The Annealing of tRNA 3Lys to Human Immunodeficiency Virus Type 1 Primer Binding Site Is Critically Dependent on the NCp7 Zinc Fingers Structure*

The nucleocapsid protein NCp7 of the human immunodeficiency virus type 1 contains two zinc fingers of the C X 2 C X 4 H X 4 C type, flanked by several basic residues, and plays a major role in viral infectivity. Thus, NCp7 was shown to promote annealing of the tRNA 3Lys to the primer binding site, a key step in reverse transcription. However, previous in vitro experiments were unable to clarify the role of the zinc fingers in this process, due to nucleic acid aggregation induced by the basic N- and C-terminal domains of NCp7. We show here that deletion of these sequences in (12–53)NCp7 strongly reduces the formation of aggregates and allows a direct visualization of the binary or ternary complexes between NCp7 and nucleic acids by gel electrophoresis. (12–53)NCp7 is able to induce hybridization of the 33 P tRNA 3Lys and the human immunodeficiency virus

Reverse transcription is a key step in the retroviral life cycle, allowing conversion of the single-stranded RNA genome into double-stranded DNA (1). The initiation of this process begins with the annealing of a primer tRNA to the viral RNA primer binding site (PBS). 1 In HIV-1, the PBS sequence, located at the 5Ј end of the genomic RNA (position 179 -196) in the leader region and referred to as the region (2), has a strict complementarity with the 18 nucleotides of tRNA 3 Lys end (3). A secondary structure model of the HIV-1⅐tRNA 3 Lys complex has been constructed (4) and shows that the interaction between the two RNAs is more extended than expected, involving a highly compact and complex structure.
The annealing of the tRNA primer to the PBS is promoted by nucleocapsid (NC) proteins (5)(6)(7)(8)(9). In vitro, NCp7 was found to be necessary for the dimerization and the encapsidation of the containing genomic RNA, the annealing of the tRNA primer to the PBS, and the initial strand transfer event (review in Ref. 10). In the virions, viral RNAs and NCps are tightly associated to form the nucleocapsid (11). In the case of HIV-1, the mature NC product, NCp7 (12), is a small basic protein (72 amino acids) containing two copies of a conserved zinc finger-like domain with the sequence Cys-X 2 -Cys-X 4 -His-X 4 -Cys, designated as the CCHC motif (13). NCp7 has been synthesized by a stepwise solid phase method (14,15), and the synthetic protein shown to possess all the in vitro biological activities of the native protein extracted from virions (14). NMR studies have shown that, whereas the N-and C-terminal parts of the protein are flexible, the finger domains are folded around the divalent ion (16,17). Moreover, the two zinc complexed domains are spatially close, a property that has been confirmed as being biologically important (17,18). Indeed, mutations of the zinc binding amino acids, or replacement of amino acids in the short sequence linking the CCHC boxes, which alter the NCp7 structure, abolish the virus infectivity in vivo (18 -21).
In apparent discrepancy with the results of mutation experiments, the basic regions of the NCp7, rather than the zinc fingers, were proposed to be involved in its promoting effect of annealing complementary nucleic acids "in vitro" (7,22). With the aim of investigating more precisely the role of the zinc finger domains in the hybridase activity of NCp7, we have developed a method allowing the free and complexed nucleic acids to be directly visualized by gel electrophoresis and eliminating the usual extraction of the protein by a phenol-chloroform treatment (6,7). Using (12-53)NCp7 and various derivatives, our results show that the presence of both zinc fingers is necessary and sufficient to promote the annealing of the radiolabeled 33 P tRNA 3 Lys with the 77-257 domain of HIV-1 RNA, which contains the PBS sequence. The presence of the N-and C-terminal parts of NCp7 induced aggregation, precluding visualization of the hybrid complex. Moreover, the conformational rearrangement of the two finger domains observed in the Cys 23 NCp7 mutant (21) led to a severe loss of in vitro annealing activity, while the replacement of the Trp 37 residue by Leu completely inhibited this activity, although interaction with nucleic acids was preserved in both mutants.
These findings demonstrate unambiguously the critical importance for the annealing activity of NCp7 of a well structured zinc finger domain and of an aromatic residue (Trp) in position * This work was supported by Association National Recherche contre le SIDA and SIDACTION. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: 37. They give structural explanations for the loss of virus infectivity observed in vivo following point mutations in the CCHC boxes and emphasize the interest of the zinc finger domain as a possible target for antiviral agents.

EXPERIMENTAL PROCEDURES
Peptide Synthesis-NCp7 and derived peptides were synthesized on a 433 automated peptide synthesizer (Applied Biosystem) using the hydroxymethylphenoxy-methyl-polystyrene resin as already described (7,14). Amino acid side chains were protected by t-butyl or trityl groups and the NH 2 group by Fmoc (N-(9-fluorenyl)methoxycarbonyl). Amino acids were incorporated using dicyclohexylcarbodiimine/hydroxybenzotriazole as coupling reagents. At the end of the synthesis, the peptidyl-resin was treated for 2 h with trifluoroacetic acid in the presence of scavengers to obtain the crude peptide. Purification was performed by reverse-phase HPLC with acetonitrile-water gradients. The purity of the peptides was checked by electrospray mass spectroscopy. NCp7 and NCp7 derivatives (13-30, 34 -51, 12-53, Cys 23 (12-53), Leu 37 (12-53)) were lyophilized in presence of 1.5 eq of ZnCl 2 per zinc finger (14). The (13-56)NCp7dd in which the two zinc fingers have been replaced by a Gly-Gly unit has the following sequence: VKGGRAPRKKGGGTERQANF.
Assay for 33 P-tRNA 3 Lys Annealing to RNA-(77-257)-pDM3/BssHII and pTL9/BanI plasmids were used for the in vitro HIV-1 RNA-(77-257) and tRNA 3 Lys transcription, respectively. The latter was kindly supplied by Dr. J. L. Darlix and C. Gabus (INSERM 412, Lyon). Ribo-Max™ large scale RNA production system T7 was used, following the supplier's instructions (Promega). The synthesis of tRNA 3 Lys was performed by using [␣-33 P]UTP as a marker (250 Ci per 10 g of plasmid). The RNAs were purified by an 8% urea-polyacrylamide gel, and their concentrations were determined by UV measurements, using ⑀ 260 ϭ 10,800 M Ϫ1 cm Ϫ1 , the ratio at 260 and 280 nm was around 2. RNAprotein interactions were initiated by incubation for 15 min at 37°C in 15 l of a buffer containing 25 mM Tris-HCl (pH 7.5), 80 mM NaCl, 0.1 mM MgCl 2 . The different RNAs used were heat-denatured by heating for 2 min at 95°C and then chilled at 0°C for 2 min prior to use. The amount of RNA, 5.10 Ϫ12 mol per assay, was kept constant. Different concentrations of protein were used depending on the experiment. The reaction was stopped by two addition of 1 l of 20% glycerol containing 0.01% bromphenol blue. RNA-protein complexes were analyzed by native 7% polyacrylamide gel electrophoresis in 100 mM Tris borate buffer (pH 8.0). The gel was run at 35 V/cm and 4°C for 2 h and stained with ethidium bromide. The bands were visualized with a Polaroid MP-4 Land Camera on a Bio-Profil imager (Vilber Lourmat, Paris). The gel was then fixed with 10% acetic acid, 20% ethanol, dried, and autoradiographed.

RESULTS
Reverse transcription of HIV-1 genomic RNA is primed by tRNA 3 Lys , whose 3Ј end 18 nucleotides are complementary to the viral PBS (3). In vitro studies have shown that the nucleocapsid protein NCp7 is an important factor in promoting this hybridization process (7). To determine the role of the different parts of NCp7 in this activity, annealing of [ 33 P]tRNA 3 Lys to RNA-(77-257), which contains the PBS, was measured in presence of NCp7 or various peptides derived from (12-53)NCp7, a fragment that encompasses the two zinc fingers (Fig. 1). RNAs and the peptide were co-incubated, for 15 min at 37°C, as described under "Experimental Procedures." The samples were then directly analyzed by native polyacrylamide gel electrophoresis and visualized using both ethidium bromide staining and autoradiography. Due to its construction, RNA-(77-257) has a tendency to spontaneously dimerize ( Fig. 2A, lane 1) (23), and the experiment must be carried out at room temperature to obtain almost exclusively the monomeric form of RNA (Fig. 2B, lane 1). However, a comparison of the experiments made at 4°C and at room temperature (Fig. 2, A and B) shows that RNA dimerization did not interfere with the results, and further experiments were performed at 4°C to reduce RNA degradation. As depicted in Fig. 2D, preliminary experiments were carried out by using NCp7, but even at low concentrations (10 molar equivalents) the protein induced the formation of large aggregates unable to penetrate the gel, thus preventing the characterization of a RNA⅐tRNA hybrid complex.
Therefore, NCp7 was replaced by (12-53)NCp7, which has the major structural elements (finger motifs and basic linker domain) shown in vivo to be essential for its biological functions (2,18,20,21,24), but is devoid of the highly basic flexible terminal parts that are mainly responsible for nucleic acid aggregation through nonspecific electrostatic interactions (25).
When tRNA 3 Lys and RNA(77-257) were co-incubated in the presence of increasing (12-53)NCp7 concentrations, several features occurred depending on the ratio of protein/nucleic acids. At low concentrations of peptide ( Fig. 2A, lanes 2), a shift of the two RNAs was observed corresponding to the fixation of the peptide on both tRNA 3 Lys and RNA-(77-257). Addition of larger amounts of (12-53)NCp7 ( Fig. 2A, lane 3) led to a decrease of complexed tRNA 3 Lys and the appearance of a new band (noted c on Fig. 2A) revealed both by ethidium bromide staining and autoradiography, which was attributed to the formation of a tRNA⅐RNA complex. The annealing activity of (12-53)NCp7 was maximum for 70 molar equivalents corresponding to one peptide for a total of 4 -5 nucleotides. The hybrid complex coexisted with complexes formed between (12-53)NCp7 and both types of RNA, suggesting that the reaction is not complete in these conditions. In contrast  1-6, respectively) or 0, 10, 50 molar equivalents of NCp7 (lanes 7-9, respectively). Samples were analyzed by native 7% polyacrylamide gel electrophoresis at 4°C. A, complexed and uncomplexed nucleic acids are visualized by ethidium bromide staining. B, same conditions as in A but the migration was carried out at room temperature. C, autoradiogram of experiment A. D, autoradiogram of an assay using NCp7 in place of (12-53)NCp7. Arrows indicate: h, high molecular weight complex; m and d, monomeric and dimeric RNA-(77-257), respectively; and c, ternary complex made of tRNA 3 Lys , RNA-(77-257), and (12-53)NCp7.
NCp7 Zinc Fingers Are Required for tRNA Annealing 4820 to the large band corresponding to RNA⅐(12-53)NCp7 complexes, the ternary tRNA 3 Lys ⅐RNA⅐(12-53)NCp7 complex migrated under the form of a fine band, suggesting a finite number of bound NCp7 molecules and a well defined conformation for this complex. When the peptide concentration was increased further, the binary complexes RNA⅐(12-53)NCp7 disappeared to the profit of both the ternary complex and high molecular weight aggregates containing the radiolabeled tRNA 3 Lys in the deposing wells. No gel shift was observed for the ternary complex (Fig. 2, band c), suggesting that the number of (12-53)NCp7 molecules bound to the complex is constant whatever the peptide concentration used.
To determine if a particular domain of (12-53)NCp7 could direct the peptide hybridase activity, different fragments were tested. They correspond to the structural elements assumed to be involved in NCp7 functions: (13-30)NCp7 (first finger), (34 -51)NCp7 (second finger), and (13-56)NCp7dd (basic linker). When the two fingers were used separately, no gel shift was observed (Fig. 3, lanes 1-7). The (13-56)NCp7dd interacts with nucleic acids, as illustrated by a gel shift, but was unable to induce the annealing of tRNA 3 Lys to the PBS sequence of RNA-(77-257) (Fig. 3, lanes 8 -10). This suggests that the presence of the three elements is necessary for NCp7 hybridase activity.
The role of the conformational behavior of the finger domains of NCp7 was then studied by using two mutants of the (12-53)NCp7. The Cys 23 (12-53)NCp7 corresponds to the mutation of His 23 of the first CCHC box to Cys. In NCp7, this mutation was shown to induce a rearrangement of the residues around the zinc ion and increase the distance between the two finger motifs (21). The Cys 23 (12-53)NCp7 interacts with nucleic acids, since a gel shift was observed (Fig. 4, lanes 12-14), and promotes the formation of the ternary complex (Fig. 4, lane 13). However, the annealing activity of the mutant was considerably reduced as shown by the weakness of the hybrid band (band c, lane 3) obtained for a higher concentration of peptide. Leu 37 (12-53)NCp7 corresponds to the replacement of Trp 37 in the second finger by a leucine. No structural data are yet available for this mutant, but Trp 37 was shown in vivo and in vitro to be important for the interaction with nucleic acids, very likely through intercalation (19, 26 -28, 35). Fig. 4 shows that, despite its ability to bind nucleic acids, this mutant peptide has completely lost the annealing activity of NCp7 as illustrated by the absence of a ternary complex (lanes 8 -11). Taken together these results show that not only the presence of the fingers domain, but also their spatial arrangement and some specific amino acids (Trp 37 ) are necessary for its hybridase activity. DISCUSSION The retroviral NC proteins are highly basic molecules that contain one or two well conserved CX 2 CX 4 HX 4 C motifs capable of coordinating a zinc ion with a very high affinity (13,29,30). In HIV-1 virions, NCp7 contains two zinc fingers flanked by several basic residues. Mutation experiments have shown that the presence of the two fingers are required for all the in vivo functions of NCp7 (19 -21). This has been strongly reinforced by point mutations of NCp7, such as the replacement of His 23 by Cys or of Pro 31 by Leu, which were shown to modify the structure of the zinc finger domain of NCp7 and to result in the complete loss of viral infectivity of the mutants (18,21,24). However, in vitro experiments showed that annealing activity was apparently due mainly to the presence of a large number of basic amino acids in the flexible N-and C-terminal sequences surrounding the neutral zinc finger domain. Thus deletion of this domain and replacement of each CX 2 CX 4 HX 4 C box by a Gly-Gly moiety did not inhibit the formation of RNA⅐tRNA 3 Lys complexes (7). However, the conditions used in these experiments promoted the formation of high molecular weight aggregates also found to be induced by polylysine (23) through nonspecific ionic interactions between basic amino acids of the protein and the negatively charged ribose phosphate backbone of nucleic acids. This avoided the direct visualization of tRNA⅐RNA hybrid complex in the gel as illustrated in this study (Fig. 2D). Therefore, with the aim of studying the role of the zinc finger of NCp7, the N-and C-terminal parts of this highly basic protein were removed, and the resulting (12-53)NCp7 peptide was used in a new assay in which it was incubated with 33 P tRNA 3 Lys and a shorter RNA-(77-257) fragment, which contains the PBS. The mixture was then directly analyzed by native gel electrophoresis without preliminary treatment with phenol to remove the protein (6,7,23). At low concentrations of (12-53)NCp7, the protein interacts with tRNA 3 Lys and RNA-(77-257) in agreement with previous results showing that NCp7 has a similar affinity for different types of RNA (27). In these experiments we cannot determine the exact number of peptide molecules bound on each species, but it is interesting to notice that in presence of 40 molar equivalents of NCp7, the ternary complex tRNA 3 Lys ⅐RNA-(77-257)⅐(12-53)NCp7 was already formed and that the migration of the corresponding band was observed in its final position (in the presence of an excess of (12-53)NCp7). Moreover, the large reduction in the migration of the bands corresponding to binary complexes formed with tRNA 3 Lys and RNA-(77-257) suggest that most of the (12-53)NCp7 are bound to these RNAs and that only a small number of (12-53)NCp7 molecules are necessary to induce the annealing of tRNA 3 Lys to the PBS. Different approaches allowing the quantification of NCp7 molecules bound on the ternary complexes are required to answer this question. Another interesting observation is the shape of the band corresponding to the NCp7 Zinc Fingers Are Required for tRNA Annealing 4821 ternary complex, which is relatively discrete, suggesting a well defined conformation for this complex. The annealing of tRNA onto RNA-(77-257) requires the presence of both zinc fingers of NCp7, as shown by the lack of ternary complex formation observed with the isolated N-or C-terminal zinc fingers or with a peptide in which these domains have been replaced by a Gly-Gly linker. In the latter case, a shift of the tRNA 3 Lys band is in agreement with the binding of the basic linear peptide, indicating that ionic interactions involving the basic spacer plays a role in (12-53)NCp7 nucleic acids recognition.
The presence of high molecular weight complexes embedding the tRNA 3 Lys was observed for high concentrations of (12-53)NCp7, a result which emphasizes the nucleic acids aggregating properties through nonspecific electrostatic interactions of highly basic peptides. This phenomenon, which was also observed with polylysine (23), is dependent on the number of positively charged amino acids in the peptide (25,31) as clearly shown here when we compare the aggregation induced by NCp7 or its central  domain. The nonspecific RNA binding activity of the N-and C-terminal domains was previously observed in the binding of fusion proteins constituted by GST and NCp7 or various fragments to HIV-1 RNA domains (31). The requirement of the zinc fingers to ensure a specificity to this latter interaction was proposed to be due to complementary structural particularities in both NCp7 and HIV-1 RNA (31).
The specificity of the annealing activity of the NCp7 zinc fingers is clearly illustrated by a comparison of the formation and position in the gel of the ternary complexes generated by the (12-53)NCp7 and its mutants. The replacement of His 23 by Cys in NCp7 was shown to induce changes in the folding around the zinc atom in the proximal finger and a loss of the spatial proximity between the two zinc fingers (21). The important loss of ternary complex formation observed here for this mutant demonstrates that the spatial arrangement of the central domain of the NCp7 encompassing the two zinc fingers and the short linker are critical for the hybridase activity. Interestingly, the migration of the ternary complex is different from that of the complex formed with the wild-type (12-53)NCp7, suggesting that either a smaller number of Cys 23 (12-53)NCp7 molecules are involved in the complex formation or that the complexes have structural differences. The biological relevance of the NCp7 folded conformation was demonstrated in vivo by mutations such as His 23 3 Cys or Pro 31 3 Leu, which give mutants with a complete loss of infectivity (18,21,24). This was attributed, in the case of the His 23 3 Cys mutant, to a defect in reverse transcription and/or NCp-dependent steps leading to proviral integration (8,21,32,33). This could be due to a severe reduction in the hybridization of the tRNA 3 Lys to the PBS observed here and/or to other steps requiring nucleic acids hybridization, such as DNA strand transfer.
In almost all retroviruses, aromatic amino acids are present either in each finger (HIV) or in one finger (Rous sarcoma virus, Moloney murine leukemia virus) (13,24,34). Moreover, several studies have underlined the importance of Trp 37 in HIV-1 (19,26,27), its replacement by a nonaromatic amino acid leading to a loss of viral infectivity. When, Trp 37 was replaced by a hydrophobic amino acid such as leucine in Leu 37 (12-53)NCp7, no ternary complex was observed, illustrating the crucial importance of Trp for the NCp7 hybridase activity. This could be related to the intercalation observed by NMR of Trp between two successive bases of short oligonucleotides such as d(ACGCC) (35).
The aim of this work was to characterize the domains of the HIV-1 NCp7 necessary for its hybridase activity. Our results show that the zinc fingers are necessary and sufficient for the NCp7-promoted annealing of tRNA 3 Lys to the PBS and under-line the critical role of the three-dimensional structure of the peptide. The basic residues of NCp7 could be the driving force for NCp7⅐RNA complexation, whereas the fingers structure could ensure a proper orientation of the protein allowing for instance intercalation of Trp between two successive bases of single-or double-stranded nucleic acids (36). Therefore because of their requirement for NC protein activity, the zinc fingers represent an interesting target for the design of compounds aimed at selectively inhibiting the annealing of tRNA 3 Lys to viral RNA, a critical step for HIV-1 virus replication.