The function of S₁₀₉, S₁₄₉ and S₁₇₈ residues, positioned in the N-terminus, the interdomain linker and the C-terminus of CA respectively (see location in Figure 1), was analyzed using NL4.3 virions bearing an alanine substitution at each position. Viruses were produced by transfection experiments of 293T cells with pNL4.3 or S₁₀₉A, S₁₄₉A and S₁₇₈A mutated molecular clones and used to infect the MAGIC-5B indicator cell line (Figure 2A). All three mutants were found to be poorly infectious compared to the NL4.3 wild-type (WT) viruses when viral input was normalized according to reverse transcriptase (RTase) activity. These data confirm replication defects previously reported for S₁₀₉A, S₁₄₉A and S₁₇₈A mutants 27. Infectivity of HIV-1 mutants characterized by post-entry blocks has been previously reported to be greatly influenced by the route of viral entry. Pseudotyping with envelope from other viruses enables some HIV-1 mutants to bypass early post entry blocks 293031. Incorporation of VSV-G, which allows viruses to enter the target cell using the endocytic low pH pathway, was found to restore infectivity of viruses bearing mutations/deletions in the Nef accessory gene 30 or of those lacking the ability to incorporate cyclophilin A (CypA) 31. For testing similar effects, S₁₀₉A, S₁₄₉A and S₁₇₈A mutants pseudotyped with VSV-G envelope were generated by co-transfection of proviral clones with a plasmid expressing VSV-G. VSV-G-pseudotyped S₁₀₉A, S₁₄₉A and S₁₇₈A viruses (referred below as VSV-G-S₁₀₉A, VSV-G-S₁₄₉A and VSV-G-S₁₇₈A respectively) were normalized for RTase content and used in single-round infectivity assays of the MAGIC-5B cell line (Figure 2C). As expected, when pseudotyped with VSV-G, a strong enhancement of infectivity was observed for WT virions as compared to non-pseudotyped viruses (data not shown). Interestingly, pseudotyping with VSV-G significantly restored infectious properties of S₁₄₉A and S₁₇₈A mutants to levels observed for VSV-G-WT viruses. In contrast, VSV-G-S₁₀₉A viruses remained weakly infectious. Similar results were obtained using an extended range of viral input (Figure 2D and [Additional file 1]). At lower doses (100 to 1,000 cpm of RTase activity), VSV-G pseudotyped viruses did not saturated the cell culture, as less than 90% of the cells were found to be infected using a direct β-galactosidase staining assay (data not shown). Accordingly, the infectivity rescue observed for the VSV-G-S₁₄₉A and VSV-G-S₁₇₈A viruses cannot be ascribed to an overestimation due to the use of saturating doses of VSV-G-WT. MAGIC-5B cells were next infected with S₁₄₉A or S₁₇₈A viruses expressing either VSV-G or HIV Env using doses adjusted to generate comparable levels of strong-stop DNA copies in the infected cells as defined by qPCR experiments. In these cells, β-galactosidase activity was observed to be dramatically enhanced when S₁₄₉A or S₁₇₈A particles were delivered to the cell by the endocytic pathway [Additional file 2]. Finally, replication of VSV-G-S₁₄₉A and VSV-G-S₁₇₈A was abolished when MAGIC-5B cells were maintained in the presence of 20 μM AZT indicating that LTR-transactivation is not due to a pseudo-transduction artefact. Similar pseudotyping experiments were performed using the amphotropic murine leukaemia virus (MLV) glycoprotein, which allows infection of the target cell through a pH-independent fusion with the plasma membrane 32. In these conditions, infectivity of MLV Env-S₁₀₉A and MLV Env-S₁₄₉A was comparable to that of the corresponding mutants expressing HIV Env. Indeed, β-galactosidase activity generated in MAGIC-5B cells was found to be 2% vs 4.3% for MLV Env-S₁₀₉A and non pseudotyped S₁₀₉A viruses respectively or 6% vs 4% for MLV Env-S₁₄₉A and non-pseudotyped S₁₄₉A viruses respectively when compared to WT virus expressing the corresponding envelope glycoproteins. (Figure 2A and 2B). Infectivity of S₁₇₈A mutant was found slightly increased when pseudotyped with MLV Env (19% compared to 13% observed for MLV Env-S₁₇₈A and non pseudotyped S₁₇₈A viruses respectively). Infectivity defects associated with substitutions in CA can thus be rescued to variable extent by incorporation of different envelopes. Infectivity of S₁₄₉A and S₁₇₈A mutants was efficiently restored following VSV-G incorporation. Differences in infectivity observed between HIV Env expressing viruses and VSV-G pseudotypes suggest that shunting the viral core in the target cell through a pH-dependent endocytic pathway may bypass post-entry defects generated by mutation of S₁₄₉ and S₁₇₈ residues in CA.

Next, we examined the ability of CA mutants to accomplish early post entry events (i.e. RT). First of all, we tested whether mutations in CA impaired the RT machinery incorporated within viral particles using endogenous reverse transcription (ERT) experiments. This approach was successfully used to show alterations generated by CA mutations in Rous sarcoma virus 33. Normalized amounts of cell free virions, determined by exogenous RTase activity, were permeabilized and incubated in the presence of dNTPs to promote DNA synthesis primed by the endogenous tRNA primer using the RNA genome as a template. Viral DNA synthesis was then monitored by qPCR detection of strong-stop and second-strand transfer DNAs. RT efficiency was established by calculating the fraction of second-strand transfer products generated relative to strong-stop DNA copies expressed as a percentage. As shown in Figure 3A, ERT activity occurred for all viruses tested indicating that early and late HIV-1 DNAs were produced at least as efficiently by CA mutants as WT viruses when viral cores were permeabilized through the use of detergent. Unexpectedly, ERT level was increased by 2.5 fold for the S₁₄₉A mutant. Thus, alanine substitutions in CA did not markedly impair the formation of a functional nucleoprotein complex within the virions. We next analyzed RT capacities of CA mutants in the host cell. Total DNA was prepared from MAGIC-5B cells infected for 24 hours with normalized amounts of WT or CA mutant viruses and subjected to qPCR amplification using a specific set of primers allowing detection of RT intermediates. As shown in Figure 3B, strong-stop DNA was present at a similar ratio in cells infected with WT (2.9 × 10⁶ copies/10⁶ cells) or CA mutant viruses (from 2.7 to 3.9 × 10⁶ copies/10⁶ cells). All viruses tested were thus competent for fusion with the target cell membrane and inhibition of replication observed for S₁₀₉A, S₁₄₉A and S₁₇₈A mutants occurred at a post-fusion step, as previously proposed 27. Reverse transcription intermediates were efficiently detected in cells infected with WT virus (first-strand transfer, full-length minus strand and second-strand transfer DNAs were 8 × 10⁵; 2.5 × 10⁵ and 2 × 10⁵ copies/10⁶ cells). In cells infected with the S₁₇₈A mutant, first-strand transfer, full-length minus strand and second-strand transfer DNAs copies ranged from 70 to 95% levels quantified from cells infected by WT viruses. In contrast, RT was found most drastically altered in cells infected with S₁₄₉A virus, as synthesis of full-length minus strand and second-strand transfer DNAs was reduced by nearly 70% compared to control conditions. Finally, level of first strand transfer DNA was significantly decreased and full-length minus strand DNA synthesis was almost abolished in cells infected with S₁₀₉A viruses indicating that early reverse transcription was drastically reduced in these cells. We next tested the presence of 2-LTR circles in infected cells. 2-LTR circles are unproductive forms of viral DNA created by end-to-end ligation, that can be used as a reporter for nuclear import of the viral genome, since it localizes predominantly in the nucleus of infected cells 34. Using primers that specifically amplify the LTR-LTR junction, we found that all CA mutants were impaired for 2-LTR circle formation (Figure 3C). Altogether, these data indicate that CA mutations impair early replication at different steps. S₁₀₉A mutant was unable to accomplish RT. In contrast, S₁₄₉A and S₁₇₈A mutants produced different levels of second-strand transfer DNA and were impaired in their ability to produce 2-LTR circles.

Having demonstrated that VSV-G incorporation rescues infectivity of S₁₄₉A and S₁₇₈A mutant particles, we investigated the ability of these pseudotyped mutants to synthesize proviral DNA. Strong-stop and second-strand transfer DNAs were quantified from infected cells by qPCR experiments and RT efficiency was calculated as described for ERT experiments (see above). Similar RT efficiency was observed in cells infected with VSV-G-S₁₄₉A, VSV-G-S₁₇₈A or VSV-G-WT viruses (Figure 3D). This indicates that RT progressed efficiently for these viruses. In contrast, RT progression remained dramatically impaired in cells infected with VSV-G-S₁₀₉A viruses. These data were next compared to RT efficiency calculated from cells infected with viruses expressing HIV Env. Following VSV-G incorporation, RT efficiency was increased by 11 and 15-fold in cells infected with WT and S₁₇₈A viruses respectively (Figure 3D). This stimulation was also observed for VSV-G-S₁₀₉A, despite proviral DNA synthesis remaining dramatically inefficient. Interestingly, RT efficiency was enhanced by 38-fold when S₁₄₉A viral particles contained VSV-G. Formation of 2-LTR DNA was next investigated in cells infected with VSV-G-S₁₄₉A or VSV-G-S₁₇₈A viruses (figure 3E). Consistent with infectivity assays, 2-LTR DNA was produced efficiently in cells infected with VSV-G-S₁₄₉A and VSV-G-S₁₇₈A. As expected, 2-LTR circles could not be amplified from cells infected with VSV-G-S₁₀₉A. Altogether, our data indicate that infection through the endocytic pathway restored the ability of S₁₄₉A and S₁₇₈A mutants to produce 2-LTR DNA. Moreover, progression of RT from early to late steps was stimulated upon incorporation of VSV-G for all viruses studied, with a stronger effect observed for the S₁₄₉A mutant. In conclusion, delivering the viral genome through a different route may enhance RT progression or possibly steps that allow nuclear import of the viral genome.

We next examined the capacity of S₁₀₉A, S₁₄₉A and S₁₇₈A mutants to saturate the TRIM family proteins. TRIM proteins expressed in some Old World monkey cells efficiently restrict post-entry steps of HIV-1 replication 910. While the precise mechanisms of this restriction remain unclear, the restrictive properties of TRIM factors are correlated with their capacity to interact with the incoming viral cores 3536. TRIM recognition is sensitive to the maturation of the CA-p2-NC junctions, to the stability of the capsid shell and finally to the organization and folding of exposed surfaces of the assembled core 113637. We thus took advantage of these characteristics to evaluate the impact of S₁₀₉A, S₁₄₉A and S₁₇₈A substitutions on structural properties of the corresponding cores. HIV-1 infection in Cos7 cells is restricted by TRIM5α. This restriction can be saturated in the presence of virus-like particles or elevated amounts of saturating virus that displays properly folded cores with an appropriate stability 38. To evaluate the impact of S₁₀₉A, S₁₄₉A and S₁₇₈A mutations on core integrity, Cos7 cells were infected with increasing amounts of VSV-G pseudotyped WT or S₁₀₉A or S₁₄₉A or S₁₇₈A virions and challenged with a fixed amount (200 ng CA protein defined by anti-CA ELISA assay) of an HIV-1 reporter virus bearing a GFP sequence in place of the nef gene (HIV-1_R7-GFP) 39. This dose was previously ascertained to allow expression of GFP at less than 1% in OMK cells (data not shown). When cells were co-infected with increasing amounts of pseudotyped WT virus, an increasing percentage of GFP-positive cells could be detected by FACS analysis indicating a dose-dependent enhancement of HIV-1_R7-GFP reporter virus replication (Figure 4). When similar experiments were performed saturating Cos7 cells with increasing amounts of S₁₀₉A, S₁₄₉A, S₁₇₈A viruses, GFP expression levels remained extremely reduced, indicating that TRIM5α-mediated restriction of HIV-1_R7-GFP replication could not be overcome by the presence in the infected cells, of cores bearing S₁₀₉A, S₁₄₉A or S₁₇₈A mutations. Similar results were obtained using the OMK cell line that expresses the TRIMCyp restriction factor, which results from a retrotransposition event of the cyclophilin A pseudogene into the TRIM5 locus 1040 (data not shown). In conclusion, CA mutants were thus poorly recognized by TRIM5α in Cos7 cells or TRIMCyp in OMK cells. As TRIM recognition of the incoming viral cores requires the proper intermolecular packing of adjacent CA molecules, our results indicate that substitution of serine residues in CA generates modifications in the capsid shell that render viral cores resistant to the cellular restriction machinery.

The effect of CA mutations on Gag assembly was investigated by analyzing cell free viral particles morphology in electron microscopy experiments. As shown in Figure 5A and 5B, the presence of normal-sized mature virions was observed in preparations of WT, S₁₄₉A and S₁₇₈A particles. In contrast, S₁₀₉A particles displayed a significant decrease in size. No modification of the sphericity index was observed for any mutant tested (data not shown). The presence of unambiguous cone-shaped nucleoids was observed in WT and S₁₇₈A viruses. In contrast, S₁₄₉A particles displayed mild morphological defects characterized by irregularly shaped cores. When S₁₀₉A viruses were examined, no core was observed and viruses contained aggregates that were acentric and presented a lobular aspect.

The Gag expression and processing pattern was further characterized at the level of cell-associated proteins and cell free particles in immunoblotting experiments. When viral lysates of 293T transfected cells were reacted with anti-CA mAbs, expression and processing of the Gag structural precursor were found to be similar for cells expressing WT and mutants viruses (Figure 6A). Analyzing cell free virions normalized according to RTase activity (Figure 6B), the expected ratio of Gag precursor, p41 intermediate and mature CA were observed for S₁₄₉A and S₁₇₈A virions as compared to WT. In contrast, S₁₀₉A virions presented an accumulation of unprocessed Gag and processing intermediates, including p41 and p25. Hence, Gag maturation is compromised by S₁₀₉A mutation in CA. All mutant viruses were found to package processed Pol (p51 and p66) proteins at a normal level relative to WT virions (Figure 6C). Finally, no significant difference in the levels of CypA incorporation was detected from WT and CA mutants (Figure 6D). Altogether these data indicate that the S₁₇₈A mutation does not compromise the ability of CA to assemble in vivo. The S₁₄₉A mutation generates subtle core morphological defects despite efficient maturation of CA. Finally, production of viral particles with normally assembled and mature cores strictly depends upon S₁₀₉ integrity.

Stability of WT and mutant cores was addressed using an in vitro dissociation assay. The corresponding cell-free particles were envelope-stripped by ultracentrifugation through a sucrose cushion overlaid with detergent as described in Materials and Methods. Preparations of WT cores processed for negative staining and electron microscopy revealed recognizable intact, cone-shaped particles with a small fraction of unstructured aggregates and spherical structures, typical of CA monomers (Figure 7A). Cone shaped structures were also predominantly observed from preparations of S₁₇₈A cores and more rarely from S₁₄₉A samples. On the contrary, aberrant aggregates, but no core, were isolated from S₁₀₉A mutants. These results are consistent with data obtained from the biochemical and morphological analyses of entire S₁₀₉A particles. Due to the aberrant morphology of the structures purified, the S₁₀₉A mutant was excluded from subsequent experiments. Preparations of WT, S₁₄₉A and S₁₇₈A cores were then incubated for 2 h at 37°C in conditions that were previously reported to allow core destabilisation 26. Uncoating efficiency was determined after centrifugation of the reaction mixture and a measure of CA concentration in the soluble (monomeric CA) and pellet fractions (assembled cores) was made using an anti-CA ELISA assay. In these experimental conditions, incubation at 37°C efficiently promoted dissociation compared to incubation at 4°C (data not shown). When mutant cores were assayed, no significant variation in uncoating efficiency was observed for S₁₄₉A cores compared to WT (Figure 7B). Similar incubation of S₁₇₈A core particles revealed an increased dissociation rate. Thus alanine substitution of the S₁₇₈ residue in CA decreases the stability of HIV-1 cores.

pNL4.3 and pR7-GFP HIV-1 molecular clones 39, pHEF-VSV-G 53 and SV-A-MLV-env 54 vectors encoding envelope glycoproteins of VSV and MLV, respectively, were obtained through the AIDS Research and Reference Reagent Program, NIAID, NIH. pNL4.3_S109A, pNL4.3_S149A, and pNL4.3_S178A molecular clones have been described elsewhere 27.

Viruses were produced by transfection of 293T cells with HIV-1 molecular clones using the JetPei transfection reagent (QBiogen). Pseudotyping of HIV-1 viruses was achieved by co-transfection with the pHEF-VSV-G or the SV-A-MLV-env plasmid. Two days after transfection, virus-containing supernatants were collected, filtered onto 0.22 μM membranes, aliquoted and stored at -80°C.

MAGIC-5B indicator cells, which stably express the β-galactosidase reporter gene cloned downstream of the HIV-1 LTR promoter, were plated at 8 × 10⁴ cells per well, in 24-well plates and exposed to HIV-1 stock solutions normalized according to RTase activity determined as previously reported 55. Forty-eight hours post-infection, viral infectivity was monitored by quantification of o-nitrophenyl β-D-galactopyranoside hydrolysis from cell lysates as previously described 55. β-galactosidase activity was evaluated by measuring absorbance at 405 nm and was normalized according to total protein content in the cell lysate.

Total DNA was extracted from MAGIC-5B cells (8 × 10⁴) infected for 24 h with normalized amounts (4.5 × 10⁴ cpm RTase activity) of DNAseI-treated virus. The presence of contaminating pNL4.3 plasmid DNA was checked as previously described 27. HIV-1 DNA synthesis was then monitored by qPCR as follows: 100 ng total DNA sample were added to the reaction mix containing 0.4 μM of each primer, and 2 μl SYBR Green master amplification mix (Fast start DNA Master plus SYBR Green I amplification kit, Roche). For each amplification, a control reaction was performed in which DNA sample was replaced by water. Reactions were subjected to a first cycle of 10 min at 95°C followed by 40 amplification cycles of 15 s at 95°C; 15 s at 65°C and 20 s at 72°C on the RotorGene system (Labgene). Fluorescence signal was recorded at the end of each cycle. A standard curve was generated from 10 to 100,000 copies of pNL4.3 plasmid. The copy numbers of HIV-1 DNA were normalized to that of the GAPDH DNA quantified in parallel as an endogenous control. Primers used for amplification were: strong-stop DNA: 5'-AAGCAGTGGGTTCCCTAGTTAG-3' and 5'-GGTCTCTCTGGTTAGACCA-3'; first-strand transfer: 5'-AGCAGCTGCTTTTTGCCTGTACT-3' and 5'-ACACAACAGACGGGCACACAC-3'; full-length minus strand, 5'-CAAGTAGTGTGTGCCCGTCTGTT-3' and 5'-CCTGCGTCGAGAGAGCTCCTCTGG-3' and second-strand transfer 5'-AGCAGCTGCTTTTTGCCTGTACT-3' and 5'-CCTGCGTCGAGAGAGCTCCTCTGG-3'. Amplification of 2-LTR circles was performed as previously reported 56.

Sucrose cushion-purified virus particles (normalized according to exogenous RTase activity to 4.5 × 10⁴ cpm) were incubated for 18 h at 37°C in the presence of 0.2 mM Triton X-100, 10 mM Tris, pH 7.4, 10 mM MgCl₂, and 200 μM of each dNTP in a final volume of 50 μl. A no-nucleotide control reaction mixture was always included. Nucleic acids were then extracted once with phenol:chloroform:isoamyl alcohol (25:24:1) and once with chloroform, and precipitated with ethanol. Purified products were resuspended in 50 μl of water and used (2.5 μl) in qPCR experiment as described above.

5 × 10⁴ OMK or Cos7 cells were plated in 24-well plates the day before infection. The cells were co-infected with VSV-pseudotyped HIV-1 or CA mutant particles and superinfected 3 hours later with an appropriate dose (200 ng CA) of VSV-pseudotyped HIV_R7-GFP reporter virus. Two days after inoculation, cells were detached using trypsin and analyzed for GFP expression using an EPICS PROFILE XL4C cytometer (Beckman Coulter). Values were reported as the percentage of GFP+ cells in the culture. VSV-G-pseudotyped HIV-GFP reporter particles were titrated onto each cell line to determine the appropriate dose for use in the restriction assay.

Virus-producing cells were processed for thin-layer electron microscopy as described elsewhere 55. For negative staining, 20 μl sample core suspensions were applied to Formvar-coated grids (mesh size, 200) and stained with 2% uranyl acetate for 1 min. Preparations were examined with a Hitachi H.7100 transmission electron microscope.

Cell or cell-free virions pelleted by ultracentrifugation (350,000 g, 5 min at 4°C) were solubilized in SDS-PAGE sample buffer and separated on a 12.5% SDS-PAGE. Proteins transferred to PVDF membrane (Millipore) were revealed using a rabbit polyclonal serum directed to RT (kindly provided by J. L. Darlix, ENS, Lyon, France) or CypA (Biomol Research Laboratories Inc.), and mAbs raised against CA (IOT34A clone; Abcam) or actin (C4 clone; MP Biomedicals). Secondary antibodies conjugated to horseradish peroxidase were revealed by enhanced chemiluminescent detection (Pierce Biotechnology, Inc.).

Viral cores were purified by a spin-through technique. Briefly, cell free virions were loaded onto the top of a discontinuous sucrose density gradient composed of 1 ml 50% sucrose at the bottom covered by 1 ml Triton 0.1% in 10% sucrose and centrifuged at 100,000 g in a SW50.1 rotor (Beckman) for 2 h at 4°C. Cores were then resuspended in PBS and stored at -80°C. In vitro uncoating was assayed by incubating purified cores diluted in 10 mM Tris HCl pH 7.4, 100 mM NaCl, 1 mM EDTA for 2 h at 4°C or at 37°C. After ultracentrifugation for 20 min at 13,800 g, 4°C, the extent of core dissociation was calculated by the ratio of CA detected in the supernatant (oligomeric CA) to that present in pellets (assembled cores) by anti-CA ELISA assay.