INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is important for virion infectivity. Vif is required for HIV-1 replication in peripheral blood mononuclear cells, and its function is likely to be essential in vivo (12, 19, 20, 24, 56, 58, 61). vif-defective viruses can enter cells normally and initiate reverse transcription but are defective in the ability to complete proviral DNA synthesis (13, 26, 42, 53, 57, 61). This results from an effect of Vif in the virus-producing cell, most likely during events involved in virus assembly (24, 48, 61). A low level of Vif, estimated at between 1 and 40 molecules per virion, is associated with the core of HIV-1 particles (11, 15, 22, 29, 39, 54). However, virion incorporation may be nonspecific and does not appear to be required for Vif function (15, 54). Although Vif is required for HIV-1 replication in primary T cells and monocytes/macrophages, the requirement for Vif differs among cell lines (19, 24, 48, 57, 61). This finding has led to the classification of cell lines as nonpermissive or permissive for the replication of vif-defective viruses. The cellular or viral targets for Vif function have not been identified. Recent studies suggest that Vif may act to overcome an inhibitory factor present in nonpermissive cells (40, 52). An alternative model is that Vif may compensate for a cellular factor(s) that is required for the production of infectious virus particles but is present only in permissive cells.

Several lines of evidence suggest that the action of Vif affects the virion core. The major structural proteins of the mature HIV-1 virion are proteolytic cleavage products of the HIV-1 p55 Gag precursor protein, matrix (p17), capsid (p24), nucleocapsid (p7), and p6 (reviewed in reference 60). The cone-shaped core contains p24, p7, the pol gene products reverse transcriptase (RT), integrase (IN), and protease, the accessory proteins Vpr and Nef, a small fraction of p17, tRNA^Lys, and the viral RNA genome (2, 33, 62). The p24 capsid protein forms the cone-shaped shell, and the other proteins and RNA genome are localized to the core interior (60). The p7 nucleocapsid protein is required for packaging of genomic RNA and is complexed with the RNA in the internal nucleocapsid. Surrounding the core is a protein layer of the p17 matrix protein apposed to the lipid envelope, which contains the gp120 and gp41 Env glycoproteins. vif-defective virions exhibit structural abnormalities in the virus core. Tightly packed electron-dense material is found only in the broad end of the cone-shaped core, while the material inside the narrow end appears transparent (5, 8, 27). Furthermore, vif-defective virions are impaired in the ability to synthesize viral DNA in the endogenous RT reaction, an in vitro assay in which virions synthesize viral DNA by using endogenous RNA as a template (26, 42). Moreover, vif-defective virions can initiate reverse transcription in acutely infected cells, but the reverse transcripts appear to be prematurely degraded (53). These findings suggest that a component of the virion core is likely to be a target for Vif function. However, most studies have not found a consistent difference between wild-type and vif-defective virions in the virion composition of Env or Gag proteins, viral enzymes, or packaged viral RNA (8, 22, 43).

Little is known about the early postentry steps that lead to uncoating and disassembly of the virion core. Fusion and virus entry are followed by a poorly defined uncoating process in which the p24 shell is released from the core to form the uncoated viral nucleoprotein complex (also called the reverse transcription complex [RT complex]), which contains the viral genome, tRNA^Lys primer, RT, integrase p7, p17, Vpr, and host cell proteins (reviewed in reference 60). After reverse transcription of the viral genome, a preintegration complex is formed that is actively transported to the nucleus. These observations together with previous studies described above suggest that Vif may play a role during virus assembly which affects the stability and uncoating of the internalized HIV-1 core.

In this study, we examined the effect of Vif on the stability of the HIV-1 core in permeabilized virions and purified cores. The integrity of the core was examined following virion permeabilization or removal of the lipid envelope and treatment with various triggers, including S100 cytosol, deoxynucleoside triphosphates (dNTPs), detergents, NaCl, and buffers of different pH, to mimic aspects of the uncoating and disassembly process. Under all conditions tested, vif mutant virions produced in nonpermissive cells released increased amounts of core components (p24, RT, and p7) to the soluble fraction compared to wild-type virions. Purified cores could be isolated from wild-type but not vif mutant virions by sedimentation through detergent-treated gradients. These results demonstrate that Vif increases the stability of virion cores and suggest that the core of vif-defective viruses may be degraded prematurely or disassemble abnormally after virus entry.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Virus and cell culture. A cocultivation method was used to produce large quantities of wild-type and vif mutant virus in nonpermissive CEM, HUT78, and H9 cells or permissive SupT1 cells (26). Infection of CEM cells was initiated by cocultivation with 293T cells transfected with 10 µg of wild-type or vif mutant HXB2 DNA by the calcium phosphate method from 24 to 48 h after transfection. Infection of HUT78, H9, and SupT1 cells was initiated by cocultivation with 293T cells cotransfected with 1 µg of pHCMV-G, which expresses the vesicular stomatitis virus envelope glycoprotein, and 10 µg of wild-type or vif mutant HXB2 DNA by the calcium phosphate method from 24 to 48 h after transfection. The HIV-1 vif mutant viral DNA was made by changing the HXB2 sequence encoding Vif amino acids 21 and 22 to two in-frame stop codons (24). Cultures were maintained in RPMI medium plus 10% fetal calf serum, with medium changes every 1 or 2 days. Virions were harvested from 24-h culture supernatants from days 4 to 10 after infection. The culture supernatants were clarified by centrifugation at 2,000 × g for 10 min and filtration through a 0.45-µm-pore-size Millipore filter prior to virion pelleting by centrifugation through 20% sucrose in a phosphate-buffered saline (PBS) cushion at 125,000 × g for 90 min. Pelleted wild-type and vif mutant virions were resuspended in 50 mM Tris (pH 7.4) and normalized for the same amount of exogenous RT activity by incorporation of [³H]dTTP into an artificial poly(A)(dT)₁₅ template as described elsewhere (26).

Endogenous RT assay. The standard endogenous reaction was performed as described previously (26) in a 50-µl volume containing 500,000 cpm of exogenous RT units of HIV-1, 50 mM Tris-HCl (pH 7.4), 2 mM dithiothreitol, 2 mM magnesium acetate, 0.1 mM three dNTPs (dATP, dCTP, and dGTP), 50 µCi of [³H]dTTP, and the indicated detergent for 20 h at 37°C. In initial experiments, virions were permeabilized with the following concentrations of detergents for 10 min at room temperature prior addition of reaction buffer: 5 to 20 µg of melittin (Sigma) per ml, 0.01 to 0.04% NP-40 (Sigma), 0.01 to 0.04% Cymal-6 (cyclohexyl-hexyl--D-maltoside; Anatrace), and 0.01 to 0.04% Triton X-100 (Sigma). For subsequent experiments, 10 µg of melittin per ml was used for virion permeabilization. For some reactions, the reaction buffer contained a final concentration of 50, 150, or 500 mM NaCl or 50 mM Tris-HCl buffer with pH 5.0, 7.0, or 9.0. The reactions were terminated by addition of 1/10 volume of stop buffer (final concentrations, 50 mM Tris-HCl [pH 8] and 1% sodium dodecyl sulfate [SDS]) and spotting onto DE81 filters for quantitation by liquid scintillation counting.

Treatment of virions with chemical triggers or S100 cytosol. Wild-type and vif mutant virions (200,000 cpm of exogenous RT units) were permeabilized with 5 to 20 µg of melittin per ml, 0.01 to 0.04% NP-40, 0.01 to 0.04% Cymal-6, or 0.01 to 0.04% Triton X-100 for 10 min at room temperature prior to addition of 50 mM Tris-HCl (pH 7.4; to a final reaction volume of 50 µl) and incubation for 1 h at 37°C. The samples were then centrifuged at 14,000 × g in an Eppendorf Microfuge for 1 h. The supernatant and pellet fractions were separated and loaded onto an SDS-16% polyacrylamide gel and analyzed by Western blotting. In separate experiments, 200,000 cpm of exogenous RT units of wild-type or vif mutant virions was permeabilized with 10 µg of melittin per ml for 10 min at room temperature prior to addition of 50 mM Tris-HCl (pH 7.4) with 50 to 500 mM NaCl, 50 mM Tris-HCl buffered to different pH values, or 1 to 10 µg of S100 cytosol proteins and incubated at 37°C for 1 h. The supernatant and pellet fractions were separated and analyzed by Western blotting as described above.

Preparation of S100 cytosol. H9 cells were resuspended in hypotonic buffer (10 mM Tris-HCl [pH 7.4], 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM dithiothreitol) in the presence of protease inhibitors (leupeptin [5 µg/ml], antipain [50 µg/ml], aprotinin [10 µg/ml], pepstatin [5 µg/ml], and phenylmethylsulfonyl fluoride [100 µg/ml]), incubated on ice for 10 min, homogenized with 20 strokes of a glass Dounce homogenizer (type B), and centrifuged at 500 × g for 5 min to pellet nuclei. A cytosol fraction (S100) was prepared by centrifugation of the supernatant at 100,000 × g for 1 h. Total protein concentration was determined by the Bio-Rad DC assay, and aliquots of S100 fractions were stored at 80°C.

Sucrose gradient analysis. Wild-type and vif mutant virions (500,000 cpm of exogenous RT units) were permeabilized with 10 µg of melittin per ml, treated with 500 mM NaCl in 50 mM Tris-HCl (pH 7.4), 20 µg of S100 cytosol, and 2 mM dNTPs plus 2 mM magnesium acetate, or 10 µg of RNase (Boehringer Mannheim) per ml, incubated for 1 h at 37°C, and layered over a 1-ml 20 to 60% sucrose gradient. The gradients were centrifuged at 14,000 × g for 3 h at 4°C, and 125-µl fractions were collected from the top. The pellet was resuspended in 125 µl of 20% sucrose. One-third of each fraction was used for SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis.

Western blot analysis. Samples were separated by SDS-PAGE on 12 or 16% polyacrylamide gels under reducing conditions, transferred to polyvinylidene difluoride membranes (Millipore), and probed with mouse monoclonal anti-p7 antibody 4D1, mouse monoclonal anti-p17 (Advanced Biotechnologies Inc.), rabbit anti-p24 (Intracel), and mouse anti-RT (Intracel) antibodies, and human monoclonal HIV-1 gp41 antibody 2F5 (obtained from Hermann Katinger through the NIH AIDS Research and Reference Reagent Program) (10, 44, 45). Blots used for multiple hybridizations were stripped of antibodies by washing for 30 min at 50°C in stripping buffer (62.5 mM Tris-HCl [pH 6.8], 2% SDS, 100 mM -mercaptoethanol) (22). Blots were visualized by horseradish peroxidase-conjugated anti-human (Cappel), anti-rabbit, or anti-mouse (Amersham) immunoglobulin G antibodies and enhanced chemiluminescence (Renaissance; NEN).

Preparation of virion cores. Wild-type and vif mutant virion cores were prepared by a modification of the "spin-thru" method previously used for HIV-1 (2, 33) and HIV-2 (30). A sucrose step gradient was prepared by layering 400 µl of 60% sucrose in PBS, 400 µl of 40% sucrose, 200 µl of 20% sucrose containing 0.5% Triton X-100, and 150 µl of 10% sucrose as a barrier between virus and detergent prior to centrifugation; 50 µl of concentrated virions (2 × 10⁶ cpm of exogenous RT units) was layered on top of the gradient and centrifuged at 14,000 × g for 3 h at 4°C. Eight fractions of 150 µl were collected from the top of the gradient, and the pellet was resuspended in 150 µl of 20% sucrose. Fractions were analyzed by SDS-PAGE, Western blot analysis, and electron microscopy.

Electron microscopy. Virions were permeabilized and treated as described above prior to fixation with 2.5% glutaraldehyde in PBS and postfixation with 1% OsO₄. The fixed material was embedded in Epon-Araldite, sectioned, and poststained with 1% uranyl acetate. Virions and cores in sucrose gradient fractions were analyzed by negative staining. The sucrose fractions were diluted with 1 ml of 2.5% glutaraldehyde and centrifuged for 1 h at 14,000 × g. The pellets were resuspended in 0.5% glutaraldehyde, applied onto carbon-coated grids, and stained with 1% uranyl acetate. Sections and negatively stained samples were analyzed at an accelerating voltage of 60 kV with a JEOL 1200EX electron microscope.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Detergent stability of virions. To examine the effect of Vif on virion core stability, we treated wild-type and vif mutant virions with detergent and performed in vitro assays to assess the functional and structural integrity of virion cores. For endogenous RT assays of detergent-treated virions, we used the nonionic detergents NP-40, Triton X-100, and Cymal-6. We also tested melittin, an amphipathic polypeptide derived from bee venom which forms membrane pores with an average size of 3 nm and can accommodate the release of 50.7-kDa size markers from permeabilized vesicles (34). Melittin has been reported to allow synthesis of more full-length cDNA in the endogenous RT reaction compared to nonionic detergents (4, 6, 14, 66).

View larger version (21K): [in this window] [in a new window]   FIG. 1.   Endogenous RT activity in wild-type and vif mutant virions. Wild-type and vif mutant virions produced in CEM cells were used for the endogenous RT reaction. Virions normalized for the same value of exogenous RT activity (500,000 cpm per sample) were permeabilized with different concentrations of melittin, NP-40, Cymal-6, or Triton X-100 prior to addition of reaction buffer, and the endogenous RT activity was measured by incorporation of [3H]dTTP for 20 h at 37°C. Values shown are means ± standard deviations (n = 2) and are representative of three or four independent experiments using different preparations of virions.

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Release of core proteins into the soluble fraction after detergent treatment of wild-type and vif mutant virions. Wild-type and vif mutant virions (200,000 cpm of exogenous RT units per sample) produced in CEM cells were permeabilized with melittin (0, 5, 10, and 20 µg/ml), NP-40 (0, 0.01, 0.02, and 0.04%), Cymal-6 (0, 0.01, 0.02, and 0.04%), or Triton X-100 (0, 0.01, 0.02, and 0.04%) prior to addition of buffer and incubation for 1 h at 37°C. The samples were centrifuged, and the supernatant and pellet fractions were separated by SDS-PAGE and analyzed by Western blotting. The blots were used for multiple hybridizations and were initially hybridized with a rabbit anti-p24 antibody. The blots were then stripped and reprobed sequentially with mouse anti-RT followed by a mixture of mouse anti-p17 and anti-p7 monoclonal antibodies. Results are representative of two or three independent experiments using different preparations of virions.

Effects of NaCl, pH, and S100 cytosol on virion cores. To further examine the effect of Vif on the stability of virion cores, we analyzed the sensitivity of wild-type and vif mutant virions to treatment with NaCl or different pH conditions. Virions produced in nonpermissive HUT78 cells were permeabilized with 10 µg of melittin per ml prior to addition of endogenous RT reaction buffer containing 0 to 500 mM NaCl or 50 mM Tris-HCl buffered to different pH values. For both wild-type and vif mutant virions, optimal endogenous RT activity was detected at 0 to 50 mM NaCl (Fig. 3A). NaCl concentrations above 50 mM consistently decreased endogenous RT activity, consistent with a previous study (66). An increase in NaCl concentration from 50 mM to 150 mM markedly reduced endogenous RT activity for vif mutant but not wild-type virions. Thus, the endogenous RT activity of vif mutant virions is more sensitive to high salt concentrations than that of wild-type virions. The endogenous RT reaction had a narrow optimal range for pH (Fig. 3A). The optimal pH for the endogenous RT reaction was 8, consistent with previous reports (6, 66). Both wild-type and vif mutant virions showed reduced endogenous RT activity at more acidic or basic pH. At neutral or slightly basic pH (pH 7 to 8), the endogenous RT activity of vif mutant virions was 53 to 55% of that of the wild type. At very low (pH 5 and 6) or high (pH 9) pH, the endogenous RT activity of vif mutant virions was 36 to 38% of that of the wild type.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   Release of core proteins into the soluble fraction after treatment of wild-type and vif mutant virions with NaCl, different pH conditions, or S100 cytosol. Wild-type and vif mutant virions produced in HUT78 cells were permeabilized with melittin (10 µg/ml) prior to addition of reaction buffer containing 50 mM Tris-HCl (pH 7.4) with 0, 50, 150, or 500 mM NaCl, 50 mM Tris-HCl buffered to 7.4 (control pH), 5, 7, or 9, or 0, 1, 5, or 10 µg of S100 cytosol proteins resuspended in 50 mM Tris-HCl (pH 7.4). (A) Endogenous RT activity of wild-type (closed circles) and vif mutant (open circles) (500,000 cpm of exogenous RT units per sample) was measured as described in the legend to Fig. 1. (B) The supernatant and pellet fractions of treated wild-type and vif mutant virions (200,000 cpm of exogenous RT units per sample) were analyzed by Western blotting as described in the legend to Fig. 2. Results are representative of two or three independent experiments using different preparations of virions.

Sucrose gradient analysis of virion core stability. To further investigate the effect of Vif on virion core stability and to detect more subtle alterations in virion integrity, wild-type and vif mutant virions produced in HUT78 cells were analyzed on 20 to 60% sucrose gradients following treatment with melittin and other triggers (Fig. 4). After melittin treatment alone, the gradient distributions of viral proteins were similar for wild-type and vif mutant virions, as determined by analysis of gradient fractions by Western blotting. The peak of p24, p17, and p7 proteins was found in fractions 4 to 6, which correspond to a density of 1.16 to 1.20 g/ml (Fig. 4), similar to the density of intact HIV-1 virions. This finding provides further evidence that treatment of virions with 10 µg of melittin per ml does not grossly disrupt virion integrity. Treatment with 500 mM NaCl resulted in detection of p24 and p7 proteins in the top fractions (1 to 3), most likely corresponding to free or partially solubilized proteins. A larger fraction of p24 and p7 proteins was released from vif mutant virions than from wild-type virions. A small amount of p17, which may represent a minor fraction of partially solubilized viral membranes, was detected in fraction 3 after treatment of virions with NaCl. Treatment of virions with S100 cytosol and dNTPs had similar effects as treatment with NaCl (Fig. 4). Treatment of virions with 10 µg of RNase per ml resulted in release of a small fraction of p7 into the top fractions to similar extents in wild-type and vif mutant virions, whereas release of p24 into the top fractions was greater for the vif mutant (Fig. 4). In endogenous RT assays, 10 µg of RNase per ml resulted in 60 and 100% decreases in endogenous RT activity of wild-type virions and vif mutant virions, respectively (data not shown). To determine the effects of the various treatments on the association of RT with virion cores, the gradient distribution of RT was analyzed. Western blotting with the anti-RT antibody was not sufficiently sensitive to detect minor changes in the quantity of RT, most likely because of the low level of RT in virions. We therefore performed exogenous RT assays of the gradient fractions. After melittin treatment alone, the gradient distribution of exogenous RT activity paralleled that of other virion proteins, with peak levels detected in fractions 4 to 6 (Table 1). Following treatment of virions with NaCl, dNTPs, or S100 cytosol, the amount of exogenous RT activity detected in top fractions 1 to 3 was 1.4- to 3.4-fold higher for vif mutant virions than for wild-type virions under all conditions tested except treatment with RNase. Together with the Western blot analyses, these results demonstrate that a larger fraction of p24, p7, and RT is released from vif mutant virions than from wild-type virions after treatment with NaCl, dNTPs, or S100 cytosol.

View larger version (29K): [in this window] [in a new window]   FIG. 4.   Sucrose gradient analysis of wild-type and vif mutant virions produced in HUT78 cells. Virions (500,000 cpm of exogenous RT units per sample) were permeabilized with melittin (10 µg/ml), treated with 500 mM NaCl in 50 mM Tris-HCl (pH 7.4), 20 µg of S100 cytosol proteins, and 2 mM dNTPs together with 2 mM magnesium acetate, or 10 µg of RNase per ml, and then analyzed on 20 to 60% sucrose gradients. Fractions were collected from the top of the gradient. Lanes P represent the pellet. The gradient fractions were analyzed by Western blotting as described in the legend to Fig. 2. Results are representative of two or three independent experiments using different preparations of virions.

View larger version (22K): [in this window] [in a new window]   FIG. 5.   Sucrose gradient analysis of wild-type and vif mutant virions produced in permissive SupT1 cells. Virions (500,000 cpm of exogenous RT units per sample) were treated and analyzed as described in the legend to Fig. 4.

Electron microscopy analysis of virion integrity and core structures. To examine the effect of RNase and S100 cytosol on virion integrity and core structures, wild-type and vif mutant virions produced in HUT78 cells were treated with melittin alone or in combination with RNase and S100 cytosol as described above and then analyzed by electron microscopy (Fig. 6). Untreated morphologically mature wild-type virions exhibited dense cone-shaped or round cores (Fig. 6). In contrast, untreated mature vif mutant virions exhibited nonhomogenous packing of the core (Fig. 6), consistent with previous studies (5, 8, 27). A fraction of untreated wild-type and vif mutant virions exhibited an immature morphology with a dense spherical shell along the inner surface of the envelope. Treatment with melittin alone reduced the frequency of cone-shaped cores by 40% for wild-type and vif mutant virions and increased the frequency of mature wild-type and vif mutant virions with round cores (data not shown). Occasional disruptions of the viral membrane were also detected.

View larger version (73K): [in this window] [in a new window]   FIG. 6.   Electron microscopy analysis of virions after treatment with RNase or S100 cytosol. Wild-type and vif mutant virions produced in HUT78 cells were resuspended in buffer (50 mM Tris-HCl [pH 7.4]) (Untreated) or treated with melittin (10 µg/ml) in combination with 10 µg of RNase per ml (RNase) or 20 µg of S100 cytosol (S100) for 1 h at 37°C prior to embedding and analysis by electron microscopy. Size bar indicates 100 nm.

Stability of purified virion cores. To analyze the effect of Vif on the stability of virion cores in the absence of the surrounding viral envelope, cores from wild-type and vif mutant virions produced in HUT78 cells were purified by a modification of the spin-thru method previously used for wild-type HIV-1 (2, 33) and HIV-2 (30). Concentrated wild-type and vif mutant virions were layered on top of a sucrose gradient containing a detergent layer of 0.5% Triton X-100 and centrifuged for 3 h at 14,000 × g. Eight fractions and the resuspended pellet were analyzed for viral proteins by Western blotting (Fig. 7). The distributions of viral proteins for wild-type and vif mutant virions were very similar after sedimentation through control sucrose gradients without a detergent layer. The peak of viral proteins was detected in fractions 2 to 5, which correspond to densities of 1.08 to 1.19 mg/ml. A 15-kDa band was detected by the anti-p7 antibody in both wild-type and vif mutant preparations, most likely corresponding to the uncleaved p7-p6 Gag protein.

View larger version (38K): [in this window] [in a new window]   FIG. 7.   Western blot analysis of purified cores. Wild-type and vif mutant virions produced in HUT78 cells were layered on top of 10 to 60% sucrose gradients containing a layer of 20% sucrose without any detergent (Untreated), 20% sucrose with 0.5% Triton X-100 (Detergent), or 20% sucrose with 0.5% Triton X-100 and 500 mM NaCl (Detergent-NaCl). The gradients were centrifuged for 3 h at 14,000 × g, and fractions were collected from the top. Lanes P represent the pellet. The distribution of viral proteins was analyzed by Western blotting of the fractions. Blots were used for multiple hybridizations and were stripped and reprobed as described in the legend to Fig. 2.

View larger version (66K): [in this window] [in a new window]   FIG. 8.   Electron microscopy analysis of purified wild-type cores. Fraction 4 from the sucrose gradient shown in Fig. 7 without detergent (Untreated Fraction 4) or the combined fraction 8 and pellet from gradients containing detergent (Detergent Pellet), or detergent and NaCl (Detergent+NaCl Pellet) were analyzed by negative staining. Only fractions from gradients layered with wild-type virions are shown. Size bar indicates 100 nm.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we demonstrate that Vif is important for the stability of HIV-1 virion cores. vif mutant cores were more sensitive to disruption by detergents, high salt, basic pH, S100 cytosol, dNTPs, and RNase than wild-type cores, as determined by endogenous RT assays, biochemical analyses, and electron microscopy. We also found that RT and p7 are released more readily from vif mutant virions than from wild-type virions after treatment with detergents, high NaCl and S100 cytosol, and other triggers, indicating that the internal nucleocapsid may be less stably packaged in the absence of Vif. Consistent with this model, previous studies have shown that vif mutant virions exhibit redistribution of electron-dense core material representing the nucleocapsid (5, 8, 27) and are defective in the ability to synthesize viral DNA in the endogenous RT reaction and in acutely infected cells (13, 26, 53, 57, 61). The membrane-associated p17 protein was more readily released from vif mutant virions than from wild-type virions in some experiments, but this finding was inconsistent and was not observed using other methods (Fig. 4 and 7), suggesting that this result may reflect cellular microvesicles that can cosediment with virions (15). These findings suggest that Vif increases the stability of virion cores. This may permit efficient viral DNA synthesis by preventing premature degradation or disassembly of RT complexes during early events after virus entry.

Our studies provide biochemical and structural evidence suggesting that a component of the virion core is likely to be a target for Vif function. One possibility is that Vif may affect the p55 Gag or p160 Gag-Pol precursor proteins, their interactions during virus assembly, or their processing during virus maturation. However, several studies have not demonstrated any difference in the virion composition of Gag or Pol proteins in wild-type or vif mutant virions (8, 22, 43, 48), raising the possibility that Vif function may modify one of these proteins indirectly or may influence interactions between Gag proteins. In vitro studies suggested that Vif may interact with the p55 Gag precursor, possibly by interacting with p7 (7, 28). However, studies thus far have not detected a direct interaction between Vif and Gag proteins in HIV-1-infected cells (50). Other studies have suggested that Vif may affect Gag or Gag-Pol processing (32, 49). However, effects of Vif on Gag or Gag-Pol processing are not consistently observed in vif mutant virions (8, 22, 43, 48), suggesting that this is unlikely to be its major mechanism of action. It is also possible that a component of the core other than a Gag or Pol protein (2, 33, 62) may be a target for Vif function. The Nef and Vpr proteins are unlikely candidates since these genes are defective in the HXB2 viruses used in this and other studies (24, 26, 27, 53). Cyclophilin A, a cellular protein that is associated with HIV-1 cores (23, 59), is also an unlikely candidate, since cores of mutant viruses that are deficient for its incorporation into virions are more stable than those of wild-type virus (25, 65). Moreover, its pattern of cell dependency is different from that of Vif (3, 65). Although Vif does not appear to affect the packaging of HIV-1 RNA into virions (61) or dimerization of the genomic RNA (26), a recent study suggested that Vif may bind to the genomic RNA (70). Further studies are required to identify the target(s) for Vif function in the virion core.

The molecular mechanism for the action of Vif in the virus-producing cell and how this is manifested during early events after virus entry are not known. During assembly, newly synthesized Gag molecules form cytoplasmic complexes prior to transport to the plasma membrane (36, 37). Vif in the cytoplasm might associate with these virus assembly intermediates (50, 51), either directly or via a cellular cofactor (40, 52), and thereby influence virus assembly. Consistent with this model, Vif and Gag have been shown to bind independently of each other to detergent-insoluble cytoplasmic complexes in the virus-producing cell (50). Recent studies suggest that the action of Vif may be mediated through a cellular factor in a species-specific manner (40, 52, 55). In view of this, it will be of interest to determine whether Vif neutralizes an endogenous cellular inhibitor (40, 52) that interferes with the production of stable virion cores. Reverse transcription in vivo takes place after virus entry within a partially disassembled core termed the RT complex (60, 68). The RT complex must allow access of cellular dNTPs for reverse transcription of the viral genome while retaining the association between RT, the viral genome, and other viral and cellular factors (60). The increased stability of virion cores produced in the presence of Vif may be important during steps of the uncoating process which result in release of the internal nucleocapsid and formation of stable RT complexes. Consistent with this model, a recent study showed that treatment of vif mutant virions with high dNTP concentrations in the absence of detergents increases virion infectivity (17), possibly by allowing initiation of reverse transcription in a cell-free environment (69).

Studies on mechanisms involved in HIV-1 virion uncoating and disassembly have been limited by a lack of in vitro assays. One reason these early events are difficult to study is the low fraction of infectious particles in HIV-1 virus preparations, estimated to be 10⁴ to 10⁷ (35). Furthermore, the rapid fusion between the viral and cellular membranes and the inherent instability of the HIV-1 core (62) provide further obstacles to detailed characterization of early postentry events. Several studies have been published on the purification and biochemical characterization of lentivirus cores (2, 30, 33, 38, 47, 62, 67). In the present study, we found that purified cores contain RT, p24, p17, and p7, but not gp41, consistent with previous studies (2, 33, 62). We further demonstrated that purified HIV-1 cores can be used to demonstrate effects of mutations and various triggers on core stability and disassembly. Interestingly, equine infectious anemia virus lacks a Vif protein (41) and has an unusually stable core (6, 47). However, HIV-1 and other lentivirus cores are unstable and easily disintegrate after removal of the viral membrane (2, 30, 33, 62). Therefore, in vitro assays which are less disruptive yet render virion cores accessible to various triggers are also of interest to facilitate studies on biochemical mechanisms that regulate core stability and disassembly. Early postentry events and viral DNA synthesis can occur in the absence of fusion mediated by the HIV-1 Env (18). The endogenous RT reaction allows for synthesis of viral cDNA without completely removing the viral membrane. In this study, we developed similar assays to study the integrity of virion cores under various conditions. We found that treatment with 10 µg of melittin per ml permeabilizes HIV-1 virions while grossly preserving the structural integrity of virion cores. Permeabilization with melittin was sufficient to allow molecules (dNTP and salt) and enzymes (RNase) to enter virions and for core proteins to be released from virions. These and other in vitro cell-free assays for virion core stability and disassembly may provide new insights into early postentry events and their regulation by viral and cellular factors. Similar approaches may also facilitate identification of the component(s) of the S100 cytosol fraction which induces disassembly of HIV-1 cores.

Most replication-defective gag mutants exhibit altered core morphology with round rather than cone-shaped cores (16, 21, 31, 46). Wild-type virion preparations also contain a large fraction of virions with round cores (27, 46). These round cores might represent incorrectly packaged and noninfectious virion cores, or they may be cone-shaped cores viewed in horizontal plane of section (27). The observation that electron-dense cone-shaped cores generally correlate with the ability to perform the early steps of the viral life cycle including the synthesis of full-length cDNA (46) suggests that virions with round cores may largely represent noninfectious particles. Consistent with this prediction, our finding that S100 cytosol and RNase treatment reduced the frequency of cone-shaped but not round cores suggests that the majority of round cores may not be competent to undergo the normal uncoating process. This possibility together with the low infectivity-to-particle ratio (35) may help to explain why our assays did not detect greater differences between wild-type and vif mutant virions. It has been suggested that maturation of the HIV-1 core facilitates the formation of a structure of intermediate stability (1, 63). The mature cone-shaped core is stable enough to prevent premature degradation of the viral reverse transcription machinery but less stable than the immature core to allow for uncoating and disassembly after viral entry. It is possible that during the postentry steps of the viral life cycle, the conical p24 core shell is released and a round or irregularly shaped RT complex is formed (Fig. 6) (17). Previous studies have shown that several p24 gag mutants with reduced infectivity exhibit increased or decreased core stability (9, 21). These observations together with our studies on vif mutant cores provide support for the hypothesis that a cone-shaped core of intermediate stability may be optimal for early events that lead to release of the internal nucleocapsid and the formation of stable RT complexes.

Our results support a model for the action of Vif in which Vif acts in nonpermissive cells to allow formation of a condensed mature viral core. The stable core formed in the presence of Vif may protect the viral genome and prevent premature degradation of the viral nucleoprotein complex during uncoating in a newly infected cell. Understanding the function of Vif may provide new insights into critical steps involved in virus assembly, uncoating, reverse transcription, and other early events required for the establishment of infection and thereby may lead to the identification of potential therapeutic targets.