding to 1.25 · 10 , 2.5 · 7 7 7 7 6 Lys,2 0 0 0 0 10 and 5 · 10 M for 5 RNA and 2.5 · 10 ,5 · 10 and 10 M for 5 –3 RNA. Note that hybridization of tRNA to the 3 PBS caused an inhibition of the initiation of Gypsy reverse transcription which was severe with the TYA1-D peptide (compare lanes 2–4 with 12–14) and moderate with the homologous peptide (compare lanes 5–7 with 15–17). suggest that the Gypsy Gag resembles more that of an ancient from TY3 since the 3 PBS is not implicated in the process retrotransposon than the canonical Gag of the gammaretro- of cDNA synthesis in vitro and ex vivo (Figures 8 and 9). virus MLV, formed of the well-defined matrix, capsid Yet the exact role of the Gypsy 3 PBS could be at the and NC domains. On the opposite the Gypsy genomic level of genome maintenance or translation via formation RNA has long structured untranslated regions, the 5 - and of a circular RNA. This is presently under investigation. 3 -untranslated regions (UTRs) (Figure 3) similar to the In conclusion, these findings on the NC-like domain of MLV genomic RNA. In addition, the Gypsy 5 -UTR directs Gypsy Gag and on the existence of two PBSs indicate that translation of Gag and Env via specific internal ribosome Gypsy resembles active retrotransposons and is distinct from entry sites (IRES) that resemble the MLV Gag and Env canonical simple retroviruses, such as the MLVs and avian IRESes (44,46,47). leukosis viruses. In agreement with this conclusion, the env- Second, we characterized two distinct PBSs located at independent reverse transcription of Gypsy in D.hydei cells 0 0 the 5 and 3 ends of the Gypsy genomic RNA (Figure 5), (Figure 9) is reminiscent of the fact that the efficiency of which are reminiscent of those found in the yeast TY3 Gypsy replication in D.melanogaster females was not affected retrotransposon (7). The G-NC peptide promoted the by the frameshift disruption of the env gene (40). On the other Lys,2 0 0 0 annealing of primer tRNA to both the 5 and 3 PBSs hand, the presence of a single 5 PBS necessary for cDNA syn- (Figure 6). Furthermore, the simultaneous annealing of thesis (Figures 8 and 9), of long structured UTRs, of functional Lys,2 primer tRNA to these two PBSs (Figure 7) resulted in IRESes and Env, and the fact that cell-free Gypsy particles are 0 0 the formation of a 5 –3 RNA complex, indicating that a infectious (13,14) indicate that Gypsy should be considered as tRNA bridge can possibly generate a circular Gypsy RNA an ancient simple retrovirus, similar to the gammaretrovirus as proposed for the TY3 RNA (7). However, Gypsy differs MLV. To investigate how much the NC functions are Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 Nucleic Acids Research, 2006, Vol. 34, No. 20 5775 Figure 9. The 5 PBS is critical for Gypsy cDNA synthesis ex vivo. D.hydei cells were transfected with WT or PBS-mutated Gypsy DNA as reported in materials and methods. The 10.3 kb transfected DNA is shown at the top. The dotted line represents the vector DNA that was used to clone the Gypsy element with a 0 0 complete 3 LTR (rectangle with P, a PvuI site) and a shortened 5 LTR (square) the U3 sequence of which was replaced by a Copia enhancer (black box). Reverse transcription is expected to regenerate the missing sequence, giving rise to the full-length 7.5 kb Gypsy DNA shown at the bottom. The 2-LTR and 1-LTR circular DNA products of 7.5 and 7.0 kb, respectively, are shown. A total of 1 mg of total DNA were digested with either EcoRI (R) or NdeI (N), blotted and probed with the fragment schematized at the bottom. A unique band is expected to correspond to each category of molecules. The only exception corresponds to the integrated proviruses (not represented here), which if randomly inserted at various distances from the EcoRI or NdeI genomic restriction sites, are expected to produce a smear corresponding to a heterogeneous population of molecules larger than 6.6 and 5.7 kb, respectively. In contrast, in double digests involving PvuI (P) and either NdeI (N) or EcoRI (R), all types of cDNA products should merge into a single band, as shown in lanes 1, 5, 8 and 12). Most of the transfected plasmids have run out of the gel after being degraded into small DNA fragments by the methylation-dependent DpnI enzyme; therefore, the upper band only 0 0 corresponds to the minority of plasmids, which have lost these prokaryotic methylation tags. D5 and D3 PBS are mutants which only differ from the wild-type Gypsy sequence (WT) by the DPBS mutations described in Figure 3, at positions (proviral DNA sequence) 482–492 and 6727–6737, respectively (16). Note that 0 0 the D5 PBS, but not the D3 PBS deletion resulted in the absence of cDNA synthesis (lanes 3–4 and 9–10). conserved from Drosophila Gypsy to the MLV, substituting ACKNOWLEDGEMENTS MLV-NC by the Gypsy NC-like is now in progress using The authors thank Marc Ruff (Strasbourg) for helpful advice MLV-based vectors. on peptide expression and purification procedures. Thanks are due to Damien Ficheux (IBCP CNRS) for TYA1-D, MLV- NC and HIV-1 NC peptide synthesis and purification. Gag SUPPLEMENTARY DATA mutant plasmids were generated by Emeline Sarot with the ` ˆ Supplementary Data are available at NAR Online. technical assistance of Genevieve Payen-Groschene. Work Downloaded from https://academic.oup.com/nar/article-abstract/34/20/5764/3100468 by Ed 'DeepDyve' Gillespie user on 06 February 2018 5776 Nucleic Acids Research, 2006, Vol. 34, No. 20 supported by CNRS, ANRS and INSERM to J.L.D., by ARC melanogaster cell lines. Biochem. Biophys. Res. 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Nucleic Acids Research – Oxford University Press

Published: Nov 13, 2006

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