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Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer

Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 33, Issue of August 17, pp. 31274 –31278, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer* Received for publication, May 18, 2001, and in revised form, June 13, 2001 Published, JBC Papers in Press, June 19, 2001, DOI 10.1074/jbc.M104577200 Ken-ichi Takahashi‡§, Seiki Baba‡, Yoshio Koyanagi¶, Naoki Yamamoto, Hiroshi Takaku‡, and Gota Kawai‡** From the ‡Department of Industrial Chemistry, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-8588, the ¶Department of Virology, Tohoku University School of Medicine, 2-1 Seiryou-machi, Aoba-ku, Sendai 980-8575, and the Department of Molecular Virology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Nucleocapsid (NC) protein possesses nucleotide-an- dimeric RNA, annealing of tRNA onto primer binding site nealing activities, which are used in various processes (PBS) of the viral RNA, protection of reverse transcriptase (RT) in retroviral life cycle. As conserved characters, the NC pausing, and strand transfer during reverse transcription (for CX HX C proteins have one or two zinc fingers of CX 2 4 4 review, see Refs. 1 and 2 and references therein). These func- motif surrounded by basic amino acid sequences. Re- tions are associated with the specific binding and the annealing quirement of the zinc fingers for the annealing activities activities to viral RNA. The NC proteins of all retroviruses of NC protein remains controversial. In this study, we except those of spumaviruses have one or two zinc fingers of focused the requirement in the process of maturation of CX CX HX C motif surrounded by basic amino acid sequences. 2 4 4 dimeric viral RNA. Discrimination between immature Three-dimensional structures of NC proteins in several retro- and mature dimers of synthetic RNA corresponding to viruses are known (3–10). The crystal structure of complex of the dimerization initiation site of human immunodefi- the NC protein and packaging signal of the genomic RNA in the ciency virus type 1 (HIV-1) genomic RNA was performed 2 human immunodeficiency virus type 1 (HIV-1) demonstrated -dependent stability in gel electro- based on their Mg two zinc finger knuckles bind to G-rich loop of the RNA in a phoreses and on their distinct signal pattern from NMR stem-loop structure and N-terminal basic sequence forms a analysis of imino protons. Chaperoning activity of the 3 -helix and binds to major groove of stem (5). Alternations HIV-1 NC protein, NCp7, and its fragments for matura- 10 and deletions of the zinc fingers impaired the specific RNA tion of dimeric RNA was investigated using these exper- imental systems. We found that the two basic regions binding activity (11–13) and packaging of the viral RNA (11, flanking the N-terminal zinc finger of NCp7, which are 14), and extinguished viral infectivity (11, 14), but did not connected by two glycine residues instead of the zinc much affect nonspecific RNA binding activity (13, 15) and the finger, were sufficient, although about 10 times the annealing activities such as annealing of complementary DNA amounts of peptide were needed in comparison with and RNA (16), tRNA-PBS annealing (16 –19), viral RNA dimer- intact NCp7. Further, it was found that the amount of ization (17–19), strand transfer, (20), and reduction of RT paus- basic residues rather than the amino acid sequence it- ing (21). On the other hand, mutations and deletions of the self is important for the activity. The zinc fingers may basic regions flanking the zinc fingers impaired the annealing involve the binding affinity and/or such a possible spe- activities (16 –22), RNA binding activity (12, 15, 17, 23, 24), cific binding of NCp7 to dimerization initiation site virion formation (15), and viral infectivity (22, 24 –27). In con- dimer that leads to the maturation reaction. trast, Remy et al. (28) disagreed the dispensability of the zinc fingers for the tRNA-PBS annealing activity of NC protein. 1 Feng et al. (29) also reported on the requirement of residues Nucleocapsid (NC) protein is a component in retroviral par- within the N-terminal zinc finger, not the zinc finger structure ticles and takes various functional roles in retrovirus life cycle, itself, for maturation reaction of dimeric viral RNA. Rong et al. involving encapsidation of the genomic RNA, maturation of the (30) reported on functional difference of RNA complexes an- nealed by NC protein and its mutants. They showed that NC This is an open access article under the CC BY license. protein devoid of the zinc fingers could anneal tRNA onto the * This work was supported by Research for the Future Program Grant JSPS-RFTF97L00503 from the Japan Society for the Promotion viral RNA, but the resultant primer-template complex was not of Science and in part by a grant-in-aid for high technology research elongated by RT to the full-length negative-strand of strong- from the Japanese Ministry of Education, Science, Sports and Culture. stop DNA, unlike the primer-template complex yielded by wild- The costs of publication of this article were defrayed in part by the type NC protein. payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to Maturation of dimeric viral RNA is one of the NC protein- indicate this fact. involved reactions in retrovirus life cycles. Maturation of virion § Present address: Div. of Biological Science, Graduate School of particle occurring after release from host cell is executed Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, through cleavage of gag protein to generate NC protein by viral Japan. ** To whom all correspondence should be addressed. Tel./Fax: 81-47- protease. In this maturation event, the dimeric RNA genome in 478-0425; E-mail: [email protected]. virion particle is converted into a heat-stable dimer (31, 32). In The abbreviations used are: NC, nucleocapsid; PBS, primer binding vitro experiments showed that 5 leader sequences of viral RNA site; RT, reverse transcriptase; HIV-1, human immunodeficiency virus genome form two types of dimer with different stability, named type 1; DIS, dimerization initiation site; PAGE, polyacrylamide gel electrophoresis. as loose and tight dimers (33–37). Loose dimer is spontaneously 31274 This paper is available on line at http://www.jbc.org NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 31275 formed at physiological temperature and converted into tight dimer by incubation with NC protein (35–37). The sequence required for the spontaneous dimerization of the genomic RNA in HIV-1 was identified and named as the dimerization initia- tion site (DIS), which can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem (38, 39). The DIS was also shown to be necessary and sufficient for exhibiting the two-step dimerization involving the HIV-1 NC protein, NCp7 (37, 40). In this reaction, Zn is not required (40). The two types of secondary structure of the DIS dimer were predicted (38, 39). In the first one, base pairs are mostly within monomer to form a stem-loop structure and inter-monomer base pairs are formed only at the self-comple- mentary six nucleotides in the loop (Fig. 1A, kissing-loop dimer). In the second one, base pairs are entirely between two monomers (Fig. 1A, extended-duplex dimer). These two differ- ent topologies of the DIS dimer were confirmed by unambigu- ous discrimination between intra- and intermolecular nuclear Overhauser effect in NMR measurements of an equimolar mix- ture of N-labeled and non-labeled DIS molecules (41) and also displayed in the three-dimensional structures of the two types of the DIS dimer determined by H NMR spectroscopy (42– 44) and x-ray crystallography (45). In the present study, we investigated whether the zinc fin- gers are required for the maturation reaction of the DIS dimer. The previously established experimental system were clearly able to discriminate the two types of the DIS dimer (40, 41) and thus reveal the annealing activity of NCp7. We show here that the zinc fingers are dispensable and the basic regions sur- rounding the N-terminal zinc finger are sufficient for the mat- FIG.1. The two types of dimers of DIS39 (A) and NCp7 and its uration reaction of the DIS dimer. related peptides (B). Mutated or randomized residues in NCBR[12] EXPERIMENTAL PROCEDURES are underlined. Preparation of RNAs and Peptides—RNA oligonucleotides were synthesized either chemically by a phosphoramidite method with an trometer at a probe temperature of 25 °C. Solvent proton signal was automatic DNA/RNA synthesizer, Expedite model 8909 (PerSeptive suppressed by a jump-and-return pulse sequence (46) with a delay of 65 Biosystems Inc.) or enzymatically by an in vitro transcription method s, and 1000 –16,000 scans were accumulated. A 3-Hz exponential with AmpliScribe T7 transcription kits (Epicentre Technologies Co.). multiplication prior to the Fourier transformation and a polynomial For chemically synthesized RNAs, protection groups were removed with base-line correction were applied. ammonia and tetra-n-butylammonium fluoride. Purification with poly- acrylamide gel electrophoresis (PAGE) using 30  40-cm glass plates RESULTS (Nihon Eido Co. Ltd., Tokyo, Japan) under denaturing conditions, and To examine the RNA annealing activity of NCp7, we used our extensive desalting by ultrafiltration (Centricon YM-3, Amicon Inc.) experimental system of a 39-mer RNA (DIS39) covering the were carried out. Lyophilized samples of synthetic nucleocapsid protein NCp7 (LAV strain 72-amino acid sequence) and its related peptides whole DIS sequence. DIS39 forms the two types of dimers, the were purchased from Peptide Institute Inc. (Osaka, Japan) and Sawady kissing-loop dimer and extended-duplex dimer (Fig. 1A), whose Technology Co. Ltd. (Tokyo, Japan), respectively. base pairing topologies were confirmed by NMR analysis (41). Assay for Conformational Conversion of RNA Dimers—Assay was They can be readily discriminated by PAGE; both dimers retain performed in the following way. First, 12 M DIS39 in 4 l of water was dimeric states during flowing through polyacrylamide gel con- heated at 95 °C for 5 min and chilled on ice for 5 min, and then 4 lof 2 2 taining Mg , but in PAGE without Mg , the kissing-loop 2 PN buffer (1 PN buffer contains 10 mM sodium phosphate (pH 7.0) and 50 mM NaCl) was added. In the case of heterodimer of DIS39K1 and dimer separates into monomers, whereas the extended-duplex DIS39K2, 12 M amounts of each RNA in 2 l of water were heated dimer retains the dimeric state because of their different Mg separately at 95 °C for 5 min and chilled on ice for 5 min. After 2 lof dependent stability. As reported in the previous study (40), 2 PN buffer was added to each RNA solution, both solutions were DIS39 mostly forms the kissing-loop dimer by itself at 37 °C mixed. Second, various concentrations of NCp7 or its related peptides in (Fig. 2, lane 1) and is converted into the extended-duplex dimer 12 lof1 PN buffer were added to the RNA solutions. The molar ratio when incubated with an equimolar NCp7 at 37 °C (Fig. 2, lane of peptide to RNA was 1:1 through 10:1. In the case of experiments without peptide, 12 lof1 PN buffer was added. Third, the mixture 2). Using this experimental system, we examined the relevance solutions were incubated at 37 °C for 2.5 or 20 h and then treated with of the two basic regions of NCp7 to the RNA-annealing activity. phenol/chloroform solution, regardless of the presence or absence of First, we prepared three peptides, NCBR1, NCBR2, and peptide. Ten l of the aqueous layer containing RNA was collected and NCBR[12], which correspond to the first basic region, the mixed with 10 l of loading buffer containing glycerol and dyes. Then, second basic region, and both connected by a linker of two the solution was divided into two parts, which were separately analyzed glycine residues (Fig. 1B). Instead of intact NCp7, each of the by electrophoreses through non-denaturing polyacrylamide gels (10% or 15%) in TBM buffer (89 mM Tris, 89 mM borate, 1 mM MgCl ) and in peptides was mixed with DIS39 at 1 and 10 equivalent molar TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA), respectively, at ratios of peptide to RNA. For DIS39 incubated with 10 equiv- room temperature. After electrophoresis, the gels were stained with alent molar amount of NCBR1, the upper band in Mg -free gel Red Stain (Bio-Rad) and RNA was visualized by a ultraviolet illumina- (Mg -independent dimer) became slightly intense (Fig. 2A, tor (FAS-III, Toyobo Co. Ltd., Osaka, Japan). compare lanes 1 and 4), indicating that shift to the extended- NMR Measurements—DIS39 in 1 PN buffer was concentrated to duplex dimer in a small population was induced by NCBR1. On 0.06 – 0.7 mM by ultrafiltration, and D O was added to 5%. Final volume was 200 l. NMR spectra were recorded on a Bruker DRX-500 spec- the other hand, no difference was found in electrophoretic 31276 NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS FIG.2. Assay for conformational conversion of DIS39 by NCp7 and its related peptides. Samples incubated for 2.5 h (A)or20h(B) are shown. DIS39 was incubated alone (lane 1), with an equivalent molar NCp7 (lane 2) and with a 1 and 10 equivalent molar amount of NCBR1 (lanes 3 and 4, respectively), NCBR2 (lanes 5 and 6, respective- ly), and NCBR[12] (lanes 7 and 8, respectively). K, kissing-loop dimer; E, extended-duplex dimer. pattern between DIS39 incubated with NCBR2 and DIS39 without any peptide (Fig. 2A, compare lane 1 and lanes 5 and FIG.3. The heterodimer of DIS39 mutants, DIS39K1 and 6), indicating that NCBR2 gave no effect to DIS39. In the case DIS39K2 (A), and examination of its complex formation with of NCBR[12], although the annealing activity was weak when NCp7 and its related peptides, if any, surviving from phenol/ mixed at an equimolar ratio (Fig. 2A, lane 7), 10 equivalent chloroform treatment (B). DIS39K was incubated without any pep- molar amount of NCBR[12] clearly showed the activity; the tides (lane 3), with an equivalent molar NCp7 (lane 4), and with 1 and 2 2 lower band in Mg -free gel (monomer separated from Mg - 10 equivalent molar NCBR[12] (lanes 5 and 6, respectively). Lanes 1 and 2 are the wild-type DIS39 incubated without and with an equiva- required dimer) mostly disappeared, and the upper band lent molar NCp7. Incubation time is 2.5 h. K, kissing-loop dimer; E, (Mg -independent dimer) drastically increased (Fig. 2A, com- extended-duplex dimer. pare lanes 1 and 8), indicating complete shift from the kissing- loop dimer to the extended-duplex dimer. NCp7 and NCBR[12] seemed not to show turnover because extended incubation did not change electrophoretic patterns (Fig. 2B). There might be a possibility that the increased population of the upper band in Mg -free gel (Fig. 2A, lane 8) could not be the extended-duplex dimer but the kissing-loop dimer stabi- lized by NCBR[12] that could be survived from the phenol/ chloroform treatment. This possibility of complex formation, if any, could be examined by using DIS mutants that only form kissing-loop dimers but never extended-duplex dimers. We pre- viously designed a heterodimer of DIS39 mutants, DIS39K1 and DIS39K2, (termed DIS39K as the pair) as such an exclu- sive kissing-loop dimer (40) (Fig. 3A). As expected, DIS39K gave no upper band in Mg -free gel even if incubated with NCp7 (Fig. 3B, lane 4). Using this pair of mutants instead of the original DIS39, we carried out a similar experiment for the annealing activity assay of NCBR[12]. Consequently, no up- per band in Mg -free gel appeared for DIS39K incubated with 10 mol of NCBR[12]/mol of total RNA molecules (Fig. 3B, lane FIG.4. Assay for conformational conversion of DIS39 by 6). This indicates that the complex between the kissing-loop NCBR[12] at various peptide-to-RNA ratios. The ratio of dimer and NCBR[12] did not exist at least after the phenol/ NCBR[12] to DIS39 is 1, 2, 4, 8, and 10 in lanes 3, 4, 5, 6, and 7, respectively. Lanes 1 and 2 are DIS39 incubated without and with an chloroform treatment. Thus, it is highly probable that the ma- equivalent molar NCp7. Incubation time is 2.5 h. K, kissing-loop dimer; jor population in the upper band in lane 8 of Fig. 2B is the E, extended-duplex dimer. extended-duplex dimer of DIS39. This is confirmed by the NMR measurement as described below. To investigate amounts of NCBR[12] required for confor- extended-duplex dimer generated by heat-annealing (41), we mational conversion of a DIS39 dimer, the annealing assay was measured and compared their imino-proton NMR spectra. As carried out at NCBR[12] to DIS39 ratios between 1 and 10 (or shown in Fig. 5, the NMR spectra of DIS39 samples treated by ratios of NCBR[12] to DIS39 dimer between 2 and 20). As heat-annealing, NCp7 and NCBR[12] (A–C, respectively) are shown in Fig. 4, although 2 molecules of NCp7 on average are all identical, whereas they are different from the spectrum of enough to anneal one dimer of DIS39 (or 2 molecules of DIS39), DIS39 samples incubated in the absence of peptides (D). This 16 –20 molecules of NCBR[12] on average are required for indicates that both NCp7 and NCBR[12] treatment gave the annealing of one dimer of DIS39. same extended-duplex conformation that was previously ana- To determine whether NCp7- and NCBR[12]-converted lyzed in detail for DIS39 dimer prepared by heat-annealing DIS39 dimers take the same conformation and whether they (41). It should be noted that the sample for Fig. 5D is mainly in are structurally the same one as the previously characterized the kissing-loop dimer form but contained small amounts of the NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 31277 FIG.6. Assay for conformational conversion of DIS39 by mu- tant peptides of NCBR[12]. DIS39 was incubated with NCBR1 (lanes 1 and 2), NCBR[12] (lanes 3 and 4), and each mutant peptide of NCBR[12] in which alanine was substituted for one of the basic residues Arg-3 (M3, lanes 5 and 6), Arg-7 (M7, lanes 7 and 8), Lys-11 (M11, lanes 9 and 10), and Lys-14 (M14, lanes 11 and 12). DIS39 was also incubated with peptides having randomized sequences of NCBR[12], RD1 (lanes 13 and 14) and RD2 (lanes 15 and 16). Peptide- to-RNA molar ratios are 2 (odd-numbered lanes)and5(even-numbered lanes). Incubation time is 2.5 h. K, kissing-loop dimer; E, extended- duplex dimer. FIG.5. 500-MHz H NMR spectra of DIS39. Imino proton region is (40). In the present study, we further investigated dispensabil- shown. A, the RNA sample in 1 PN buffer was incubated at 95 °C for ity of the zinc fingers themselves and found that the two basic 5 min and slowly cooled down at room temperature to form the extend- ed-duplex dimer. B–D, the RNA sample (in the kissing-loop dimer form) regions flanking the N-terminal zinc finger, which were con- was incubated at 37 °Cfor4hinthe presence of 10 equivalent molar nected by a linker of two glycine residues instead of the zinc NCBR[12] (B), in the presence of 2 equivalent molar NCp7 (C), and in finger (NCBR[12]), were sufficient for the activity although the absence of peptide (D), respectively. For B–D, the RNA concentra- about 10 times the amount of peptide was needed in compari- tion during the incubation was same as that in the PAGE experiments in Figs. 2– 4. Each RNA sample was treated by phenol/chloroform and son with intact NCp7 to convert the same amount of DIS39 then concentrated by ultrafiltration. The final concentrations were dimer (Fig. 2). The possibility that complexes between the 0.25, 0.12, 0.06, and 0.14 mM, and the number of scans were 1000, 4000, kissing-loop dimer and the basic peptide behave like the ex- 16,000, and 4000 for A–D, respectively. tended-duplex dimer in gel electrophoreses was excluded by using a pair of DIS39 mutants that forms only a kissing-loop extended-duplex dimer, which gave the same signal with spec- dimer (DIS39K, Fig. 3) and, more directly, by NMR measure- tra A, B, and C. ments of DIS39 dimers converted by the basic peptide NCBR[12] was active as described above. Which residue, NCBR[12] and intact NCp7. then, is important for the activity? We examined the annealing Remy et al. (28) reported on requirement of the zinc fingers for activity of mutant peptides of NCBR[12]. Basic residues the tRNA-PBS annealing activity of NC protein. They eliminated Arg-3, Arg-7, Lys-11, and Lys-14 in NCBR[12] were individ- the phenol/chloroform treatment; thus, there is still be a possi- ually replaced with alanine (M3, M7, M11, and M14, respec- bility that they just observed the ternary complex of tRNA, PBS- tively, as shown in Fig. 1B). Furthermore, the amino acid containing viral RNA, and NCp7 without annealing. Feng et al. sequence of NCBR[12] was randomized without alteration of (29) have investigated the NCp7-induced maturation of dimeric amino acid composition; the N-terminal 14 residues and the RNA of Harvey sarcoma virus. They measured the heat stability whole amino acid residues were shuffled (RD1 and RD2, re- of RNA dimer and discriminated the two types of RNA dimer. spectively, as shown in Fig. 1B). Basically, all these mutant Their results showed the requirement of the basic residues flank- peptides showed the annealing activity to an extent similar to ing the N-terminal zinc finger for the activity of NCp7. However, that of the original NCBR[12] (Fig. 6). Some point mutants they also showed that the mutation of the aromatic residue showed a little reduced activity. This result indicates that an within the N-terminal zinc finger as well as the removal of the important factor for the activity is a large amount of basic N-terminal zinc finger lost the maturation activity, although the residues rather than the amino acid sequence itself of cysteine residues in the zinc fingers could be mutated without NCBR[12]. the loss of the activity. We note the possibility that an increasing DISCUSSION amount of their NCp7 mutants devoid of the zinc finger could There are controversial reports on dispensability or require- lead to the detection of the maturation activity, like in our case of ment of the zinc fingers for the RNA annealing activities of NC NCBR[12]. protein. Regarding one of the annealing activities, the matura- Approximately 10 molecules of NCBR[12]/DIS39 molecule tion reaction of the dimeric viral RNA, we previously established on average were required for the maturation activity. In the a simple in vitro assay system using a short RNA fragment case of an equimolar mixture between NCBR[12] and DIS39, (DIS39) corresponding to the DIS of the HIV-1 (40). The imma- a prolonged incubation did not change the fraction of the ma- ture dimer (loose dimer) and NCp7-converted mature dimer ture dimer of DIS39, as shown in Fig. 2B. However, an increas- (tight dimer) of DIS39 can be easily distinguished by their ing amount of NCBR[12] raised the fraction of the mature different Mg -dependent stabilities when they are subjected dimer linearly in an incubation time of 2.5 h (Fig. 4). The to gel electrophoreses with and without Mg . This electro- binding affinity of NCBR[12] may be decreased in comparison phoretic method was originally developed by Laughrea and with intact NCp7, and the fraction of a possible specific binding Jette (33). We have also confirmed by NMR measurements that that leads to the maturation reaction may be decreased by the the two types of DIS39 dimer take the secondary structures competition with the nonspecific, and non-active, binding. The proposed in the kissing-loop model (38, 39), the kissing-loop zinc finger domains of NCp7 may take a role to bind DIS39 dimer and extended-duplex dimer, respectively (Fig. 1A) (41). specifically and locate the basic regions in the best position on Using this assay system, we demonstrated previously that DIS39 for the maturation reaction. NCp7 did not require Zn to generate mature DIS39 dimer Although NC proteins of various retroviruses commonly con- 31278 NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 3046 –3057 tain basic regions, their amino acid sequences themselves are 16. Lapadat-Tapolsky, M., Pernelle, C., Borie, C., and Darlix, J. L. (1995) Nucleic not conserved (2). Our study demonstrated that peptides with Acids Res. 23, 2434 –2441 17. Prats, A. C., Housset, V., de Billy, G., Cornille, F., Prats, H., Roques, B., Darlix, randomized sequences of NCBR[12] had the same level of the J. L. (1991) Nucleic Acids Res. 19, 3533–3541 annealing activity as that of the wild type sequence of 18. de Rocquigny, H., Ficheux, D., Gabus, C., Allain, B., Fournie-Zaluski, M. C., NCBR[12]. On the hand, little and no activities were seen in Darlix, J. L., and Roques, B. P. (1993) Nucleic Acids Res. 21, 823– 829 19. de Rocquigny, H., Gabus, C., Vincent, A., Fournie-Zaluski, M. C., Roques, B., the shorter peptides NCBR1 and NCBR2, respectively. These and Darlix, J. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6472– 6476 indicate that some number of basic residues is required for the 20. Hsu, M., Rong, L., de Rocquigny, H., Roques, B. P., and Wainberg, M. A. (2000) annealing activity. If NCBR[12] is assumed to form a helical Nucleic Acids Res. 28, 1724 –1729 21. Wu, W., Henderson, L. E., Copeland, T. D., Gorelick, R. J., Bosche, W. J., Rein, structure, a constructed three-dimensional model of A., and Levin, J. G. (1996) J. Virol. 70, 7132–7142 NCBR[12] shows that positive electrostatic potential on the 22. Morellet, N., de Rocquigny, H., Mely, Y., Jullian, N., Demene, H., Ottmann, M., Gerard, D., Darlix, J. L., Fournie-Zaluski, M. C., and Roques, B. P. molecular surface is localized in one side of the helix. It should (1994) J. Mol. Biol. 235, 287–301 be noted that, unexpectedly, the two randomized peptides also 23. 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Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer

Takahashi, Ken-ichi; Baba, Seiki; Koyanagi, Yoshio; Yamamoto, Naoki; Takaku, Hiroshi; Kawai, Gota
Journal of Biological Chemistry – Aug 1, 2001
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 33, Issue of August 17, pp. 31274 –31278, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Two Basic Regions of NCp7 Are Sufficient for Conformational Conversion of HIV-1 Dimerization Initiation Site from Kissing-loop Dimer to Extended-duplex Dimer* Received for publication, May 18, 2001, and in revised form, June 13, 2001 Published, JBC Papers in Press, June 19, 2001, DOI 10.1074/jbc.M104577200 Ken-ichi Takahashi‡§, Seiki Baba‡, Yoshio Koyanagi¶, Naoki Yamamoto, Hiroshi Takaku‡, and Gota Kawai‡** From the ‡Department of Industrial Chemistry, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba 275-8588, the ¶Department of Virology, Tohoku University School of Medicine, 2-1 Seiryou-machi, Aoba-ku, Sendai 980-8575, and the Department of Molecular Virology, Faculty of Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan Nucleocapsid (NC) protein possesses nucleotide-an- dimeric RNA, annealing of tRNA onto primer binding site nealing activities, which are used in various processes (PBS) of the viral RNA, protection of reverse transcriptase (RT) in retroviral life cycle. As conserved characters, the NC pausing, and strand transfer during reverse transcription (for CX HX C proteins have one or two zinc fingers of CX 2 4 4 review, see Refs. 1 and 2 and references therein). These func- motif surrounded by basic amino acid sequences. Re- tions are associated with the specific binding and the annealing quirement of the zinc fingers for the annealing activities activities to viral RNA. The NC proteins of all retroviruses of NC protein remains controversial. In this study, we except those of spumaviruses have one or two zinc fingers of focused the requirement in the process of maturation of CX CX HX C motif surrounded by basic amino acid sequences. 2 4 4 dimeric viral RNA. Discrimination between immature Three-dimensional structures of NC proteins in several retro- and mature dimers of synthetic RNA corresponding to viruses are known (3–10). The crystal structure of complex of the dimerization initiation site of human immunodefi- the NC protein and packaging signal of the genomic RNA in the ciency virus type 1 (HIV-1) genomic RNA was performed 2 human immunodeficiency virus type 1 (HIV-1) demonstrated -dependent stability in gel electro- based on their Mg two zinc finger knuckles bind to G-rich loop of the RNA in a phoreses and on their distinct signal pattern from NMR stem-loop structure and N-terminal basic sequence forms a analysis of imino protons. Chaperoning activity of the 3 -helix and binds to major groove of stem (5). Alternations HIV-1 NC protein, NCp7, and its fragments for matura- 10 and deletions of the zinc fingers impaired the specific RNA tion of dimeric RNA was investigated using these exper- imental systems. We found that the two basic regions binding activity (11–13) and packaging of the viral RNA (11, flanking the N-terminal zinc finger of NCp7, which are 14), and extinguished viral infectivity (11, 14), but did not connected by two glycine residues instead of the zinc much affect nonspecific RNA binding activity (13, 15) and the finger, were sufficient, although about 10 times the annealing activities such as annealing of complementary DNA amounts of peptide were needed in comparison with and RNA (16), tRNA-PBS annealing (16 –19), viral RNA dimer- intact NCp7. Further, it was found that the amount of ization (17–19), strand transfer, (20), and reduction of RT paus- basic residues rather than the amino acid sequence it- ing (21). On the other hand, mutations and deletions of the self is important for the activity. The zinc fingers may basic regions flanking the zinc fingers impaired the annealing involve the binding affinity and/or such a possible spe- activities (16 –22), RNA binding activity (12, 15, 17, 23, 24), cific binding of NCp7 to dimerization initiation site virion formation (15), and viral infectivity (22, 24 –27). In con- dimer that leads to the maturation reaction. trast, Remy et al. (28) disagreed the dispensability of the zinc fingers for the tRNA-PBS annealing activity of NC protein. 1 Feng et al. (29) also reported on the requirement of residues Nucleocapsid (NC) protein is a component in retroviral par- within the N-terminal zinc finger, not the zinc finger structure ticles and takes various functional roles in retrovirus life cycle, itself, for maturation reaction of dimeric viral RNA. Rong et al. involving encapsidation of the genomic RNA, maturation of the (30) reported on functional difference of RNA complexes an- nealed by NC protein and its mutants. They showed that NC This is an open access article under the CC BY license. protein devoid of the zinc fingers could anneal tRNA onto the * This work was supported by Research for the Future Program Grant JSPS-RFTF97L00503 from the Japan Society for the Promotion viral RNA, but the resultant primer-template complex was not of Science and in part by a grant-in-aid for high technology research elongated by RT to the full-length negative-strand of strong- from the Japanese Ministry of Education, Science, Sports and Culture. stop DNA, unlike the primer-template complex yielded by wild- The costs of publication of this article were defrayed in part by the type NC protein. payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to Maturation of dimeric viral RNA is one of the NC protein- indicate this fact. involved reactions in retrovirus life cycles. Maturation of virion § Present address: Div. of Biological Science, Graduate School of particle occurring after release from host cell is executed Science, Nagoya University, Furo-cho, Chikusa, Nagoya 464-8602, through cleavage of gag protein to generate NC protein by viral Japan. ** To whom all correspondence should be addressed. Tel./Fax: 81-47- protease. In this maturation event, the dimeric RNA genome in 478-0425; E-mail: [email protected]. virion particle is converted into a heat-stable dimer (31, 32). In The abbreviations used are: NC, nucleocapsid; PBS, primer binding vitro experiments showed that 5 leader sequences of viral RNA site; RT, reverse transcriptase; HIV-1, human immunodeficiency virus genome form two types of dimer with different stability, named type 1; DIS, dimerization initiation site; PAGE, polyacrylamide gel electrophoresis. as loose and tight dimers (33–37). Loose dimer is spontaneously 31274 This paper is available on line at http://www.jbc.org NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 31275 formed at physiological temperature and converted into tight dimer by incubation with NC protein (35–37). The sequence required for the spontaneous dimerization of the genomic RNA in HIV-1 was identified and named as the dimerization initia- tion site (DIS), which can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem (38, 39). The DIS was also shown to be necessary and sufficient for exhibiting the two-step dimerization involving the HIV-1 NC protein, NCp7 (37, 40). In this reaction, Zn is not required (40). The two types of secondary structure of the DIS dimer were predicted (38, 39). In the first one, base pairs are mostly within monomer to form a stem-loop structure and inter-monomer base pairs are formed only at the self-comple- mentary six nucleotides in the loop (Fig. 1A, kissing-loop dimer). In the second one, base pairs are entirely between two monomers (Fig. 1A, extended-duplex dimer). These two differ- ent topologies of the DIS dimer were confirmed by unambigu- ous discrimination between intra- and intermolecular nuclear Overhauser effect in NMR measurements of an equimolar mix- ture of N-labeled and non-labeled DIS molecules (41) and also displayed in the three-dimensional structures of the two types of the DIS dimer determined by H NMR spectroscopy (42– 44) and x-ray crystallography (45). In the present study, we investigated whether the zinc fin- gers are required for the maturation reaction of the DIS dimer. The previously established experimental system were clearly able to discriminate the two types of the DIS dimer (40, 41) and thus reveal the annealing activity of NCp7. We show here that the zinc fingers are dispensable and the basic regions sur- rounding the N-terminal zinc finger are sufficient for the mat- FIG.1. The two types of dimers of DIS39 (A) and NCp7 and its uration reaction of the DIS dimer. related peptides (B). Mutated or randomized residues in NCBR[12] EXPERIMENTAL PROCEDURES are underlined. Preparation of RNAs and Peptides—RNA oligonucleotides were synthesized either chemically by a phosphoramidite method with an trometer at a probe temperature of 25 °C. Solvent proton signal was automatic DNA/RNA synthesizer, Expedite model 8909 (PerSeptive suppressed by a jump-and-return pulse sequence (46) with a delay of 65 Biosystems Inc.) or enzymatically by an in vitro transcription method s, and 1000 –16,000 scans were accumulated. A 3-Hz exponential with AmpliScribe T7 transcription kits (Epicentre Technologies Co.). multiplication prior to the Fourier transformation and a polynomial For chemically synthesized RNAs, protection groups were removed with base-line correction were applied. ammonia and tetra-n-butylammonium fluoride. Purification with poly- acrylamide gel electrophoresis (PAGE) using 30  40-cm glass plates RESULTS (Nihon Eido Co. Ltd., Tokyo, Japan) under denaturing conditions, and To examine the RNA annealing activity of NCp7, we used our extensive desalting by ultrafiltration (Centricon YM-3, Amicon Inc.) experimental system of a 39-mer RNA (DIS39) covering the were carried out. Lyophilized samples of synthetic nucleocapsid protein NCp7 (LAV strain 72-amino acid sequence) and its related peptides whole DIS sequence. DIS39 forms the two types of dimers, the were purchased from Peptide Institute Inc. (Osaka, Japan) and Sawady kissing-loop dimer and extended-duplex dimer (Fig. 1A), whose Technology Co. Ltd. (Tokyo, Japan), respectively. base pairing topologies were confirmed by NMR analysis (41). Assay for Conformational Conversion of RNA Dimers—Assay was They can be readily discriminated by PAGE; both dimers retain performed in the following way. First, 12 M DIS39 in 4 l of water was dimeric states during flowing through polyacrylamide gel con- heated at 95 °C for 5 min and chilled on ice for 5 min, and then 4 lof 2 2 taining Mg , but in PAGE without Mg , the kissing-loop 2 PN buffer (1 PN buffer contains 10 mM sodium phosphate (pH 7.0) and 50 mM NaCl) was added. In the case of heterodimer of DIS39K1 and dimer separates into monomers, whereas the extended-duplex DIS39K2, 12 M amounts of each RNA in 2 l of water were heated dimer retains the dimeric state because of their different Mg separately at 95 °C for 5 min and chilled on ice for 5 min. After 2 lof dependent stability. As reported in the previous study (40), 2 PN buffer was added to each RNA solution, both solutions were DIS39 mostly forms the kissing-loop dimer by itself at 37 °C mixed. Second, various concentrations of NCp7 or its related peptides in (Fig. 2, lane 1) and is converted into the extended-duplex dimer 12 lof1 PN buffer were added to the RNA solutions. The molar ratio when incubated with an equimolar NCp7 at 37 °C (Fig. 2, lane of peptide to RNA was 1:1 through 10:1. In the case of experiments without peptide, 12 lof1 PN buffer was added. Third, the mixture 2). Using this experimental system, we examined the relevance solutions were incubated at 37 °C for 2.5 or 20 h and then treated with of the two basic regions of NCp7 to the RNA-annealing activity. phenol/chloroform solution, regardless of the presence or absence of First, we prepared three peptides, NCBR1, NCBR2, and peptide. Ten l of the aqueous layer containing RNA was collected and NCBR[12], which correspond to the first basic region, the mixed with 10 l of loading buffer containing glycerol and dyes. Then, second basic region, and both connected by a linker of two the solution was divided into two parts, which were separately analyzed glycine residues (Fig. 1B). Instead of intact NCp7, each of the by electrophoreses through non-denaturing polyacrylamide gels (10% or 15%) in TBM buffer (89 mM Tris, 89 mM borate, 1 mM MgCl ) and in peptides was mixed with DIS39 at 1 and 10 equivalent molar TBE buffer (89 mM Tris, 89 mM borate, 2 mM EDTA), respectively, at ratios of peptide to RNA. For DIS39 incubated with 10 equiv- room temperature. After electrophoresis, the gels were stained with alent molar amount of NCBR1, the upper band in Mg -free gel Red Stain (Bio-Rad) and RNA was visualized by a ultraviolet illumina- (Mg -independent dimer) became slightly intense (Fig. 2A, tor (FAS-III, Toyobo Co. Ltd., Osaka, Japan). compare lanes 1 and 4), indicating that shift to the extended- NMR Measurements—DIS39 in 1 PN buffer was concentrated to duplex dimer in a small population was induced by NCBR1. On 0.06 – 0.7 mM by ultrafiltration, and D O was added to 5%. Final volume was 200 l. NMR spectra were recorded on a Bruker DRX-500 spec- the other hand, no difference was found in electrophoretic 31276 NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS FIG.2. Assay for conformational conversion of DIS39 by NCp7 and its related peptides. Samples incubated for 2.5 h (A)or20h(B) are shown. DIS39 was incubated alone (lane 1), with an equivalent molar NCp7 (lane 2) and with a 1 and 10 equivalent molar amount of NCBR1 (lanes 3 and 4, respectively), NCBR2 (lanes 5 and 6, respective- ly), and NCBR[12] (lanes 7 and 8, respectively). K, kissing-loop dimer; E, extended-duplex dimer. pattern between DIS39 incubated with NCBR2 and DIS39 without any peptide (Fig. 2A, compare lane 1 and lanes 5 and FIG.3. The heterodimer of DIS39 mutants, DIS39K1 and 6), indicating that NCBR2 gave no effect to DIS39. In the case DIS39K2 (A), and examination of its complex formation with of NCBR[12], although the annealing activity was weak when NCp7 and its related peptides, if any, surviving from phenol/ mixed at an equimolar ratio (Fig. 2A, lane 7), 10 equivalent chloroform treatment (B). DIS39K was incubated without any pep- molar amount of NCBR[12] clearly showed the activity; the tides (lane 3), with an equivalent molar NCp7 (lane 4), and with 1 and 2 2 lower band in Mg -free gel (monomer separated from Mg - 10 equivalent molar NCBR[12] (lanes 5 and 6, respectively). Lanes 1 and 2 are the wild-type DIS39 incubated without and with an equiva- required dimer) mostly disappeared, and the upper band lent molar NCp7. Incubation time is 2.5 h. K, kissing-loop dimer; E, (Mg -independent dimer) drastically increased (Fig. 2A, com- extended-duplex dimer. pare lanes 1 and 8), indicating complete shift from the kissing- loop dimer to the extended-duplex dimer. NCp7 and NCBR[12] seemed not to show turnover because extended incubation did not change electrophoretic patterns (Fig. 2B). There might be a possibility that the increased population of the upper band in Mg -free gel (Fig. 2A, lane 8) could not be the extended-duplex dimer but the kissing-loop dimer stabi- lized by NCBR[12] that could be survived from the phenol/ chloroform treatment. This possibility of complex formation, if any, could be examined by using DIS mutants that only form kissing-loop dimers but never extended-duplex dimers. We pre- viously designed a heterodimer of DIS39 mutants, DIS39K1 and DIS39K2, (termed DIS39K as the pair) as such an exclu- sive kissing-loop dimer (40) (Fig. 3A). As expected, DIS39K gave no upper band in Mg -free gel even if incubated with NCp7 (Fig. 3B, lane 4). Using this pair of mutants instead of the original DIS39, we carried out a similar experiment for the annealing activity assay of NCBR[12]. Consequently, no up- per band in Mg -free gel appeared for DIS39K incubated with 10 mol of NCBR[12]/mol of total RNA molecules (Fig. 3B, lane FIG.4. Assay for conformational conversion of DIS39 by 6). This indicates that the complex between the kissing-loop NCBR[12] at various peptide-to-RNA ratios. The ratio of dimer and NCBR[12] did not exist at least after the phenol/ NCBR[12] to DIS39 is 1, 2, 4, 8, and 10 in lanes 3, 4, 5, 6, and 7, respectively. Lanes 1 and 2 are DIS39 incubated without and with an chloroform treatment. Thus, it is highly probable that the ma- equivalent molar NCp7. Incubation time is 2.5 h. K, kissing-loop dimer; jor population in the upper band in lane 8 of Fig. 2B is the E, extended-duplex dimer. extended-duplex dimer of DIS39. This is confirmed by the NMR measurement as described below. To investigate amounts of NCBR[12] required for confor- extended-duplex dimer generated by heat-annealing (41), we mational conversion of a DIS39 dimer, the annealing assay was measured and compared their imino-proton NMR spectra. As carried out at NCBR[12] to DIS39 ratios between 1 and 10 (or shown in Fig. 5, the NMR spectra of DIS39 samples treated by ratios of NCBR[12] to DIS39 dimer between 2 and 20). As heat-annealing, NCp7 and NCBR[12] (A–C, respectively) are shown in Fig. 4, although 2 molecules of NCp7 on average are all identical, whereas they are different from the spectrum of enough to anneal one dimer of DIS39 (or 2 molecules of DIS39), DIS39 samples incubated in the absence of peptides (D). This 16 –20 molecules of NCBR[12] on average are required for indicates that both NCp7 and NCBR[12] treatment gave the annealing of one dimer of DIS39. same extended-duplex conformation that was previously ana- To determine whether NCp7- and NCBR[12]-converted lyzed in detail for DIS39 dimer prepared by heat-annealing DIS39 dimers take the same conformation and whether they (41). It should be noted that the sample for Fig. 5D is mainly in are structurally the same one as the previously characterized the kissing-loop dimer form but contained small amounts of the NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 31277 FIG.6. Assay for conformational conversion of DIS39 by mu- tant peptides of NCBR[12]. DIS39 was incubated with NCBR1 (lanes 1 and 2), NCBR[12] (lanes 3 and 4), and each mutant peptide of NCBR[12] in which alanine was substituted for one of the basic residues Arg-3 (M3, lanes 5 and 6), Arg-7 (M7, lanes 7 and 8), Lys-11 (M11, lanes 9 and 10), and Lys-14 (M14, lanes 11 and 12). DIS39 was also incubated with peptides having randomized sequences of NCBR[12], RD1 (lanes 13 and 14) and RD2 (lanes 15 and 16). Peptide- to-RNA molar ratios are 2 (odd-numbered lanes)and5(even-numbered lanes). Incubation time is 2.5 h. K, kissing-loop dimer; E, extended- duplex dimer. FIG.5. 500-MHz H NMR spectra of DIS39. Imino proton region is (40). In the present study, we further investigated dispensabil- shown. A, the RNA sample in 1 PN buffer was incubated at 95 °C for ity of the zinc fingers themselves and found that the two basic 5 min and slowly cooled down at room temperature to form the extend- ed-duplex dimer. B–D, the RNA sample (in the kissing-loop dimer form) regions flanking the N-terminal zinc finger, which were con- was incubated at 37 °Cfor4hinthe presence of 10 equivalent molar nected by a linker of two glycine residues instead of the zinc NCBR[12] (B), in the presence of 2 equivalent molar NCp7 (C), and in finger (NCBR[12]), were sufficient for the activity although the absence of peptide (D), respectively. For B–D, the RNA concentra- about 10 times the amount of peptide was needed in compari- tion during the incubation was same as that in the PAGE experiments in Figs. 2– 4. Each RNA sample was treated by phenol/chloroform and son with intact NCp7 to convert the same amount of DIS39 then concentrated by ultrafiltration. The final concentrations were dimer (Fig. 2). The possibility that complexes between the 0.25, 0.12, 0.06, and 0.14 mM, and the number of scans were 1000, 4000, kissing-loop dimer and the basic peptide behave like the ex- 16,000, and 4000 for A–D, respectively. tended-duplex dimer in gel electrophoreses was excluded by using a pair of DIS39 mutants that forms only a kissing-loop extended-duplex dimer, which gave the same signal with spec- dimer (DIS39K, Fig. 3) and, more directly, by NMR measure- tra A, B, and C. ments of DIS39 dimers converted by the basic peptide NCBR[12] was active as described above. Which residue, NCBR[12] and intact NCp7. then, is important for the activity? We examined the annealing Remy et al. (28) reported on requirement of the zinc fingers for activity of mutant peptides of NCBR[12]. Basic residues the tRNA-PBS annealing activity of NC protein. They eliminated Arg-3, Arg-7, Lys-11, and Lys-14 in NCBR[12] were individ- the phenol/chloroform treatment; thus, there is still be a possi- ually replaced with alanine (M3, M7, M11, and M14, respec- bility that they just observed the ternary complex of tRNA, PBS- tively, as shown in Fig. 1B). Furthermore, the amino acid containing viral RNA, and NCp7 without annealing. Feng et al. sequence of NCBR[12] was randomized without alteration of (29) have investigated the NCp7-induced maturation of dimeric amino acid composition; the N-terminal 14 residues and the RNA of Harvey sarcoma virus. They measured the heat stability whole amino acid residues were shuffled (RD1 and RD2, re- of RNA dimer and discriminated the two types of RNA dimer. spectively, as shown in Fig. 1B). Basically, all these mutant Their results showed the requirement of the basic residues flank- peptides showed the annealing activity to an extent similar to ing the N-terminal zinc finger for the activity of NCp7. However, that of the original NCBR[12] (Fig. 6). Some point mutants they also showed that the mutation of the aromatic residue showed a little reduced activity. This result indicates that an within the N-terminal zinc finger as well as the removal of the important factor for the activity is a large amount of basic N-terminal zinc finger lost the maturation activity, although the residues rather than the amino acid sequence itself of cysteine residues in the zinc fingers could be mutated without NCBR[12]. the loss of the activity. We note the possibility that an increasing DISCUSSION amount of their NCp7 mutants devoid of the zinc finger could There are controversial reports on dispensability or require- lead to the detection of the maturation activity, like in our case of ment of the zinc fingers for the RNA annealing activities of NC NCBR[12]. protein. Regarding one of the annealing activities, the matura- Approximately 10 molecules of NCBR[12]/DIS39 molecule tion reaction of the dimeric viral RNA, we previously established on average were required for the maturation activity. In the a simple in vitro assay system using a short RNA fragment case of an equimolar mixture between NCBR[12] and DIS39, (DIS39) corresponding to the DIS of the HIV-1 (40). The imma- a prolonged incubation did not change the fraction of the ma- ture dimer (loose dimer) and NCp7-converted mature dimer ture dimer of DIS39, as shown in Fig. 2B. However, an increas- (tight dimer) of DIS39 can be easily distinguished by their ing amount of NCBR[12] raised the fraction of the mature different Mg -dependent stabilities when they are subjected dimer linearly in an incubation time of 2.5 h (Fig. 4). The to gel electrophoreses with and without Mg . This electro- binding affinity of NCBR[12] may be decreased in comparison phoretic method was originally developed by Laughrea and with intact NCp7, and the fraction of a possible specific binding Jette (33). We have also confirmed by NMR measurements that that leads to the maturation reaction may be decreased by the the two types of DIS39 dimer take the secondary structures competition with the nonspecific, and non-active, binding. The proposed in the kissing-loop model (38, 39), the kissing-loop zinc finger domains of NCp7 may take a role to bind DIS39 dimer and extended-duplex dimer, respectively (Fig. 1A) (41). specifically and locate the basic regions in the best position on Using this assay system, we demonstrated previously that DIS39 for the maturation reaction. NCp7 did not require Zn to generate mature DIS39 dimer Although NC proteins of various retroviruses commonly con- 31278 NCp7 Basic Regions Convert Dimeric States of HIV-1 DIS 3046 –3057 tain basic regions, their amino acid sequences themselves are 16. Lapadat-Tapolsky, M., Pernelle, C., Borie, C., and Darlix, J. L. (1995) Nucleic not conserved (2). Our study demonstrated that peptides with Acids Res. 23, 2434 –2441 17. Prats, A. C., Housset, V., de Billy, G., Cornille, F., Prats, H., Roques, B., Darlix, randomized sequences of NCBR[12] had the same level of the J. L. (1991) Nucleic Acids Res. 19, 3533–3541 annealing activity as that of the wild type sequence of 18. de Rocquigny, H., Ficheux, D., Gabus, C., Allain, B., Fournie-Zaluski, M. C., NCBR[12]. On the hand, little and no activities were seen in Darlix, J. L., and Roques, B. P. (1993) Nucleic Acids Res. 21, 823– 829 19. de Rocquigny, H., Gabus, C., Vincent, A., Fournie-Zaluski, M. C., Roques, B., the shorter peptides NCBR1 and NCBR2, respectively. These and Darlix, J. L. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 6472– 6476 indicate that some number of basic residues is required for the 20. Hsu, M., Rong, L., de Rocquigny, H., Roques, B. P., and Wainberg, M. A. (2000) annealing activity. If NCBR[12] is assumed to form a helical Nucleic Acids Res. 28, 1724 –1729 21. Wu, W., Henderson, L. E., Copeland, T. D., Gorelick, R. J., Bosche, W. J., Rein, structure, a constructed three-dimensional model of A., and Levin, J. G. (1996) J. Virol. 70, 7132–7142 NCBR[12] shows that positive electrostatic potential on the 22. Morellet, N., de Rocquigny, H., Mely, Y., Jullian, N., Demene, H., Ottmann, M., Gerard, D., Darlix, J. L., Fournie-Zaluski, M. C., and Roques, B. P. molecular surface is localized in one side of the helix. It should (1994) J. Mol. Biol. 235, 287–301 be noted that, unexpectedly, the two randomized peptides also 23. Schmalzbauer, E., Strack, B., Dannull, J., Guehmann, S., and Moelling, K. show a similar localization of positive electrostatic potential in (1996) J. Virol. 70, 771–777 24. Fu, X. D., Katz, R. A., Skalka, A. M., and Leis, J. (1988) J. Biol. Chem. 263, a helical structure model (data not shown). This possible com- 2140 –2145 mon structural feature may be an important factor to anneal 25. Berthoux, L., Pechoux, C., Ottmann, M., Morel, G., and Darlix, J. L. (1997) nucleotide duplexes. Further mutational analysis of the basic J. Virol. 71, 6973– 6981 26. Ottmann, M., Gabus, C., and Darlix, J. L. (1995) J. Virol. 69, 1778 –1784 peptide NCBR[12] and structural analysis of a complex be- 27. Housset, V., de Rocquigny, H., Roques, B. P., and Darlix, J. L. (1993) J. Virol. tween NCBR[12] and DIS39 are required to understand de- 67, 2537–2545 28. 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Published: Aug 1, 2001

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