INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Human immunodeficiency virus type 1 (HIV-1) encapsidates two copies of full-length viral RNA that form a dimer through noncovalent linkage at the 5' end (5). The cis-acting elements that are involved in the specific packaging and dimerization of viral RNA are located in the 5' viral RNA leader sequence. Complex secondary structures have been proposed to exist in the leader region, among which SL1 and SL3 can bind to nucleocapsid (NC) protein with high affinity and are thought to be the major RNA elements responsible for viral RNA encapsidation (2, 7, 17, 19, 23, 27, 28). SL1 has also been shown to function as the dimerization initiation site (DIS) for viral RNA (1, 3, 9, 13, 19-22, 26, 30-34, 37). In addition, although the TAR and poly(A) hairpins, which are located at the 5' end of the leader sequence, bind to NC protein with low affinity, they have also been shown to contribute to the packaging process (10, 11, 16, 17, 29).

We have been particularly interested in the roles of RNA sequences, located upstream of the 5' major splice donor (SD) site, in encapsidation of viral RNA, since these RNA sequences are contained in both spliced and unspliced viral RNA, yet help the virus to selectively recruit unspliced RNA while excluding spliced RNA. To shed light on this issue, a number of deletion mutations, termed BH-D1, BH-D2, and BH-LD3, have been constructed to selectively remove RNA sequences in this region (23-25). These deletions had adverse impacts on both viral RNA encapsidation and viral replication. Interestingly, when the mutated viruses were cultured for a prolonged period, revertants with wild-type replication kinetics arose, and the results of sequencing analysis revealed two substitutional mutations, i.e., MP2 (T12I in p2) and MNC (T24I in NC), that can rescue such mutated viruses. We believe that exploration of the mechanism by which the MP2 and MNC mutations are able to compensate for the functions of the deleted RNA sequences will add new insights into the roles of RNA sequences upstream of the 5' SD site in viral RNA encapsidation and viral replication.

The first question we sought to answer was whether changes of nucleotides by the MP2 and MNC point mutations had altered cis-acting RNA signals in the gag coding region and whether the compensatory effects obtained were controlled in a cis-acting manner. This subject was pursued by insertion of stop codons into the gag gene in LD3, LD3-MP2-MNC, and BH10 to block Gag production, in order to enable a comparison of packaging efficiency among these three viral RNA transcripts. The second issue was whether the I residue is the only amino acid at either position 12 in p2 or at position 24 in NC that can rescue the above deletions. Answers to this question will shed light on the strictness of the steric interactions at these two sites for the rescue process. This subject was approached by substituting either the T12 in p2 or the T24 in NC by each of 19 other amino acids and screening for those residues that are able to rescue the deletions described above.

	MATERIALS AND METHODS

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HIV-1 DNA mutagenesis. The BH10 clone of infectious HIV-1 cDNA was employed as starting material to generate the following mutant constructs. The BH-D1, BH-D2, and BH-LD3 deletions eliminated sequences at nucleotides (nt) +200 to +226, +200 to +233, and +238 to +253, respectively, and were constructed as previously described (23, 25). To prevent translation of Gag proteins, stop codons were inserted at both the 6th and 8th amino acid positions in matrix (MA) protein and the 82nd and 85th amino acid positions in capsid (CA) protein (Fig. 1A). The former two stop codons in MA were engineered by PCR through use of primer pair pBssH-S (5'-CTGAAGCGCGCACGGCAAGAGG-3' [positions 706 to 727])-p800 (5'-CTAATTCTCCCCCGCTTCATACTCACGCTCTCGCACCC-3' [positions 829 to 792]); the latter two stop codons in CA were inserted by PCR through use of primer pair pBssH-S-p1430 (5'-GCCCTGCATGCACTTAATGCACTCAATCCCATTCTGC-3' [positions 1453 to 1417]). The entire DIS sequences at nt +243 to +277 were deleted in construct DIS that was generated by PCR using the primer pair pHpa-S (5'-CTGCAGTTAACTGGAAGGGCTAATTCACTCCC-3' [positions 1 to 21])-pDIS (5'-TACTCACCAGTCGCCGCCCTCCTGCGTCGAGAGAGC-3' [positions 750 to 680]).

View larger version (23K): [in this window] [in a new window]   FIG. 1.   (A) Schematic illustration of the inserted stop codons in the gag gene. The mutated nucleotides are underlined. (B) Analysis of viral protein expression from various constructs in transfected COS-7 cells. The top, middle, and bottom panels represent Western blots performed with MAbs against HIV-1 CA (p24), MA (p17), and Env proteins, respectively. A fusion protein of ~25 kDa that contains MA and truncated CA sequences is indicated by a star in the middle panel. Protein markers are shown on the right of the gels. BH-MA contains stop codons in MA. BH-CA contains stop codons in CA. BH-DG contains stop codons in both MA and CA. LD3-DG contains the LD3 deletion, as well as stop codons in MA and CA. LD3-MP2-MNC-DG contains the LD3 deletion and the MP2 and MNC point mutations, as well as stop codons in MA and CA.

Cell culture, transfection, and infection. COS-7 and MT-2 cells were grown in Dulbecco modified Eagle medium (DMEM) and RPMI 1640 medium, respectively, each supplemented with 10% fetal calf serum. COS-7 cells were transfected with HIV-1 DNA constructs in the presence of Lipofectamine (Gibco-BRL, Montreal, Quebec, Canada). Progeny virus was harvested 48 h after transfection and quantified by measuring levels of viral CA antigen (Ag) by enzyme-linked immunosorbent assay (Abbott Laboratories, Abbott Park, Ill.).

Radiolabeling and immunoprecipitation assays. COS-7 cells that had been transfected with HIV-1 recombinant DNA constructs were starved in DMEM without L-Met and L-Cys at 37°C for 1 h, after which the cells were metabolically radiolabeled with [³⁵S]-L-Met and [³⁵S]-L-Cys (ICN) at a concentration of 100 µCi/ml for 30 min at 37°C. After an extensive washing with complete DMEM, supplemented with 30 µg of L-Met and 60 µg of L-Cys per ml, the cells were cultured for 1 h. Virus particles from culture fluids were pelleted by ultracentrifugation and analyzed on sodium dodecyl sulfate (SDS)-12% polyacrylamide gels. The cells were washed twice with cold phosphate-buffered saline and lysed with 1 ml of NP-40 lysis buffer (50 mM Tris-Cl [pH 8.0], 150 mM NaCl, 0.02% sodium azide, 100 µg of phenylmethysulfonyl fluoride per ml, 1 µg of aprotinin per ml, and 1% NP-40). The cell lysates were clarified in a bench-top Eppendorf centrifuge at 13,000 rpm for 10 min at 4°C and then incubated with anti-HIV-1 CA monoclonal antibody (MAb) for 1 h at 4°C, after which 5 µl of protein-A linked Sepharose 4B (Pharmacia, Montreal, Quebec, Canada) was added for a further 30 min of incubation. The Sepharose 4B was then centrifuged and washed in turn with NET-gel buffer and Tris-NP-40 buffer (35). Pellets were suspended in 20 µl of 1× SDS-containing gel-loading buffer, boiled, and analyzed by SDS-12% polyacrylamide gels. Viral proteins were visualized by exposure to X-ray films.

Encapsidation of viral RNA by RT-PCR. Supernatants from COS-7 cells that had been transfected with various HIV-1 recombinant DNA constructs were clarified in a Beckman GS-6R centrifuge at 3,000 rpm for 30 min at 4°C and quantified on the basis of CA Ag levels. Viral RNA was purified from an amount of virus containing 2 ng of CA Ag using an RNA extraction kit (Qiagen). Viral RNA was dissolved in 50 µl of diethyl pyrocarbonate-treated water and treated with 20 U of RNase-free DNase (Gibco-BRL, Montreal, Quebec, Canada) at 37°C for 30 min to remove any DNA contamination. Five-microliter volumes of viral RNA were amplified for 20 cycles with a Titan One-Tube RT-PCR system (Boehringer Mannheim, Mannheim, Germany) using primer pair pGAG1-pST to analyze full-length viral RNA (23). DNA products were analyzed on 5% native polyacrylamide gels and visualized following exposure to X-ray films.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

The MP2 and MNC point mutations do not exert their compensatory effects via a cis-acting mechanism. Since MP2 and MNC compensate for cis-acting viral RNA packaging signals, it may be speculated that these two mutations act by changing cis-acting RNA signals in the gag-coding region. It was shown that MP2 and MNC restored the diminished content of viral RNA in LD3 to wild-type levels (23). We now asked whether the MP2 and MNC point mutations might act in cis to enable the LD3-MP2-MNC RNA to be more efficiently incorporated by wild-type Gag proteins than LD3 viral RNA. To pursue this subject, Gag production was eliminated from LD3 and from LD3-MP2-MNC, such that the potential cis effects of MP2 and MNC on viral RNA packaging could be assessed. For this purpose, two stop codons were inserted at the sixth and eighth amino acid positions in the MA protein to yield construct BH-MA (Fig. 1A). Surprisingly, substantial levels of the Gag-derived proteins, p40 and p24, but not Pr55^Gag, were detected in transfected COS-7 cells by Western blots (Fig. 1B, lane 1). It was later realized that the continuous presence of p40 and p24 was due to reinitiation of translation from an "AUG" codon at the tenth amino acid position in the CA coding region, an event that results in the appearance of a p40 Gag product (6). To entirely terminate Gag production, two stop codons were inserted at the 82nd and 85th amino acid positions in the CA protein. As expected, no Gag expression was now detected with BH-CA in the transfected COS-7 cells (Fig. 1B, lane 2). However, normal production of Env protein was seen (lane 2), indicating efficient transfection and normal viral gene transcription. The aforementioned four stop codons were then inserted into each of the BH10, LD3, and LD3-MP2-MNC constructs in order to engineer clones BH-DG, LD3-DG and LD3-MP2-MNC-DG, which were all defective in Gag production and yet active in the generation of Env proteins, as seen on Western blots (Fig. 1B, lanes 3, 4, and 5).

View larger version (49K): [in this window] [in a new window]   FIG. 2.   (A) Schematic illustration of the LD3 and DIS deletions. The DIS sequences are underlined. Deleted nucleotides are indicated by dashed lines. RT-PCR using primer pair pD-pA yields DNA products of 166 bp from wild-type BH10 RNA, 150 bp from LD3 RNA, and 131 bp from DIS RNA. (B) PCR using the BH10, LD3, and DIS plasmid DNA. Each of the three types of plasmid DNA was first quantified individually in separate reactions using primer pair pGAG1-pST (lanes 1 to 9) (23). The same plasmid samples were then mixed and subjected to reactions using primer pair pD-pA (lanes 10 to 12). Three dilutions were included to ensure the linear range of reactions. Lanes 13 and 14 represent negative controls with primer pairs pGAG1-pST and pD-pA, respectively. (C) RT-PCR analysis of viral RNA in transfected COS-7 cells. Total cellular RNA that was extracted from cell lysates equivalent to 100 pg of HIV-1 p24 was subjected to RT-PCR. Three dilutions, i.e., 1:1 (lane 1), 1:3 (lane 2), and 1:9 (lane 3), of each RNA sample were measured. RNase A digestion was also performed as a negative control to rule out any possible plasmid DNA contamination (lane 4). Wild-type viral DNA standards of 101, 102, 103, and 104 copies were used in RT-PCR to determine the linear range of the reactions. Combinations of constructs in each transfection experiment are shown at the top of the gels. (D) RT-PCR analysis of RNA that was extracted from virus particles. Labeling of the lanes refers to that in panel C.

Substitutions of the T amino acid at position 12 in p2 with V, L, I, C, or M can dramatically increase the infectiousness of BH-D1 mutant viruses. We have previously shown that the BH-D1 deletion mutation, eliminating HIV sequences at nt +200 to +226, attenuated viral replication. Long-term culture of the mutated virus in MT-2 cells led to a revertant containing a mutation at position 12 in p2, i.e., T12I (MP2), that helped to restore viral infectiousness to near wild-type levels (25). We now sought to answer whether other amino acids at position 12 in p2 were also able to rescue the BH-D1 deletion. Toward this end, the T12 in p2 in BH-D1 was substituted by each of 19 other amino acids (Fig. 3) (Table 1). Relevant recombinant DNA constructs were transfected into COS-7 cells, and the progeny viruses thus generated were quantified on the basis of CA Ag levels. Equivalent amounts of virus were used to infect MT-2 cells and the infectiousness of each preparation was determined by the formation of syncytia and RT activity in culture fluids. The results of Table 1 show that four amino acids, including V, L, C, and M, in addition to the previously detected I, can help to correct the adverse effects of BH-D1 on virus replication. This subject was further evaluated by replication kinetic studies (Fig. 4A). Viruses containing either a G, K, D, or E at position 12 in p2 were barely infectious. In contrast, when T12 was replaced with either V, L, I, C, or M, the recombinant viruses thus generated were able to generate high levels of RT activity (Fig. 4A). Among the five amino acids that could play this role, C was the least efficient.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   Schematic illustration of mutations in the U5 region, CA, p2, and NC. The amino acid sequence of p2, as well as parts of the CA and NC proteins, is shown. The arrows indicate viral protease cleavage sites that result in formation of p2. The substituted nucleotide in U5 or amino residues in CA, p2, and NC are underlined. Nucleotides in U5 are numbered from the beginning of the U3 region. The numbering of the amino acid positions starts from the first residue of each protein. Secondary structural domains in the amino terminus of CA protein (amino acids 1 to 151) are illustrated that include a -hairpin (), seven -helixes (H1 to H7), and a cyclophilin A (Cyp A) binding site located between H4 and H5 (14). The V27I substitution in CA exists in H1. A sketch of the first Zn finger motif, in which some of the second-site mutations are located, is shown at the top of the figure. Interactions between the C, C, H, and C amino residues and the Zn ion are indicated; moreover, the underlined V13, F16, T24, and A25 amino acid residues are close in tertiary structure and form a hydrophobic cleft that can interact with the loop nucleotides of the region of viral genomic RNA SL3 (12).

View larger version (24K): [in this window] [in a new window]   FIG. 4.   (A) Replication kinetics of wild-type BH10 and mutated viruses containing the BH-D1 deletion and substitutions of T12 in p2. (B) Infectiousness of mutated viruses containing the BH-D2 deletion, substitutions of T12 in p2, and a T24I point mutation in NC. (C) Growth curves of mutated viruses possessing the BH-LD3 deletion, substitutions of T12 in p2 and a T24I point mutation in NC. In these studies, 5 × 105 MT-2 cells were infected by viruses equivalent to 3 ng of p24 Ag. RT activity in culture fluids was determined at various times. The D2-MP2-MNC and LD3-MP2-MNC constructs, both containing a T12I substitution in p2 and a T24I mutation in NC, were generated previously (24, 25) and served as positive controls. Mock infections represent negative controls performed with heat-inactivated BH10 virus.

The W, D, and E amino acids at position 12 in p2 dramatically decreased infectiousness of wild-type virus. We next studied the effects of other substitutions at position 12 in p2 on the replication potential of wild-type BH10 virus. Accordingly, T12 in BH10 was changed to each of 19 other amino acids. These various constructs were transfected into COS-7 cells, and the progeny viruses thus generated were quantified according to the levels of CA Ag. The infectiousness of the viruses produced by these constructs was examined by infection of MT-2 cells. The data showed that the T12W substitution in p2 abolished viral replication and that T12D and T12E dramatically decreased viral infectiousness (Fig. 5). Substitutions to either L or F markedly diminished viral replication and yet were permissive for production of high levels of RT activity after 10 days, while I, P, and Y each only slightly delayed viral replication (Fig. 5). Therefore, in terms of impact on viral replication, the various substitutions can be ranked as follows: W>D, E>L, F>I, P, Y>G, A, V, S, C, M, H, K, R, N, Q. 

View larger version (35K): [in this window] [in a new window]   FIG. 5.   Replication capacities of wild-type BH10 and mutated viruses containing substitutions of T12 in p2. Mutations are represented by single-letter abbreviations of amino acids. UAG is a stop codon that was inserted at position 12 in p2. (A) Growth curves of wild-type and mutated viruses after infection of MT-2 cells. (B) RT activity in culture supernatants at day 7 after infection. Mock represents a negative control performed with heat-inactivated wild-type virus.

View larger version (46K): [in this window] [in a new window]   FIG. 6.   Processing of Gag precursor protein Pr55Gag in wild-type BH10 and mutated viruses containing substitutions of the T12 amino acid in p2. Mutations are represented by single-letter abbreviations of substituted amino acids. (A) Transfected COS-7 cells were labeled with L-[35S]Met and L-[35S]Cys for 30 min and cultured for 1 h. Cells were then lysed, and the lysates were subjected to immunoprecipitation through the use of MAb against CA (p24) Ag. The observed viral proteins, including Pr55Gag, p41, p39, p25, and p24, are labeled on the right of the gels. To clearly visualize the p25 and p24 proteins in certain mutants, enlarged portions of the gels are shown. Mock-transfected COS-7 cells were also labeled with L-[35S]Met and L-[35S]Cys and serve as a negative control. (B) The intensities of the viral protein bands were quantified through the use of the NIH Image Program, and the percentages of each viral protein in mutant or wild-type virus were plotted. (C) After 1 h in culture, labeled virus particles were pelleted from the supernatants and analyzed on SDS-12% polyacrylamide gels. A portion of the gel was enlarged to clearly show accumulation of p25 protein in the W mutant.

View larger version (83K): [in this window] [in a new window]   FIG. 7.   Levels of viral RNA in wild-type virus and in mutated viruses harboring substitutions at position 12 in p2. Levels of full-length viral RNA were assessed by RT-PCR through the use of primer pair pGAG1-pST (23). RNA samples were treated with DNase to remove any contaminating DNA. RNA samples were also digested with RNase A and then subjected to RT-PCR as a negative control to exclude the possibility of DNA contamination. A negative control of wild-type BH10 is shown in lane 1. RNA obtained from wild-type virus was diluted 1:2 and 1:4 before RT-PCR to ensure a linear range of these reactions (lanes 23 and 24). Additional controls were performed with wild-type DNA of 101, 102, 103, and 104 copies to determine the linear range of the reaction (lanes 25 to 28). The results represent three independent experiments. Band intensities were quantified with the NIH Image Program and plotted. The amount of BH10 RNA was arbitrarily set at 1.0.

A V27I mutation in CA, together with a G575A substitution in the U5 region, can partially correct the attenuated infectiousness caused by the T12D substitution in p2. The T12W, T12D, and T12E mutations severely impaired infectiousness of wild-type virus. To further study these mutations, relevant viruses were cultured in MT-2 cells over prolonged periods to generate revertant viruses with increased growth potential. In the case of the construct containing the T12W substitution, no formation of syncytia was observed after 8 weeks in culture. In contrast, the T12D and T12E mutants showed rapid syncytium formation and high levels of RT activity after 6 weeks in culture. Conceivably, these latter substitutions may have served to stimulate the occurrence of other compensatory mutations that, in turn, enhanced viral replication.

View larger version (19K): [in this window] [in a new window]   FIG. 8.   The V27I mutation in CA, together with the G575A substitution in the U5 region, stimulates growth of the BH-p2-12D mutant. For details, see Fig. 4.

The V, L, M, F, and Y amino acid residues can play the same role as I at position 24 in NC to rescue the BH-D2 mutated virus. MNC was frequently identified together with MP2 in tissue culture able to rescue the BH-D2 and BH-LD3 deletion (25). It is intriguing to know whether similar findings to those obtained with MP2 would be observed in the case of MNC. Toward this end, T24 was changed to each of 19 other amino acids to generate constructs D2-MP2-NC24X (Fig. 3). The infectiousness of the viruses from these constructs was examined by infecting MT-2 cells. The results of Fig. 9 show that V, L, and M at position 24 in NC were able to rescue the BH-D2 mutated virus as well as did an I substitution. F and Y were less efficient in this regard. Therefore, amino residues with long hydrophobic side chains are favored at position 24 in NC to compensate for the BH-D2 deletion.

View larger version (28K): [in this window] [in a new window]   FIG. 9.   (A) Effects of various substitutions of the T24 residue in NC on the infectiousness of the BH-D2 mutated viruses. Each substitution is represented by the single-letter abbreviation of amino acids. (B) The RT activity of each virus at day 7 after infection is plotted to clearly show the difference of infectiousness between viruses. For details, refer to Fig. 4.

The S, D, and N amino residues at position 24 in NC dramatically decreased the infectiousness of wild-type virus. Next, we inserted the above substitutions of T24 into the wild-type virus to examine their effects on viral replication. The results of Fig. 10 show that most of the substitutions did not affect or only moderately affected viral infectivity. The S and N mutations substantially decreased viral replication, and D eliminated viral infectiousness. Therefore, the wild-type virus can use most of the 20 amino acids at position 24 in NC without affecting viral replication. Of note, the presence of either C or H at position 24 in NC did not affect viral growth. This result implies that the presence of C or H at position 24 in NC does not interfere with the binding of Zn²⁺ ion by the CCHC motif in the first zinc finger.

View larger version (34K): [in this window] [in a new window]   FIG. 10.   (A) Growth curves of the BH10 viruses containing substitutions of T24 in NC in MT-2 cells. (B) RT activity of each virus at day 6 after infection is plotted as a bar graph to demonstrate the difference of infectivity between viruses. For details, refer to Fig. 4.

View larger version (74K): [in this window] [in a new window]   FIG. 11.   Viral genomic RNA levels in the BH10 viruses containing various substitutions of the T24 residue in NC. Substitutions are represented by the single letters of amino acids. Viral RNA from viruses equivalent to 200 pg of CA antigen was subjected to RT-PCR using primer pair pGAG1-pST (23). The intensity of each band was quantified by the NIH Image program and plotted.

View larger version (24K): [in this window] [in a new window]   FIG. 12.   (A) Infectiousness of the BH10 wild-type virus and the BH-T24N, as well as BH-V13G-T24N mutated viruses in MT-2 cells. (B) Viral genomic RNA levels in the wild-type and mutated viruses determined by RT-PCR using primer pair pGAG1-pST. RNA samples of each virus were diluted 1:2 and 1:4 to ensure the linear range of the reactions. Wild-type viral DNA standards of 101, 102, 103, and 104 copies were used to determine the linear range of reactions. Lane 1, viral RNA from viruses equivalent to 200 pg of CA Ag; lane 2, 100 pg of CA Ag; lane 3, 50 pg of CA Ag. Intensity of RNA signals was quantified using the NIH Image program with the levels of wild-type BH10 arbitrarily set at 1.0.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have previously deleted various sequences between the primer-binding site and the 5' SD site that impact on viral RNA packaging and dimerization. Interestingly, long-term culture of all the mutated viruses gave rise to the MP2 and MNC compensatory mutations in the Gag protein (23, 25). In this study, we further showed that, in addition to the MP2 (T12I in p2) substitution that was identified in culture, amino acids V, L, M, and C at position 12 in p2 can also rescue the aforementioned deletions. Among them, V, L, I, and M rescued the impaired infectivity of the mutated viruses to similar levels, while C acted in this regard to a lower extent. Similar findings were made with the MNC mutation (T24I in NC). Screening studies identified six amino acids, including V, L, I, M, F, and Y, at position 24 in NC that can help to rescue the deletions; among these, F and Y are the least efficient.

Our data argue that a nucleotide change from C to T in the MP2 and MNC mutations did not exert compensatory effects in a cis manner. The results of cotransfection experiments demonstrated that the nucleotide change from C to T in MP2 and MNC did not overcome the crippled packaging efficiency of the LD3 viral RNA (Fig. 2). Furthermore, the V, L, I, and M amino acids are coded for by different nucleotides, yet they all can rescue the LD3 deletions when present at position 12 in p2 and at position 24 in NC. Therefore, it is the hydrophobic nature of the substituted amino residues at position 12 in p2 and at position 24 in NC, rather than the altered nucleotides themselves, that rescued the deletion mutations.

One question concerning these findings is why only the I residue was identified in culture if the V, L, and M amino acids can also replace the T12 in p2 or T24 in NC to rescue the mutated viruses. This may simply be because only a single nucleotide change, i.e., ACCAUC, is required to achieve a T-to-I substitution; however, each of the other amino acid changes require mutations at two or three base positions. It is also noted from the above results that the V, L, I, and M residues are identified both in p2 and in NC as the most efficient ones in the rescue process. Since V, L, I, and M are selected from 20 amino acids and all contain long hydrophobic side chains, we hypothize that specific interactions of a hydrophobic nature involving these residues in p2 and NC with other sequence of Gag or with viral RNA are essential to the rescue of the BH-D1, BH-D2, and BH-LD3 mutated viruses.

In addition to SL1, several other RNA elements are also involved in HIV-1 RNA packaging. Based on their affinity to the Gag protein, as assessed by in vitro binding experiments, the involved RNA elements can be divided into two groups. The TAR and poly(A) hairpin show low affinity for Gag, while SL1 and SL3 exhibit high affinity (5, 8). It is understood that SL1 and SL3 contribute to the packaging process through their tight binding to Gag, yet the mechanism remains elusive by which TAR and the poly(A) hairpin affect RNA packaging. Because of the multipartite nature of packaging signals, mutation of either one of these signals cannot totally exclude viral RNA from virus particles. Moreover, the mutated viruses may revert by modifying the Gag protein to increase its binding affinity to the unchanged packaging signals, so that the RNA packaging in the mutated viruses can be restored to wild-type levels. Our compensation analysis of the SL1 sequence may be an example of this kind.

The nuclear magnetic resonance (NMR) structure of the NL4-3 NC and the SL3 RNA complex indicated a direct contact between the I24 residue of NC and SL3 RNA, which implies that I24 participates in packaging (12). However, the 24th position of NC in BH10 is occupied by a T residue. Since multiple RNA elements are involved in the packaging process, although the NC of BH10 contains a T24 residue and may show a low affinity to SL3, interactions of NC with SL1 or other viral RNA elements may still allow the virus to recruit wild-type levels of viral RNA. However, when the SL1 RNA sequence is deleted, a T24I change is necessary to strengthen the interactions between NC and SL3 RNA, such that the mutant virus can package wild-type levels of viral RNA. Since V, L, and M contain similar hydrophobic side chains to I, they may establish similar interactions with SL3 RNA when present at position 24 in NC and correct the defective viral RNA packaging in the mutated viruses.

Another interesting finding is that a substitution of T24N in the first zinc finger motif of NC in BH10 can be compensated for by another point mutation, i.e., V13A in the same zinc finger. According to the NMR structure of NC and the SL3 RNA complex, the V13 and I24 amino acid residues, together with F16 and A25, form a hydrophobic cleft to which the G⁹ of SL3 can bind. Moreover, V13 and I24 contribute to the formation of a compact structure of NC by hydrophobic interactions with F6 (12). Therefore, V13 and I24 are important residues in NC. It is possible that substitution of T24 in BH10 with N may introduce steric interference with the V13 residue and further lead to distortion of NC conformation. The G amino acid contains an -H as its side chain and therefore can release the possible steric hindrance when substituted for V13. This issue needs to be tested by NMR analysis of the mutated NC protein binding to the SL3 RNA.

It is also interesting that the V27I substitution in CA can stimulate growth of the BH-p2-12D mutant. V27 is located in a region that forms "helix 1" in the CA protein (14, 15) (Fig. 3). Both crystallographic and mutagenesis studies suggest that proteolysis between MA and CA is followed by refolding and that the helices of two distinct CA molecules, including "helix 1," then create a new CA-CA interface essential for the formation of a condensed conical core (36). The V27I mutation may adjust this process to correct deficits caused by the T12D mutation in p2. On the other hand, identification of the V27I mutation in CA also suggests possible interactions of p2 with certain regions of CA.

Proteolytic cleavage of Gag gives rise to four distinct proteins, i.e., MA, CA, NC, and p6. Although the structure of each of these proteins has been determined, their conformation within the context of the Gag protein is not fully resolved. The fact that wild-type virus can successfully accommodate 15 of 20 amino acids at position 12 in p2 suggests that this residue is not normally involved in crucial interactions with other regions of Gag. However, the presence of some amino acids at this position, e.g., W, D, or E, may conceivably exert negative effects on Gag conformation and may, therefore, result in abnormal Gag processing, RNA packaging, and viral replication. The V, L, I, and M substitutions may have established novel interactions that are crucial for the viability of the deleted virus but insignificant in regard to wild-type virus.