In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55 Gag Multimerization*

Transcription

1 THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 38, Issue of Septmeber 17, pp , by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. In Vitro Identification and Characterization of an Early Complex Linking HIV-1 Genomic RNA Recognition and Pr55 Gag Multimerization* Received for publication, May 20, 2004, and in revised form, July 6, 2004 Published, JBC Papers in Press, July 9, 2004, DOI /jbc.M Ariel Roldan, Rodney S. Russell, Bruno Marchand, Matthias Götte, Chen Liang, and Mark A. Wainberg From McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada and the Departments of Experimental Medicine and Microbiology & Immunology, McGill University, Montreal, Quebec H3A 2B4, Canada The minimal protein requirements that drive viruslike particle formation of human immunodeficiency virus type 1 (HIV-1) have been established. The C-terminal domain of capsid (CTD-CA) and nucleocapsid (NC) are the most important domains in a so-called minimal Gag protein (mgag). The CTD is essential for Gag oligomerization. NC is known to bind and encapsidate HIV-1 genomic RNA. The spacer peptide, SP1, located between CA and NC is important for the multimerization process, viral maturation and recognition of HIV-1 genomic RNA by NC. In this study, we show that NC in the context of an mgag protein binds HIV-1 genomic RNA with almost 10-fold higher affinity. The protein region encompassing the 11th -helix of CA and the proposed -helix in the CA/SP1 boundary region play important roles in this increased binding capacity. Furthermore, sequences downstream from stem loop 4 of the HIV-1 genomic RNA are also important for this RNA-protein interaction. In gel shift assays using purified mgag and a model RNA spanning the region from 223 to 506 of HIV-1 genomic RNA, we have identified an early complex (EC) formation between 2 proteins and 1 RNA molecule. This EC was not present in experiments performed with a mutant mgag protein, which contains a CTD dimerization mutation (M318A). These data suggest that the dimerization interface of the CTD plays an important role in EC formation, and, as a consequence, in RNA-protein association and multimerization. We propose a model for the RNA-protein interaction, based on previous results and those presented in this study. Human immunodeficiency virus type 1 (HIV-1) 1 Gag polyprotein contains all the necessary domains to: 1) encapsidate two identical copies of viral genomic RNA, 2) coordinate the assembly and budding of viral particles, and 3) organize the envelope protein on the virion surface (for reviews see Refs. * This research was supported by the Canadian Institutes of Health Research. 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: McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, 3755 Cote Ste-Catherine, Montreal, Quebec H3T 1E2, Canada. Tel.: ; Fax: ; mark.wainberg@mcgill.ca. 1 The abbreviations used are: HIV-1, human immunodeficiency virus 1; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; wt, wild type; mgag, minimal Gag; CA, capsid; EC, early complex; NC, nucleocapsid; CTD, C-terminal domain; NTD, N-terminal domain; nt, nucleotides ). During viral maturation, this precursor is processed by the viral protease (PR) to yield mature viral proteins: matrix (MA), capsid (CA), nucleocapsid (NC), p6, as well as two spacer peptides, SP1 and SP2. The external and N-terminal domain, MA, is responsible for the membrane association of the precursor (6 8). CA is composed of two domains, an N-terminal domain (NTD) and a C-terminal domain (CTD), and mediates important Gag-Gag interactions. NC is a small and basic peptide that contains two zinc finger motifs, and, through them, is responsible for the binding and encapsidation of genomic viral RNA. The p6 peptide is important at a late stage in the budding process (9, 10). SP1 has been shown to be critical for proper conformation of virus-like particles (VLPs) (11), while the role of SP2 is less well characterized. However, little is known about early steps in the multimerization process and how HIV-1 genomic RNA is recognized and preferentially encapsidated. It is also not known whether these two events are linked. Although MA has important roles in the HIV-1 life cycle, it is dispensable for in vitro generation of VLPs (12 14). Almost all the required protein-protein interactions that drive particle formation are governed by CA and NC (15 20). CA hexamerizes in solution and the main interaction for this hexamer is thought to be with the NTD (21, 22). CA also has the ability to dimerize because of a specific pair of amino acids located in the CTD (23). It has been hypothesized that the CTD along with SP1 forms an assembly domain that drives the formation of spheres (11, 24 27). The minimal domains required to generate VLPs in vivo are a myristylation signal, the CTD, SP1, NC, and p6 (28, 29). It has been shown that deletion of SP1 abolishes the ability to form spheres, resulting instead in either cones or cylinders (25). Our group and others have proposed that SP1 is involved in Gag multimerization (26, 30), and this small peptide has also been shown to influence packaging (31). Recently, we have also shown that SP1 is involved in the specific recognition of HIV-1 genomic RNA but not spliced forms of viral RNA (32). On the other hand, NC is required for virion assembly (33 35) and promotes Gag-Gag interactions, mainly through its ability to bind nucleic acids (16, 36 38). There are two hypotheses as to how NC performs its function. One is that the binding of NC to nucleic acids neutralizes its charge, giving rise to proteinprotein interactions. The other is that the RNA serves as a scaffold to which NC binds and on which Pr55 Gag is accumulated, making possible the necessary protein-protein interactions. NC binds to the encapsidation signal (see below) and it has been shown that sequences up to nt 500 are important in genomic RNA recognition (39 44). It is accepted that the first bases of viral HIV-1 This paper is available on line at

2 HIV-1 RNA Encapsidation and Gag Multimerization genomic RNA are structured. Several models have been proposed for the folding of this region, with alternative conformers, but the overall conformation of this 5 -leader region (5 -LR) remains to be fully defined (45). Several structures within the 5 -LR are well characterized, i.e. the Tat transactivation response element (TAR), poly(a), the primer binding site (PBS) (46 48). The HIV-1 packaging signal ( ) is multipartite containing four stem loops (SL1 4) (39, 49). SL1 promotes the formation of the RNA dimer through a palindromic sequence and, accordingly, is also known as the dimerization initiation site (DIS) (50 52). NC binds to the four stem loops of, but it has been shown that it binds much more strongly to SL2 and SL3 than to SL1 or SL4 (53 55). The high resolution structure of the complex formed between SL2/NC (56) and SL3/NC (57) has been resolved by NMR. SL4, with a weaker affinity for NC, has been proposed to stabilize the structure formed by SL2 and SL3 (58). Various authors have proposed long distance interactions between 5 -regions (between the poly(a) region and the PBS) with 3 -counterpart sequences (generally downstream of SL4) thought to promote a more stable conformer (59, 60). Taking into account the participation of SP1 in each of the multimerization process (26, 30, 61), viral maturation (24, 25), and recognition of the HIV-1 site by NC (31, 32), we decided to study relevant RNA-protein interactions in vitro using a minimal Pr55 Gag (mgag) protein. This recombinant protein includes amino acids 2 7 of MA (the myristyl anchor), the CTD of CA, SP1, and NC (28). Using this protein, we have analyzed the binding and multimerization abilities of native NC compared with NC in the context of the precursor (mgag), using probes containing different extensions of the 5 -leader region (5 -LR). Here, we report that NC in the context of Pr55 Gag has a much higher affinity for the 5 -LR and that this higher affinity correlates with the formation/occurrence of an early complex (EC) that involves two proteins (mgag) that are bound to 5 -LR RNA. This complex was not present in experiments performed with mature NC alone. Dissection of this EC reveals that amino acids at the N terminus of NC are involved in the protein-rna association and, furthermore, that N terminus extended versions of NC also acquire the ability to multimerize. Moreover, we have identified sequences downstream of SL4 that are involved in the recognition of the 5 -LR by mgag, as they increase the affinity of this interaction and enhance the formation of the EC. These results suggest that recognition of the 5 -LR and Gag dimerization (and eventually multimerization) are linked. Based on previous and our own data, we propose a model for the recognition of HIV-1 genomic RNA. MATERIALS AND METHODS Expression Vectors and Purification of Recombinant Proteins Unless otherwise stated, all expression vectors were constructed by PCR amplification of the encompassing DNA sequence of relevant fragments of Pr55 Gag from an HBX2 plasmid clone. These fragments were cloned into the expression vector Topo 100-D (Invitrogen) (Fig. 1). This vector has a T7 expression cassette with a 36-amino acid N-terminal extension containing a 6-histidine motif, a FLAG motif, and an enterokinase site. Site-directed mutagenesis was performed to produce M318A from the wild-type (wt) mgag vector. For cloning procedures Top10 cells (Invitrogen) were used, while Bl21 (DE3) cells (Novagen) were employed for protein expression. The different recombinant proteins were purified using Ni-NTA resin (Qiagen) in a batch purification protocol under denaturing conditions. Briefly, transformed bacteria were grown and induced for 3hat37 C. Cells were pelleted by centrifugation and lysed with buffer B (6 M GdnHCl, 0.1 M NaH 2 PO 4, 0.01 M Tris-HCl, ph 8.0, 20 mm -mercaptoethanol). Complete lysis was achieved by stirring for 1 h (h) at room temperature and the lysate was then clarified by centrifugation at 10,000 g for 30 min. The supernatant was mixed with previously equilibrated Ni-NTA resin in buffer B, and binding was performed for 90 min with gentle shaking. The lysate resin was loaded in empty 5-ml columns, allowed to drain, and washed twice with buffer C (same as B but ph 6.3). Finally, the protein was eluted in buffer E (same as B but ph 4.5). The buffer of the proteins was exchanged using centrifugal filter devices (Amicon) to 7 M urea, 50 mm Tris-HCl, ph 8.5 for further purification of the protein, and removal of nucleic acids by anion exchange chromatography using Q-Sepharose resin (Amersham Biosciences) in a batch procedure. Proteins were collected from the unbound material and redissolved to mg/ml. Refolding was accomplished through protein dialysis against 0.5 M Tris-HCl, ph 8.0, 50 mm NaCl, 10 M ZnCl 2,5mM/0.5 mm reduced/oxidized glutathione, 0.01% CHAPS at 4 C for 3 h. Two buffer exchanges were completed, and this was followed by a final dialysis step against storage buffer (same as dialysis but 50 mm Tris-HCl, ph 8.0 and no CHAPS). Removal of unfolded proteins was accomplished by ultracentrifugation at 100,000 g for1h. Synthesis and Labeling of RNA To produce probes for gel shift analysis, various DNA fragments encoding portions of the 5 -LR were amplified by PCR (Fig. 2). For sense probes, the T7 promoter was included at the 5 -end of the forward primer, whereas the same promoter was similarly added into the reverse primer in the case of antisense probes. Radiolabeled RNAs were transcribed using the Megashortscript kit (Ambion) following recommendations of the manufacturer, but in the presence of 96 M UTP and 50 Ci of [ 32 P]UTP (3000 Ci/mmol, ICN) for 2 h at 37 C. The DNA template was removed by the addition of 2 units of DNase I and further incubation at 37 C for 15 min. Radiolabeled probes were purified on denaturing polyacrylamide gels. Finally, the probe was dissolved in 20 mm Tris-HCl, 100 mm NaCl, and renatured by heating at 70 C for 5 min and cooling slowly to room temperature. Gel Shift Assay Gel shift assays were done as previously described with minor modifications (62). Binding reactions were performed in 10 l containing 0.2 nm of the radiolabeled probe, 10 mm HEPES ph 7.5, 100 mm KCl, 1 mm MgCl 2, 0.5 mm EDTA, 1 mm dithiothreitol, 20 ng/ l Poly I Poly C, 7.5% Ficoll and 0 3 M of recombinant protein. After incubation for 15 min on ice, the reaction mixtures were electrophoresed through nondenaturing 6% polyacrylamide gels in 0.5 Tris borate/ EDTA at 120 V for hs at 4 C. Gels were dried before exposure to Bio-Max Kodak films at 80 C. Cross-linking Experiments Proteins were dialyzed for 2 h against a storage buffer containing 50 mm HEPES ph 7.5, and ultracentrifuged at 100,000 g for 1 h. Binding reactions were performed as for gel shifts and then irradiated by UV (UV Crosslinker 2400, Stratagene). Complexes were resolved by electrophoresis on denaturing 5% polyacrylamide gels, which were dried and exposed. Filter Binding Assay The filter binding protocol was derived from previous work (Ref. 63 and references therein). Similar binding reactions as for gel shifts were performed in a volume of 20 l using 1 nm radiolabeled probe. After incubation for 15 min on ice, the samples were filtered through prewet 0.45 M nitrocellulose disks (Millipore). Disks were washed twice with storage buffer, dried, and the radioactivity retained by the filter was quantified by liquid scintillation counting. Data were corrected for background binding and are expressed as percentage of counts; 100% represents the saturation of the probe within each experiment. Protein Ultracentrifugation Preliminary binding experiments were done after centrifugation of the recombinant proteins at 10,000 g for 15 min. However, no interpretable data were obtained in either filter binding assays or in gel shift assays (data not shown). In contrast, ultracentrifugation of the protein at 100,000 g for1hat4 C yielded interpretable data by both techniques. Analysis of the ultracentrifuged protein through gel filtration chromatography columns (Superdex 200, 30/10 HR; Amersham Biosciences) revealed a narrow peak at an elution volume corresponding to 22 kda, the expected size of the protein. Centrifugation at 10,000 g revealed the same peak, but a shoulder thought to be misfolded protein was also present. All subsequent experiments were performed after ultracentrifugation of the protein at 100,000 g for1h. RESULTS Differential Binding of NC and mgag Binding to HIV-1 Genomic RNA Probes Our laboratory has generated in vivo evidence that SP1 is involved in the recognition of HIV-1 genomic RNA (32); therefore, we decided to also study this protein-rna interaction in vitro. For this purpose, we designed and purified a minimal Gag protein (mgag), which possesses all the domains including NC, that are needed to generate

3 39888 HIV-1 RNA Encapsidation and Gag Multimerization FIG. 1. Schematic representation and analysis of peptides used. a, top, schematic representation of Pr55 Gag. Numbers correspond to the last amino acids of each peptide within the precursor. A black line denotes amino acids 2 7, which are included in mgag. Below, fragments present in the different recombinant proteins used. An asterisk in mgag denotes the position of the M318A mutation. The gray represents the N-terminal histidine tag of the vector used. b, SDS-PAGE analysis of the different proteins used. Approximately 5 g of protein were loaded in each lane, separated by electrophoresis, and visualized by staining with Coomassie Brilliant Blue. Two different protein ladders are shown (first and last lane), and the molecular masses of the bands are given on the side in kda. The identity of the proteins employed is given below each lane. VLPs (Fig. 1). The first gel shift assays were performed with these proteins, and a nucleic acid probe containing the fragment from nt 223 to 506 (1/6) ( 1 is capping site; strain HXB2) (Fig. 2), based on evidence that these sequences are important in RNA packaging (39, 64). As reported by others, a small percentage of the probe migrated very slowly, and this band was thought to represent RNA dimers (Fig. 3) (39, 51, 64 67). In support of this idea, the use of probes lacking SL1 did not yield this band (see also Fig. 7, below and compare panel C with the other panels in this figure). We also observed multiple bands in our gel shift assays as reported by others (39, 53, 64). The interactions of proteins with the probe differed in regard to both affinity and potential for multimerization. Gel shift assays performed in the presence of 50 mm NaCl, 20 ng/ l of Poly I Poly C and mgag revealed a 4 6-fold stronger affinity for the 1/6 probe than that observed with NC (Fig. 3, compare lines 8 and 17). This was not expected, since it had been assumed that NC and Pr55 Gag have similar binding affinity for HIV-1 genomic RNA (39, 64). Our gel shift assays also showed that mgag has a higher ability to multimerize even at low protein concentration, while NC binds to the probe in a more gradual and limited fashion (Fig. 3). In order to further characterize the different affinities and the complexes observed, we studied this protein-rna interaction in greater detail at the protein and RNA levels. mgag Binds to the 5 -LR as a Dimer While NC Does So as a Monomer In order to determine the identity of the different bands in the gel shift assays, the RNA-protein complexes were cross-linked and resolved in denaturing gels. As expected, NC bound to the probe as a monomer (Fig. 4B). At the lowest concentration of NC, a 1:1 RNA-protein association was observed. As the protein concentration was increased, a second and a third shifted band were seen, with molecular masses corresponding to 2 and 3 proteins bound to each probe, respectively. In contrast, in the same type of experiments performed with mgag, the first shifted band corresponded to the presence of two proteins that were bound to the RNA probe (probe, 90 kda, mgag, 20 kda) (Fig. 4A). It is important to note that this is not a RNA dimer, since it would have migrated at 180 kda. A second and a third shifted band were also present, with molecular masses corresponding to the binding of four and six proteins to one molecule of RNA, respectively. These experiments suggest that NC binds to the RNA probe as a monomer while mgag does so as a dimer. We then reanalyzed our gel shift assay results with this in mind and noted the absence of any intermediate 1:1 RNAprotein association. This, together with the stronger affinity of mgag than NC for the RNA probe suggested that an EC might have formed. To examine this hypothesis, we asked whether an EC might be observed in the presence of M318A, a mutation known to disrupt the CTD dimerization interface. We used site-directed mutagenesis to generate M318A mgag and showed that presence of the EC was completely voided by this mutation, as was the pattern of multimerization (Fig. 5). These experiments confirm that wt mgag binds to the 5 -LR as a dimer, and that this association requires the dimerization interface of the CTD. Binding Properties of N-terminal Extensions of NC to 5 -LR RNA Probes To analyze which fragments were involved in the strongest binding of the protein to the RNA, we produced different clones expressing N-terminal-extended versions of NC. This led to the generation of SP1-NC, the complete putative CA-SP1 -helix-nc (354-NC), and the 9th, 10th, and 11th -helices of the CTD of CA-NC (308-NC, 327-NC, and 339-NC, respectively) (Fig. 1). Due to the pattern of multimerization, it was difficult to study binding affinities by gel shift. For this reason, the binding affinities of these proteins for HIV-1 genomic RNA probes were studied by filter binding assays under various conditions as described under Materials and Methods. We first analyzed the binding properties of wt mgag, M318A mgag, SP1-NC, and NC to the 1/6 probe. As observed with the gel shift assays, NC showed a remarkably higher dissociation constant than mgag. The difference between proteins was 7.5- fold in the presence of competitor and high salt concentration 250 mm NaCl (i.e. K d NC: 0.75 M; K d mgag: 0.1 M) (Fig. 6A) and approximately one order of magnitude in the absence of competitor and low salt concentration 50 mm- (i.e. K d NC: 40 nm; K d mgag: 4 nm). Under all conditions, NC and SP1-NC bound with similar affinities, suggesting that SP1 does not provide binding capabilities to the protein. On the other hand, M318A mgag possessed different properties, based on the presence or absence of competition. In the absence of competitor, M318A showed a K d 40 60% higher than that observed for wt mgag (Fig. 6B). However, when a nonspecific RNA was added

4 HIV-1 RNA Encapsidation and Gag Multimerization FIG Leader region and description of probes used: Top, illustration of secondary structures of the HIV-1 non-coding leader region. The structure shown for ECP was generated from an M-fold analysis of that region. Below, small arrows indicate the position of the PCR primers for preparation of the riboprobes. The name by which the riboprobes are cited in the text is given at the right followed by the primer pair used in each case. FIG. 3. Multimerization and binding properties of mgag and NC studied by gel shift. Gel shift assay using 1/6c probe and wt mgag (left) and NC (right). The arrows on the right show the position of the free probe and for the dimer probe. The arrows on the left indicate the position of the 2, 4, 6, and 8 mgag proteins bound to the probe. The upper bands represent the association of n number of proteins with n number of probes. The protein concentration is as follows: lanes 1 and 10,3 M; 2 and 11,2 M; 3 and 12, 1 M; 4 and 13, 0.75 M; 5 and 14, 0.5 M; 6 and 15, 0.4 M; 7 and 16, 0.3 M; 8 and 17, 0.2 M; 9 and 18, no protein. to the reaction (i.e. Poly I Poly C), the K d observed almost doubled. These results suggest the possibility that the dimerization interface of wt mgag plays an important role in specific binding to the 5 -LR. In contrast, gel shift analyses using SP1-NC and the 1/6 probe showed a different binding pattern than that observed for NC. SP1-NC protein showed evidence of more multimerization capabilities than NC. This is strongly supported by the fact that higher concentrations of SP1-NC resulted in large aggregates that could not enter the gel, which was never the case for NC. These results suggest that SP1 provides an interface that is required for protein-protein interactions. When we included the other intermediate proteins in the analysis, we found that the complete putative -helix CA-SP1 (354-NC) increased affinity by 20 25%, while the 11th -helix of CA (339-NC) had 100% higher affinity than that observed for NC (Fig. 6A) (K d - M-; NC: 0.75, 354-NC: 0.57, 327-NC: 0.33). In the absence of a nonspecific RNA, the affinity of 339-NC was almost the same as that observed for M318A mgag, while in the presence of Poly I Poly C, the K d of 339-NC was higher than that observed for M318A mgag. This suggests that 339-NC possesses all of the domains necessary for nucleic acid binding, and that the hydrophobic core of the CTD is important for binding when a RNA competitor is present, possibly because of a conformational requirement. SP1-NC does not increase this affinity whereas 354-NC does. This might be explained by the presence of the complete putative -helix in 354-NC. These results, together with the gel shift SP1-NC data, suggest that the COOH half of the putative -helix interacts with the same motif within the protein dimer of the EC, whereas the NH 2 half of the -helix interacts with viral RNA. We then analyzed the behavior of 339-NC in regard to salt concentration. We found that low salt concentrations resulted in an almost doubling of affinity of 339-NC than were attained at high salt concentrations (Fig. 6E). This suggests that the hydrophobic nature of the amino acids in this region might play an important role in RNA binding. On the other hand, 327-NC and 308-NC possessed an affinity similar to or lower than NC. We have no clear explanation for this result, but it is possible that the presence of these -helices in the absence of the complete CTD core might lead to a conformational disturbance in this region. Binding Properties of mgag for Different Probes In order to

5 39890 HIV-1 RNA Encapsidation and Gag Multimerization FIG. 4.Cross-linking of mgag and NC to the 1/6c probe. Crosslinking assay using 1/6 as probe with mgag (A) and NC (B). Numbers on the left represent the molecular mass (MW) of the ladder employed in kda. The MW of the probe, mgag and NC are 91, 20, and 10 kda respectively. A, lane 1, molecular mass ladder; 2, mgag, 0.5 M; 3, no protein. B, lane 1, molecular mass ladder; 2 5, decreasing amounts of NC (2, 1, 0.5, 0.25 M, respectively). The arrows indicate the free probe, its association with 2 and 4 molecules of mgag (A), and its association with 1, 2, and 3 molecules of NC (B). FIG. 5.Effects of M318A on formation of EC. Gel shift assay using 1/6c probe and wt mgag (left) and M318A mgag (right). The arrows on the left show the position of the free probe and of the dimer probe. The arrows on the right indicate the position of the 2, 4, 6 mgag proteins bound to the probe. The protein concentrations are as follows: lanes 1 and 7, 1 M; 2 and 8, 0.75 M; 3 and 9, 0.5 M; 4 and 10, 0.4 M; 5 and 11, 0.3 M; 6 and 12, 0.2 M; 13, no protein. characterize the role of viral RNA in its interaction with protein, we analyzed both the multimerization and affinity properties of probes encompassing different domains of the 5 -LR (Fig. 2). The probe lacking SL1 (i.e. 2/6) had the same ability as 1/6 to generate the EC and to multimerize (Fig. 7, A and C) but, interestingly, the affinity was only about 40% of that observed for 1/6 (Fig. 6C). Another difference between these two probes is the absence of high mass complexes, seen with 1/6 but not with 2/6, suggesting the possibility that NC promotes probe dimerization only at a certain protein concentration. The presence of EC was much more sensitive to reduction of the probe from the 3 -end. M-fold analysis of the nt region of the HBX2 strain of HIV-1 suggests the possibility of a similar conformation at the analogue position reported for HIV- 1 Mal (59). Interestingly, when probes lacking this region were used (i.e. 1/4 or 1/5), the EC was generated but only at higher protein concentrations (Fig. 7, D and E) and, moreover, multimerization ability was dramatically reduced. Probes containing part of this structure (1/6i) restored both EC formation and multimerization ability to a certain extent (Fig. 7B), supporting the concept that this particular structure is involved in proper protein-rna interactions. Furthermore, the K d for these incomplete probes was about 70% of that of 1/6c, while the 1/4 and 1/5 probes showed a marked reduction in affinity ( 26%). To rule out the possibility that the size of the probe was the crucial element in these interactions, we performed the same assays with a probe of a similar size but spanning from the PBS to downstream of SL4. This probe, PB/4, possessed an affinity similar to that observed for 2/6 (Fig. 6C), but EC formation and the multimerization pattern were reduced to that observed for 1/4 and 1/5. As a control, we also verified the affinity of NC for the different probes. In contrast to our findings with mgag, no significant differences were observed, showing that NC alone is incapable of discerning between the probes (Fig. 6D). These results suggest an association between EC formation (by mgag) and the increased binding capabilities of this protein for the probe. Furthermore, it is possible that this interaction might facilitate the initial recognition and specific packaging of the 5 -LR of genomic RNA in vivo. DISCUSSION Much effort has gone into a better understanding of RNA packaging and the participation of Gag domains in virus assembly and release. We have designed an in vitro assay to study early events in viral protein-rna interactions and the role of these interactions in viral assembly. Based on previous and our own data, we propose a model for the protein-genomic RNA interaction (Fig. 8). Our first important finding is that NC, in the context of Pr55 Gag, binds with much higher affinity to the 5 LR than does NC alone (Fig. 6, A and B). Note that the former represents the immature form of the protein whereas the latter represents the mature version. This was not expected, since it had been assumed that NC and Pr55 Gag have similar binding affinity for HIV-1 genomic RNA (39, 64). Several explanations include our preliminary data using Gag p6 and CA-NC, showing a lower affinity for the 1/6 probe than mgag. Others have employed full-length MA sequences that do not acquire proper conformation in a prokaryotic environment (68, 69). In addition, others have compared the binding activities of both proteins with a 178/383 probe comprising SL1, 2, 3, and 4, and we show here that sequences downstream of nucleotide 383 are crucial for the differential binding of mgag (39). Other investigators also used GST-tagged proteins, and a GST tag is larger than that which we employed (GST 28 kda, His 3 kda) (39, 64). Our data suggest that the 11th -helix of CA and the putative CA/SP1 -helix provide the surfaces for the increased affinity of mgag for the HIV-1 genomic RNA probe. As shown, the region encompassing the putative -helix in CA/SP1 (354- NC) increases affinity of NC for HIV-1 genomic RNA, and 339-NC has 100% more affinity than NC alone. Furthermore, 339-NC in the absence of competition, has a similar K d to that of M318A, meaning that this extended NC already possesses all the required domains for RNA binding. In the presence of a nonspecific RNA, 339-NC shows a decrease in affinity in relation to M318A, suggesting a conformational requirement of the hydrophobic core of the CA CTD for the specific recognition of HIV-1 genomic RNA. Finally, the dimeric ability of wt mgag was responsible for the 2-fold difference in affinity in relation to M318A mgag in the presence of a nonspecific RNA. We have also shown that the stronger affinity of mgag for HIV-1 genomic RNA is dependent on structures downstream of SL2 and SL3. Indeed, the binding data suggest that SL6 is markedly more important than SL4 and SL5 (Fig. 6C). It has already been shown that sequences up to nt 500 are important for HIV-1 packaging. Pr55Gag has been shown to have strong affinity for RNA sequences extending to the AccI restric-

6 HIV-1 RNA Encapsidation and Gag Multimerization FIG. 6.Binding affinities studied by filter binding. Filter binding assay was performed as described under Materials and Methods, which is a modification of the protocol introduced by Schmalzbauer et al. (63). In A and B the inverse of the concentration is plotted against the inverse of the saturation factor. The K d and R 2 are shown between parentheses. A, in the presence of 20 g/ l of Poly I C and 250 mm of NaCl; B, no competitor, 250 mm NaCl. In C and D is plotted the ratio between the K d of the different probes with that observed for 1/6c using mgag (C) and NC (D). Similarly, in E is plotted the ratio between the K d of NC (black), 339-NC (dark gray), and mgag (light gray) inre- lation to that observed for mgag in the presence of low salt concentration (50 mm NaCl, left panel) and high salt concentration (250 mm NaCl, right panel) tion site ( 497) (39, 64, 70). Moreover, in vivo evidence for a role of genomic RNA in packaging comes from gene transfer experiments that used HIV-1 based vectors. A higher packaging rate was reported for a vector that included the coding region of Gag up to nt 1000 compared with a vector that extended only to nt 400 (42). These data were confirmed in studies showing that a region up to nt 535 contributed to increased packaging of the vector used (41). Our data show that an RNA segment between nt 400 to 500 is involved in interactions between mgag and viral RNA. Furthermore, we have shown that these stronger RNA-protein associations are related to the formation of a RNA-protein complex, including two molecules of protein and one of RNA (Figs. 4A and 5). The fact that two proteins bind to the 5 -LR is not surprising, since the encapsidation signal of the HIV-1 genomic RNA contains at least two strong affinity binding sites for NC (i.e. SL3 & SL2) and the C-terminal domain of CA has a dimer interface. The presence of a M318A mutation, which is known to impair each of CTD dimerization, proper Gag assembly, and viral infectivity (23, 71), also abolished formation of the EC. The EC apparently has an additive impact on the affinity of protein for genomic RNA since M318A has approximately half of the binding affinity of wt mgag ([K d ] K d n) but was crucial for multimerization ability. Nevertheless, this complex was present at a much lower protein concentration ( M) than the dimer equilibrium concentration for CTD (18 M) (23) suggesting that this association takes place very early in the assembly process. The EC links genomic RNA recognition, Pr55 Gag dimerization, and multimerization. We were able to elucidate a new function for the 5 -LR other than its role in protein binding and RNA dimerization, i.e. promotion of the formation of the RNA-protein complex termed the EC. Probes that lacked SL1 showed a drastic reduction in protein affinity (Fig. 6C) but did not impair EC conformation (Fig. 7, C and F). In contrast, SL6 (or EC platform -ECP-) was crucial for the formation of this EC (Fig. 7, D F), and this also had important impact on affinity (Fig. 6C). Probes lacking ECP still yielded complexes at higher protein concentrations but less efficiently (Fig. 7F). Interestingly, however, multimerization was drastically reduced. These results suggest that protein-rna binding allows a particular conformation or lattice that is important for proper Gag dimerization. Multimerization by dimer blocks has already been reported (68, 72). Furthermore, in vivo evidence for such early dimer formation has also been reported in two studies that employed cysteine specific cross-linking (73, 74). How does this protein-rna interaction take place? According to structural studies, the CA/SP1 region is unstructured in solution (23). Nevertheless, formation of almost every RNAprotein complex that has been characterized involves confor-

7 39892 HIV-1 RNA Encapsidation and Gag Multimerization FIG. 7.Multimerization and EC formation studied by gel shift using different probes. Gel shift assay using 1/6c (A), 1/6i (B), 2/6 (C), 1/5 (D), and 1/4 (E) probes with wt mgag. The arrows on the left show the position of the free probe and of the dimer probe. The arrows on the right indicate the position of the 2 and 4 mgag proteins bound to the probe. The protein concentrations are: lane 1, 0.5 M; 2, 0.25 M; 3, no protein. In F is given the protein concentration where the EC formation is maximal (ECF), and the proportion between this EC and the free probe is shown (%). FIG. 8.Proposed model for HIV-1 genomic RNA-protein interactions. The black line represents the RNA sequence from SL1 up to SL6. The protein domains are shown in colors: NC in orange, SP1/CA -helix in yellow, and the 11 th -helix of the CTD of CA in green. Light colors denote the front of the complex, while the dark colors correspond to its rearside. mational changes in the protein, the RNA or both (75, 76). It has also been shown that site-specific binding involves, at least for DNA-protein interactions, coupled changes in 2, 3, and/or 4 structure of the protein (77). Therefore, it is possible that the CA/SP1 region folds upon binding to viral RNA as suggested by the ability of the protein to form the EC in the presence of a specific structure in the probe (i.e. SL6-ECP). Computational modeling of this region predicts an -helix conformation. Our results suggest that the 11th -helix of CA and the putative CA/SP1 -helix might bind to HIV-1 genomic RNA (mainly SL6-ECP) in a helix-loop-helix (HLH) fashion. These elements are comprised of a short -helix (11th -helix of CA) and a long -helix (putative CA/SP1), connected by a flexible linker (78), that is usually followed by a highly basic domain involved in the protein: nucleic acid interaction (38, 63). In this model, a Gag dimer would interact with the 5 -LR as shown in Fig. 8: one NC domain would interact with SL3 and the adjacent HLH domain would interact with one side of the ECP, while the other NC would interact with SL2 and the adjacent HLH domain with the other side of the ECP. The CTD core and its potential for dimerization would provide a conformational advantage for specific HIV-1 genomic RNA recognition and make this complex more stable. Interestingly, HLH elements are usually involved in DNA binding as well as in protein dimer formation. We have shown that the putative CA/SP1 affords both multimerization capabilities and increases affinity. Furthermore, this increase reached its maximum when the short -helix (the complete HLH element) was present. In agreement with the formation of an HLH/RNA complex is the fact that mgag binds to the 5 -LR in a dimer fashion, as opposed to what is observed for NC, which binds as a monomer. The virus could take advantage of this induced fit model in two ways: First, the energetic cost for protein folding would be paid only by binding with the 5 -LR of HIV-1 genomic RNA, assuring mutual recognition. As a consequence only mutually recognized counterparts could multimerize in the proper way, leading to the formation of infectious virus particles. The hydrophobic sensitivity of the protein-rna interaction supports this model (77). Furthermore, given that the final conformation of the protein-rna complexes would depend on sequences from both protein and RNA, the mutual recognition between the parts is likely to be much more flexible than would be the case between rigid surfaces. Finally, according to the evolutionary conservation of the CTD-SP1-NC region among retroviruses, it is possible that the HIV-1 genomic RNA is specifically recognized in a similar way by other viruses. Others have identified a SL3 variant with greater affinity for NC than wt SL3 (79). They proposed that this SL3 variant is not present inside the virus, since a stronger affinity for the element could eventually interfere with other roles of NC in the

8 HIV-1 RNA Encapsidation and Gag Multimerization viral life cycle. Here, we report that NC in the precursor context has a one order of magnitude increase in ability to recognize a SL2/SL3/ECP element, but this high affinity is lost upon Gag cleavage during maturation, leaving NC free to perform its other functions. It is also possible that the HLH/RNA interactions provide binding support to Pr55 Gag, whereas NC is able to bind and unbind a variety of important elements (trna Lys, RT, etc). It is interesting to note that association forces become weaker in a C-terminal/N-terminal sense (NC-RNA 0.2 M 3 W317-M M 3 NTD-NTD 100 M 3 MA-MA -mm range), giving strength to the idea that proper protein-rna associations help drive the assembly process (14, 23, 80 84). The process of multimerization and assembly can be seen as a protein folding process in which two alternatives are possible: proper folding and assembly versus aggregation. The M318A mutation strongly reduced HIV-1 infectivity with reduced particle production and defective assembly of capsid and Gag (71). Our data suggest that the early association of two Gag proteins with the 5 -LR provides a conformation that is crucial for assembly and proper Gag-Gag interactions. It has recently been reported that RNA-protein interactions do not abrogate viral assembly but play a major role in the stability of the particles formed (85). This is in agreement with our hypothesis of proper protein-protein interactions that occur upon binding to the HIV-1 5 -LR. SL6 (ECP) plays an important role in our in vitro system and it is possible that HIV-1 genomic RNA possesses SL6 (ECP) analogue elements throughout its extension that could be bound/recognized by Gag during the assembly process. The ECP might then represent the first of such structures. In summary, we have shown that two proteins can bind to the 5 -LR to form an early complex (EC). 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