To address the mechanism of polarized MLV assembly toward Env/receptor-induced synapse, we applied a visual assay that could overcome two limitations of the classic approach by measuring spreading of viral infectivity. First, as already discussed, the C-tail of Env modulates the fusogenicity of Env causing cell-cell fusion. The visual approach allowed us to separate the ability of various Env mutants in directing viral assembly to cell-cell contact sites from their ability to mediate cell-cell fusion. Second, it also allowed us to study polarization of assembly and budding-incompetent Gag variants. Using this visual assay, we identified a tyrosine residue within the C-tail of Env and MA in Gag as the viral determinants required for polarized assembly. Recruitment of Gag was dependent on MA and the C-tail of Env. Myristoylation at the N terminus of MA that mediates membrane targeting was also required. In contrast, Gag lacking CA and NC were still efficiently recruited to contact sites. As such, targeting of Gag to sites of cell-cell contacts likely occurs at the cytoplasmic leaflet of the plasma membrane. Local concentration of Gag likely allows the nucleation of assembly explaining how assembly is polarized to contact sites. The absence of a role for NC, the main genome binding domain of Gag, likely excludes a possible role of viral genome in the sorting of Gag.
Various previous reports point to a role of tyrosine motifs in the cell-to-cell spread and in the coupling of Env and Gag trafficking toward the basolateral side in polarized epithelial cells (8
). In vivo
studies indicate that tyrosine mutations affect MLV latency and pathogenesis (8
). In the case of MLV, the involvement of a tyrosine motif has been assumed to point to a role in endocytic trafficking despite the fact that overall surface levels did not change, as also observed by us (8
). Indeed, our detailed analysis did not confirm a role for the classic YxxØ motif in polarized assembly in cell-cell contact condition. We also tested the possible involvement of tyrosine phosphorylation by substituting Y with F and found that this Env mutant behaved like the wild type. As such, our analysis identifies a tyrosine residue within Env C-tail that is required for polarized assembly. Our characterization of the viral determinants for polarized assembly will assist the isolation of cellular factors that directly interact with the tyrosine motif and/or are required for a C-tail and matrix-dependent recruitment of Gag to the MLV synapse.
The molecular mechanism by which Gag is recruited to Env/receptor complexes at the cell-cell interface remains unknown. The best evidence for an interaction between the C-tail of Env and MA has been presented for HIV (7
). Coupling between the trafficking of Env and Gag has also been observed for MLV (42
). The inability of myristoylation defective MLV Gag to polarize indicates that if a direct interaction between the C-tail of Env and MA plays a role, it would depend on prior membrane association. The establishment of an Env/receptor-dependent synapse precedes the recruitment of Gag (16
). At this point, we favor a model by which the C-tail of Env first modulates the cell-cell interface to generate an adhesive milieu that is required for polarized assembly. This view is supported by the correlation between the residues identified in our study as being responsible for polarity and residues that affect the cell-cell fusion activity of MLV Env (2
; J. Jin and W. Mothes, unpublished data). The generation of a stable and long-lived cell-cell interface that prevents cell-cell fusion likely precedes Gag recruitment. The molecular mechanism of MA targeting to cell-cell interface may involve a subsequent direct or indirect interaction between the C-tail and MA. The hypothesized dual function of the C-tail of Env in the modulation of adhesive interfaces and the recruitment of MA would explain why the determinants identified here are distinct from the requirements for Env incorporation into virions (28
; Marc Johnson, unpublished data). It is also worth mentioning that the adhesion-induced polarity described here is distinct from the polarity observed in primary migrating T cells, in which HIV Gag is polarized to the uropod by an Env-independent mechanism (24
By comparing Gag assembly of two different retroviruses, HIV and MLV, we documented two potential modes of virus cell-to-cell transmission. After establishment of MLV Env-receptor adhesion interface, MLV Gag can be recruited to the contact sites prior to assembly. This process depends on MLV MA-mediated membrane targeting and/or MA-Env C-tail interaction. Presumably, this recruitment is along the inner leaflet of the plasma membrane. In contrast, HIV Gag assembles along the cell surface outside of the synapse and is then recruited to MLV synapse by outer leaflet flow. These two modes of adhesion-induced transmission can both contribute to efficient virus cell-to-cell transmission. The here observed behavior of HIV Gag in the context of an MLV Env/receptor synapse is consistent with the observation of lateral movement of HIV to the virological synapse in T cells (13
). The ability of HIV to utilize surface movement may also explain independence from the cytoplasmic tail of the envelope, although a dependence has also been reported (6
). Further work will be required to understand how the virological synapse can generate flow along the membrane to promote virus cell-to-cell transmission.