Proc. Nati. Acad. Sci. USA Vol. 89, pp. 6472-6476, July 1992 Biochemistry

Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers (solid-phase synthesis/retrovirus/nucleic add binding protein)

HUGHES DE ROCQUIGNY*, CAROLINE GABUSt, ANNE VINCENTt, MARIE-CLAUDE FOURNIt-ZALUSKI*, BERNARD ROQUES*t, AND JEAN-LUC DARLIXt *Ddpartement de Chimie Organique, U266 Institut National de la Sante et de la Recherche MEdicale Unite Associe6 498, Centre National de la Recherche Scientifique, Universitd Rend Descartes, 4 avenue de l'Observatoire, 75270 Paris 06, France; and tLaboRetro CJF Institut National de la Santd et de la Recherche MWdicale, Ecole Normale Superieure de Lyon, 46 allfe d'Italie 69364 Lyon, France

Communicated by Pierre Chambon, March 12, 1992

in the case of the lymphadenopathy-associated virus (LAV) strain (7), contains the two zinc fingers flanked by several basic residues (7). In addition to wrapping the genomic RNA (2, 15, 16) within the capsid, mature NC proteins show in vitro preferential binding to single-stranded nucleic acids and the highest affinity for their corresponding viral RNA (17-19). In relation with this property, HIV-1 NC protein as well as Rous sarcoma virus (RSV) and murine leukemia virus (MuLV) NC proteins potentiate dimerization of the retroviral RNA containing the encapsidation sequence Psi and activate annealing of the replication primer tRNA onto the viral RNA (2, 9, 12, 20, 21). Mutations that destroy the first zinc finger of HIV-1 or of RSV NC protein, or the unique zinc finger of Moloney MuLV (MoMuLV) NC protein, result in a strong impairment of genomic RNA packaging (22-25). Moreover, mutations that replace the basic residues by neutral ones in the vicinity of the first zinc finger of RSV NC protein, or the unique finger of MoMuLV NC protein, also cause a genomic RNA packaging defect in virus assembly (ref. 26; V. Housset & J.-L.D., unpublished data). In vitro these latter mutations were shown to disrupt the high-affinity RNA binding as well as the RNA annealing activities of the NC protein (refs. 26 and 27; unpublished data). These results indicate that in vivo, the retroviral NC zinc fingers and the flanking basic residues probably control the encapsidation of the viral genomic RNA. In this study, the minimum amino acid sequence required for the annealing activities of the NC protein of HIV-1 LAV strain was investigated by using synthetic NCp7 and derivatives. NCp7 with or without the two zinc fingers as well as NCp7 peptides of 34 and 27 residues (positions 13-72 and 13-64 without the fingers, respectively) all have the activities of NCp15 in vitro (Fig. 1). However deletion of short sequences containing basic residues flanking the first zinc finger leads to a complete loss of NC protein activity. These findings could facilitate the rational design of small molecules endowed with antiviral potency through inhibition of HIV-1 NCp15 functions.

ABSTRACT The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNALYS,3 annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA anneling activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first flnger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and a ling activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents. In human immunodeficiency virus type 1 (HIV-1) virions, as in all known retroviruses, the genome corresponds to a 70S complex containing two identical unspliced viral RNAs (1-4). In the 70S, two major noncovalent RNA-RNA interactions take place close to the 5' end of the viral RNA. One involves the association ofthe two RNAs at the level of a region named dimer linkage structure (DLS) (3). The other corresponds to the annealing of the replication primer tRNA (1, 5), tRNALyS,3 in HIV (6, 7), at the primer binding site (PBS) of the viral RNA. In the capsid, the genomic RNA dimer is also tightly associated with nucleocapsid (NC) proteins (8), which derive from a maturation of the gag polyprotein (2, 9-12). The retroviral NC proteins are well conserved, highly basic molecules that contain one or two C-X2-C-X4-H-X4-C (CCHC) motifs capable of coordinating a zinc ion (13, 14). In HIV-1 virions the NCp15 is ultimately processed in NCp7 and p6 proteins (15). NCp7, which is formed of 72 amino acids

MATERIALS AND METHODS Synthesis and Purification of HIV-1 NCp7 and NCp7Derived Peptides. HIV-1 NCp7 protein and NC-derived peptides depicted in Fig. 1 were synthesized by solid-phase using Abbreviations: NC, nucleocapsid; RSV, Rous sarcoma virus; MuLV, murine leukemia virus; MoMuLV, Moloney MuLV; HIV, human immunodeficiency virus; HOBt, 1-hydroxybenzotriazole; DCC, dicyclohexylcarbodiimide; LAV, lymphadenopathy-associated virus; DTT, dithiothreitol; PBS, primer binding site; RT, reverse transcriptase. tTo whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 89 (1992)

PAGE). After UV irradiation the NC-HIV-1 RNA complexes were digested for 30 min at 20°C with 5 units of T1 RNase, incubated in 5 mM EDTA/1% SDS for 2 min at 80°C, and analyzed by SDS/15% PAGE. Following electrophoresis gels were autoradiographed for 2-12 hr. Assay for Viral RNA Dimerization and Primer 32P-tRNALys,3 Annealing. Assay conditions were as described for UV crosslinking except that incubation was with 50 ng of 32ptRNALyS,3 and 300 ng of HIV-1 RNA (positions 1-415) for 20 min at 37°C. Reactions were stopped by adding 3 ,u of 1% SDS/10 mM EDTA/20 mM Tris-HCI, pH 7.5/20% glycerol/ 0.01% bromophenol blue. Reactions were then extracted once with phenol and once with phenol/chloroform to eliminate NC protein. RNAs present in the supernatant phase were analyzed by 0.9% agarose gel electrophoresis in 50 mM Tris-borate (pH 8.3) as described (2, 12, 20). After electrophoresis gels were washed with water and the RNAs were visualized by ethidium bromide staining (1 ,ug/ml for 5 min). For autoradiographic analysis, the gels were fixed with 5% (wt/vol) trichloroacetic acid and then dried. Synthesis of Strong Stop cDNA. Following primer tRNALYS.3 annealing to the primer binding site (PBS) of HIV-1 RNA, 3 ,ul of the 10-pl incubation mixture containing 32P-tRNALYs.3, HIV-1 RNA, and NC protein was added to 10 ,ul of 40 mM Tris HCl, pH 8.3/60 mM NaCI/5 mM MgCl2/5 mM DTT/ 0.25 mM (each) deoxyribonucleotide triphosphate/HIV-1 reverse transcriptase (RT) (31) and incubated for 6 min at 40°C. Reactions were stopped as described above and phenol extracted; nucleic acids were denatured by heating 2 min at 100°C. Then strong stop cDNA primed by 32PAtRNALYs,3 was analyzed by 6% PAGE in 7 M urea/50 mM Tris-borate. Autoradiography was for 12 hr.

Fmoc chemistry on an Applied Biosystems model 431A peptide synthesizer (28). 1-Hydroxybenzotriazole/dicyclohexylcarbodimide (HOBt/DCC) in N-methylpyrrolidone was used for coupling. Protecting groups were 2,3,5,7,8pentamethyl chroman-6-sulfonyl (Arg), t-butyloxycarbonyl (Lys), trityl (Gln, Asn, His, Cys), t-butyl (Ser, Thr, Tyr), and t-butyl ester (Glu, Asp). The whole reaction mixture was applied to a C4 Vydac 5-mm column (220 x 10 mm) and eluted with a linear gradient of 10-90% B [70% CH3CN/30% H20/ 0.09% trifluoroacetic acid (TFA)] in 30 min at a flow rate of 3 ml/min with a detection at 214 nm using an Applied Biosystems 151A separation system. All purifications were carried out under argon atmosphere. Sequences of NCp7 of HIV-1 LAV strain and of the derived peptides are given in Fig. 1. Solutions of NCp7 and derived peptides were at 1 mg/ml in 25 mM Tris-HCl, pH 7.0/60 mM NaCI/5 mM dithiothreitol (DTT)/30 yM ZnCI2. RNA Binding Assay. The RNA binding assay was derived from the "Southwestern" assay described by Bowen et al. (29). Twenty microliters of increasing dilutions of NCp7 and NC-derived peptides in 20 mM Tris, pH 7.5/100 mM NaCl/1 mM MgCl2/5 mM DTT/20 ,ug of RNase-free bovine serum albumin per ml was incubated with 32P-labeled HIV-1 RNA (106 cpm/ml, 106 cpm/,ug) for 10 min at 20°C in the presence of 10 ,ug of Escherichia coli tRNA per ml, then spotted on a 0.1-mm nitrocellulose membrane, and finally washed for 10 min under the same conditions. After autoradiography, each spot was cut out and assayed for bound radioactivity. UV Cross-Linking Experiments with NC Proteins and HIV-1 RNA and Primer tRNALYS3. UV (252 nm) cross-linking experiments were carried out with HIV and RSV as described (2, 10). Preparation of 32P-labeled HIV-1 RNA (positions 1-415) and replication primer tRNALYS,3 has been described (2, 9, 30). Both RNAs were purified by 6% polyacrylamide gel electrophoresis in 7 M urea/50 mM Tris-borate, pH 8.3. Assays were in 10 ,ul containing 25 mM Tris-HCl (pH 7.5), 60 mM NaCl, 0.2 mM MgCI2, 5 mM DTT, either 300 ng of 32P-labeled HIV-1 RNA (positions 1-415) or 50 ng of 32plabeled tRNALyS,3 (32P-tRNALYs,3), and NCp7 or NC-derived peptides (25-100 ng). After 5 min at 37°C the complexes were irradiated for 3-5 min at 20°C using conditions reported before (2, 10, 12). The NC-tRNA complexes were then incubated in 5 mM EDTA/1% SDS for 2 min at 80°C and analyzed by 13% polyacrylamide gel electrophoresis (SDS/ C

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RESULTS In an attempt to investigate the role of the zinc fingers and flanking residues on the RNA binding and annealing activities of HIV-1 NC protein, NCp7, NCp7A (positions 29-72) with only the second finger, NCp7B and NCp7C without the two fingers (positions 1-72 and 13-72, respectively), NCp7D (positions 51-72), the zinc fingers (NCp7F1 and -F2; positions 13-30 and 34-51, respectively) and a small basic peptide corresponding to sequences flanking the first finger, NCp7E (positions 13-35 without the first finger) (Fig. 1), have been synthesized by a solid-phase method. Noncontinuous seViral RNA

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peptides is given on the right: -, no activity; +/-, activity is not higher than 15% of activity seen with NCp15 purified from HIV-1 virions or from a recombinant E. coli (7, 11); ++, activity as NCpl5; + + +, activity two to three times higher than with NCp15.

6474

Proc. Nad. Acad. Sci. USA 89 (1992)

Biochemistry: De Rocquigny et al.

quences resulting from finger deletions in NCp7B and NCp7C fragments were linked by means of glycine residues (Fig. 1). HIV-1 NCp7 and the NCp7 derivatives were purified by HPLC and their amino acid compositions and sequences were verified. The proteins were >99% pure and their sequences conformed to the expected ones. The biological substrates of HIV-1 NC protein are HIV-1 RNA and replication primer tRNALYS,3 (2, 9). RNA binding and annealing activities of NCp7 and NCp7 derivatives (Fig. 1) were therefore monitored in vitro using T7 RNA polymerase-generated HIV-1 RNA (positions 1-415) corresponding to R, U5 (containing the PBS) (7), the dimerizationencapsidation domain (2) and the initiation codon AUG of gag, and primer 32PAtRNALYs.3. This biochemical system, previously described (2, 9, 12, 30), allowed us to examine the ability of NCp7 and NCp7 fragments to interact with their RNA substrates and promote HIV-1 RNA dimerization, primer tRNALYS,3 annealing to the reverse transcription initiation site (PBS), and synthesis of strong stop cDNA in the presence of HIV-1 RT (31). NCp7 and all NCp7-derived peptides but the fingers were shown to bind 32P-labeled HIV-1 RNA and tRNALyS,3 by filter assays under conditions used to monitor the binding of NCp1O of MoMuLV to the viral RNA (29) (data not shown). To analyze only the tight interactions between NC protein and either primer tRNALyS,3 or HIV-1 RNA (positions 1-415) we used UV cross-linking, since UV at 252 nm links proteins to nucleic acids when reactive groups are no more than 0.1 nm apart (32). NC protein was incubated with 32P-labeled RNA and the complexes formed were UV cross-linked and analyzed by SDS/PAGE. As shown in Fig. 2, NCp7 and NCp7 derivatives without the fingers (NCp7B and NCp7C) tightly interacted with primer tRNALYS,3 to form NC-tRNA complexes (lanes 2, 3, 8, 9, 11, and 12). The apparent molecular mass of about 30 kDa for the major NCp7-tRNA complexes indicates that they probably correspond to one NCp7 molecule per tRNA. NCp7A, which lacks the N-terminal domain and the first zinc finger, interacted less efficiently with tRNALYS,3 [compare lane 2 (50 ng of NCp7) with lane 6 (200 ng of NCp7A)], whereas neither NCp7D (lanes 14 and 15) nor the small peptide NCp7E and a mixture of both peptides showed significant tight RNA interactions (data not shown). To analyze the interactions of NCp7 and NCp7 peptides with HIV-1 32P-labeled RNA, the RNA was digested with T1 RNase after UV cross-linking (Fig. 2B). Again, NCp7, NCp7B, and NCp7C tightly interacted with HIV-1 RNA (lanes 2, 3, 8, 10, and 11), whereas NCp7A interacted less tightly [compare lane 2 (50 ng of NCp7) with lane 6 (200 ng of NCp7A)] and NCp7D did not interact under these conditions (lane 12). HIV-1 RNA dimerization and primer tRNA annealing to the PBS were examined upon incubation of the viral RNA and 32P-tRNALys,3 with or without NCp7 or NCp7 peptides. After 20 min at 37°C, reactions were stopped with SDS and RNAs were extracted with phenol to remove NC protein and analyzed by agarose gel electrophoresis in native conditions. Fig. 3A reports that dimerization of HIV-1 RNA (positions 1-415) did not occur in absence of NC protein (lane 1). Upon addition of NCp7, RNA dimer formation was observed in a dose-dependent manner (Fig. 3A, lanes 2-4) (28). Similarly, NCp7 strongly activated annealing of primer 32P-tRNALYs,3 to the monomer and dimer forms of HIV-1 RNA (positions 1-415) (Fig. 3B, lane 1 is the control and lanes 2-4 are with NCp7). Heating of the HIV-1 RNA-primer tRNA complex to 55°C destroyed the dimer form of HIV-1 RNA [in agreement with our previous data (2)] but did not disrupt the annealing of 32P-tRNALYs.3 to the viral RNA (Fig. 3B, lane 5). These findings demonstrate that synthetic NCp7 is as active as NCp15 purified from virions or from a recombinant E. coli

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FIG. 2. UV cross-linking of NCp7 and NCp7 peptides to tRNALYS,3 and HIV-1 RNA. (A) NC-primer tRNALYS,3 interactions. Lane 1 is control primer tRNA UV irradiated; lanes 2 and 3 are with 50 and 100 ng of NCp7 and lane 4 is with 100 ng of NCp7 minus UV irradiation; lanes 5 and 6 are with 100 and 200 ng of NCp7A and lane 7 is with 200 ng of NCp7A minus UV irradiation; lanes 8 and 9 are with 50 and 100 ng of NCp7B and lane 10 is with 100 ng of NCp7B minus UV irradiation; lanes 11 and 12 are with 50 and 100 ng of NCp7C and lane 13 is with 100 ng of NCp7C minus UV irradiation; lanes 14 and 15 are with 300 ng of NCp7D with and without UV irradiation. Autoradiography was for 2 hr. Note that the major NC-tRNA complex migrated with an apparent molecular mass of about 30 kDa and free 32ptRNALYs.3 migrated at the bottom of the gel (about 18 kDa). (B) NC-HIV-1 RNA interactions. Lane 1 is control HIV-1 RNA UV irradiated; lanes 2 and 3 are with 50 and 100 ng of NCp7 and lane 4 is with 100 ng of NCp7 minus UV irradiation; lanes 5 and 6 are with 100 and 200 ng of NCp7A and lane 7 is with 200 ng of NCp7A minus UV irradiation; lane 8 is with 50 ng of NCp7B; lane 9 is with 50 ng of NCp7B plus NCp7C minus UV irradiation; lanes 10 and 11 are with 50 and 100 ng of NCp7C; lanes 12 and 13 are with 300 ng of NCp7D with or without UV irradiation. Autoradiography was for 12 hr. Note that free 32P-labeled T1 oligonucleotides migrated at the bottom of the gel. M corresponds to stained protein markers in kDa (Amersham).

strain in promoting HIV-1 RNA dimerization and replication primer tRNALYS,3 annealing to the viral RNA (2, 9, 28). Viral RNA annealing activities of NCp7-derived peptides were monitored as above. Only NCp7B and NCp7C were

found to be very active in promoting HIV-1 RNA dimerization and annealing of primer tRNALYs,3 to the PBS. This is shown in Fig. 3 A and B (lanes 6-9 for NCp7B and lanes 10-13 NCp7C). NCp7A was poorly active in HIV-1 RNA dimerization and primer tRNA annealing to the PBS (not shown). NCp7D, either alone (Fig. 3 A and B, lanes 14 and 15) or in combination with the small basic peptide (Fig. 1), was inactive as were the finger peptides (not shown; see also Fig. 1). To analyze whether NCp7, NCp7B, and NCp7C, which are able to efficiently anneal primer tRNA to the viral RNA, could promote the initiation of reverse transcription, they were incubated with HIV-1 RNA and 32P-tRNALYs,3 and then incubated with either RTp66-p51 or RTp66 and deoxyribonucleotides. In the absence of NC protein, initiation ofcDNA synthesis was not observed (Fig. 3C, lane 1). When any of the active synthetic HIV NC proteins was added to the reaction mixture together with either RTp66-p51 (lanes 2, 3, 5, 6, 8, and 9) or RTp66 (lanes 4, 7, and 10), we observed the correct

Biochemistry: De Rocquigny et al. IICp7

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Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 4. Replication primer tRNALYS.3 annealing to HIV-1 RNA by NCp7C peptides. Assay conditions, including electrophoresis and autoradiography, were exactly as described in the text and in the legend to Fig. 3 A and B. Lane 1 is the control; lanes 2-4 are with 25, 50, and 100 ng of NCp7B; lanes 5-7 are with 25, 50, and 100 ng of NCp7C(13-64); lanes 8-10 are with 50, 100, and 300 ng of NCp7C(13-56); lanes 11-13 are with 50, 100, and 300 ng of NCp7C(29-64). M and D correspond to the monomer and dimer forms of HIV-1 RNA (positions 1-415). The arrow indicates the direction of electrophoresis.

extension product of about 260 nucleotides (32P-tRNALYs.3 plus strong stop cDNA). In addition, we repeatedly found that NCp7C (positions 13-72 without the fingers) was able to promote a high level of initiation of reverse transcription in vitro (compare lanes 3 and 4 and lanes 9 and 10). Further experiments are necessary to explain this phenomenon. To analyze the influence of the N- and C-terminal domains on NC protein activity in vitro, three NCp7-derived peptides corresponding to NCp7C (positions 13-72 without the fingers) missing the N-terminal 13VKGG residues (see Fig. 1) or C-terminal 8 or 16 residues were synthesized and purified under conditions described for the other NC peptides (see Materials and Methods and Fig. 1). NCp7C(13-64), NCp7C(29-64), and NCp7C(13-56) without the fingers were assayed for their ability to activate HIV-1 RNA dimerization, tRNALYS,3 annealing to the PBS, and initiation of reverse transcription as described in the legend to Fig. 3. We found that NCp7C(13-64) of 27 amino acids was as active as NCp7B(1-72), whereas NCp7C(13-56) was completely inactive [Fig. 4 for the annealing of primer tRNALYS,3 to the PBS; compare lanes 2-4, NCp7B, lanes 5-7, NCp7C(13-64), and lanes 8-10, NCp7C(13-56); data not shown]. NCp7C(29-64) was found to be poorly active [Fig. 4, compare lane 6, 50 ng of NCp7C(13-64), and lane 13, 300 ng of NCp7C(29-64)], indicating that residues 13VKGG are important for the RNA annealing activities of NCp7. Further experiments are necessary to define more precisely how many residues of the N- and C-terminal domains are required for a full NC protein activity in vitro.

FIG. 3. Viral RNA annealing activity of NCp7 and NCp7-derived peptides. (A) Dimerization of HIV-1 RNA. An agarose gel was stained for 5 min with 0.5 /Ag of ethidium bromide per ml to visualize HIV-1 RNA in the monomer and dimer forms. (B) Primer tRNALYS.3 annealing to HIV-1 RNA. An agarose gel was fixed for 5 min with 5% trichloroacetic acid, dried, and autoradiographed for 3 hr. Lane 1 is the control; lanes 2-4 are with 25, 50, and 100 ng of NCp7; lane 5 is as lane 4 but with RNAs heated at 550C for 1 min; lanes 6-8 are with 25, 50, and 100 ng of NCp7B; lane 9 is as lane 8 but with RNAs heated at 550C for 1 min; lanes 10-12 are with 25, 50, and 100 ng of NCp7C; lane 13 is as lane 12 but with RNAs heated at 550C for 1 min; lanes 14 and 15 are with 300 ng of NCp7D without or with 1 min at 550C. M and D correspond to the monomer and dimer forms of HIV-1 RNA (see refs. 8 and 24). (C) Synthesis of strong stop cDNA. Seventyfive, 105, and 311 nucleotides correspond to tRNA and HIV-1 RNA

DISCUSSION NCp15 and NCp7 proteins have been detected in HIV-1 virions (15), and processing of Pr555ag to generate mature NC proteins appears to be necessary for virion formation (33). Results of this study clearly show that the RNA binding and annealing activities of NCp15 are essentially due to amino acids present in NCp7. Most important, in vitro viral RNA markers in nucleotides (7, 11). Lane 1 is the control without NC protein and with 200 ng RTp66-p51; lanes 2-4 are with NCp7 and 100 or 200 ng of RTp66-pS1 or 200 ng of RTp66; lanes 5-7 are with NCp7B and 100 or 200 ng of RTp66-p51 or 200 ng of RTp66; lanes 8-10 are with NCp7C and 100 or 200 ng of RTp66-p51 or 200 ng of RTp66.

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annealing activities of NCp7 are preserved upon removal of the two zinc fingers in NCp7C, in agreement with alkylation (13) or replacement of the cysteine residues involved in zinc coordination of NCp1O of MoMuLV (27). Furthermore, in addition to the zinc fingers, 'most of the N-terminal part of NCp7 (positions 1-12) and the C-terminal 8 amino acids (positions 65-72) do not appear to be required for the RNA annealing activity. However deletion of the short sequences 13VK and 29RAPRKKG35, flanking the first finger (NCp7D in Fig. 1), led to a complete loss of RNA annealing activities in vitro (Fig. 3, lanes 14 and 15). Mixing the peptide VKGGRAPRKKG (NCp7E) with the C-terminal sequence of NCp7 (NCp7D, positions 51-72) did not restore the annealing activity. This finding confirms that these two sequences must be linked to allow tight RNA interactions and viral RNA annealing activity, and such a proximity might take place in the intact NCp7 due to the folding of the NCp7 protein (39). In agreement with this, oxidation of NC proteins resulted in a drastic and irreversible modification of the zinc finger structure and the complete loss of NCp7 activity (data not shown), a feature also observed in the case of MoMuLV NCp1O (13). The in vitro biological activities of modified NCp1O proteins that have lost their affinities for zinc ions by replacement of one of several cysteines by serine residues (27) indicate that the biologically active conformations of flexible NC proteins can be adopted in the presence of viral RNA provided that the critical basic sequences are unmodified. In the human spumaretrovirus, the NC protein has no zinc motif but contains three strongly basic motifs rich in glycine and proline residues (34). Interestingly enough, these proline-containing basic motifs are present in most retroviral NC proteins and are always located on both sides of the zinc fingers (35, 36). In the present study the question of how the HIV-1 genomic RNA is recognized among the cellular RNAs has not been addressed. However, the encapsidation element Psi and the NC protein are required for this recognition in vivo (1, 12, 22-26). Point mutations that modify the zinc ligand residues of HIV-1 NC cause a genomic RNA packaging defect (22, 24). Thus, these genetic data and our biochemical findings favor the idea that the NC protein zinc finger and the flanking basic residues cooperate to direct the selective recognition of the genomic Psi+ RNA in the infected cell. Accordingly, major NC protein binding sites have been mapped in the encapsidation-dimerization element of RSV and HIV-1 genomic RNA (2, 10). Specific interactions between NC protein and the viral Psi element could be therefore the first step toward packaging of the genome. This is supported by the in vivo defect in packaging of virions (V. Housset and J.L.D., unpublished data) following the replacement of basic residues by neutral residues in the vicinity of the zinc finger of MoMuLV NC protein that parallels the observed loss of in vitro RNA dimerization (unpublished data). Because of their requirement for NC protein activity, the sequences rich in basic amino acids flanking the zinc fingers represent an interesting target for the design of compounds aimed at inhibiting the interactions of HIV-1 NC proteins with the viral RNA and primer tRNALYS,3 that are critical for the replication of HIV-1 virus (37, 38). This issue is presently under investigation in vitro and in vivo. RTp66-pSl and tRNALYS,3 were kindly provided by Stuart Le Grice (Case Western University, Cleveland) and Gerard Keith (Strasbourg), respectively. Thanks are due to Mary Lapadat for a critical reading of the manuscript and Christine Dupuis for typing it. This work was supported by the French Programme Against AIDS (Agence National de Recherches sur le Sida). 1. Coffin, J. M. (1984) in RNA Tumor Viruses, eds. Weiss, R., Teich, N., Varmus, H. & Coffin, J. (Cold Spring Harbor Lab., Cold Spring Harbor, NY), Vol. 1, pp. 261-368.

2. Darlix, J.-L., Gabus, C., Nugeyre, M.-T., Clavel, F. & BarrdSinoussi, F. (1990) J. Mol. Biol. 216, 689-699. 3. Bender, W. & Davidson, N. (1976) Cell 7, 595-607. 4. Murti, K. G., Bondurant, M. & Tereba, A. (1981) J. Virol. 37, 411-419. 5. Peters, G., Harad, F., Dahlberg, J. E., Haseltine, A. & Baltimore, D. (1977) J. Virol. 21, 1031-1041. 6. Barr6-Sinoussi, F., Chermann, J. C., Rey, F., Nugeyre, M. T.,

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