To determine whether cell-to-cell transmission of HIV requires the coordinated assembly of Gag and Env to form infectious virus particles, we prevented the incorporation of Env into virions while preserving particle release and Env fusogenicity. To this end, we inserted into the N-terminal one-third of the MA protein mutations that were previously shown to have a dramatic effect on the incorporation of Env into budding particles (18
). Env incorporation was restored by truncation of the Env CT, leading for some mutants to the production of infectious extracellular virus particles (38
). We thus compared viral clones, carrying different combinations of MA and Env alleles, for their competence at transmission following the cell-free and cell-to-cell transmission routes. Several observations from the data described here argue for a common transmission mechanism for the cell-free and cell-to-cell HIV transmission pathways, both of which rely on the production of infectious virus particles.
The first line of evidence supporting a common mechanism is that despite the difference in the magnitude of infection in the two sets of assays, only the combinations of MA and Env that produced infectious extracellular virions were also competent for cell-to-cell transmission. This finding strongly suggests that the formation of replication-competent cell-free virions is a common requirement for the two means of transmission and argues against qualitatively different mechanisms. In a previous study, analyzing the coevolution of MA and Env in an HIV-infected patient, we identified one combination of MA and Env alleles characterized by strongly impaired Env incorporation into particles (2
). As a consequence, the single-cycle infectivity of cell-free virions was dramatically decreased (although not abolished), while virus propagation in culture proceeded through cell-to-cell transmission with a limited delay (2
). In the present study, complete abrogation of cell-free virus infectivity by MA mutations prevented the potential compensation by the concentration of virus production at cell-cell contact sites and revealed the tight requirement for the assembly of infectious virions also for cell-to-cell HIV transmission.
In addition, among clones carrying truncated Env, the levels of infectivity of MA mutants, compared to that of the wild-type matrix clone, were conserved between cell-free and cell-to-cell transmission assays. MA-A displayed 15 to 30% reductions in infectivity in both experimental systems, while the residual infectivity of MA-B ranged within 30% of that of the reference strain, and those of mutants MA-C and MA-D were clearly lower ( and ). The similarity of the values obtained in the two assays, despite their obvious experimental differences, suggests that the key determinants of infectivity are common. Of note, the incomplete restoration of MA mutant infectivity consequent to Env truncation shows that the targeted MA residues are also implicated in additional early steps of HIV replication. Indeed, MA mutants incorporated higher levels of truncated Env than did the wild-type MA clone (). Accordingly, MA mutants were at least as effective at releasing virus cores into the cytoplasm of target cells as the virus with wild-type MA (C). Despite this enhanced entry process, MA mutants displayed impaired completion of early steps of virus replication (). Previous studies have proposed a role for MA in HIV nuclear import and integration and, more recently, in viral uncoating, RNA incorporation, and cytoskeleton-mediated transport (reviewed in reference 26
); some of these roles could account for the impaired replication.
Finally, the observation that MA mutations blocked HIV cell-to-cell transmission by preventing the penetration of viral cores into the cytosol also supports our conclusion that HIV cell-to-cell transmission relies on the production of infectious virus particles. Importantly, such dramatically reduced (20- to 45-fold [B]) virus core translocation due to MA mutations was observed despite efficient Env-dependent cell-cell fusion (). Thus, under conditions allowing the exchange of cytosolic material, we did not observe productive cell-to-cell transmission. Completion of this process required truncation of the Env CT, which allows the formation of cell-free infectious virus particles. Consistently, MA-mutated cell-free virions delivered virus cores into the cytoplasm of target cells only upon truncation of the Env CT (B).
Given the clear phenotypes of the combinations of MA and Env studied here, the experimental approaches we used fulfilled our expectations despite a few potential limitations. It is possible that MA mutations may have perturbed the intracellular trafficking of the Gag precursor. We show, however, that the availability of a complementary (CT-truncated) Env resulted in the colocalization of Gag and Env at budding sites and at cell-cell contact sites and gave rise to particles competent for fusion with target cells. Such colocalization is likely passive, since MA-mutated Gag precursors are not supposed to establish functional interactions with the truncated Env. In contrast, in the presence of full-length Env, mutated Gag molecules could suffer from steric hindrance with the Env CT, resulting in limited access to the cell-cell contact sites where membrane fusion was taking place. Although this phenomenon may participate in reducing the efficiency of cell-to-cell transmission, it is unlikely to determine an absolute exclusion of Gag or to explain the complete loss of virus transmission for the four different mutant viruses, in view of the very dynamic and active transport processes taking place at cell-cell contact sites. Indeed, while expression of HIV Env increases the number and stability of cell-cell contacts (50
), their formation and the accumulation of viral and cellular proteins also take place in the absence of Env in the cell types used here (20
Another issue concerned the significant reduction in infectivity due to the truncation of Env CT in the presence of a WT MA, which limited the comparisons between viruses carrying full-length versus truncated Env. However, since MA mutations fully abrogated virus infectivity when expressed with full-length Env in both cell-free and cell-to-cell systems, our analysis did not suffer from this setback. In addition, the relatively low infectivity of DCT viruses did not prevent the establishment of a clear hierarchy for the replication competences of the different MA mutants. Finally, the use of a truncated Env to complement MA mutations limited the range of cell types to those permissive for the DCT Env variant of HIV: the MT4 T-cell line and few others (1
). Although the reasons for this constrained host cell range are not completely understood, a recent report strongly suggests that this may be due to the preferential spread of this variant via cell-to-cell transmission (20
). Importantly, however, we confirmed that the extents of colocalization of MA and Env, as well as the efficiencies of Env incorporation into virus particles, were similar in MT4 cells and the unrelated HeLa cell line. Thus, the key parameters implicated in the processes studied here do not display a cell type specificity that would limit the value of our observations.
Taken together, the data presented here show that cell-to-cell transmission of HIV requires the assembly of enveloped virus particles. Thus, the increased efficiency of this infection route is likely to result from the high local concentrations of virus particles at sites of cellular contacts rather than from a qualitatively different transmission process. The active recruitment of HIV proteins at intercellular contacts relies on the regulated secretory pathway of infected cells (30
). Virus transmission from cell to cell may overcome some conditions that are restrictive for cell-free virus infection. For instance, we have reported that this means of virus propagation limits the effect of type I interferon on HIV spread in culture (58
) and that it allows the replication of viruses carrying mutations in Env that have a strong impact on the infectivity of cell-free HIV (2
). In the confined space of the virological synapses, viruses are relatively protected from the environment and find elevated receptor concentrations, favoring virus entry. Accordingly, this means of virus transmission allows the simultaneous infection of cells by multiple virions (15
). The penetration of multiple virus particles into the same area can facilitate infection by saturating some cellular restriction factors that would otherwise intercept incoming virus particles (49
) and may reduce the effectiveness of some antiretroviral therapy (55
). Further clarification of the mechanism of cell-to-cell HIV transmission may help explain some unclear aspects of HIV biology and suggest dedicated approaches to limiting virus spread.