Nucleocapsid Promotes Localization of HIV-1 Gag to Uropods That Participate in Virological Synapses between T Cells

Abstract

T cells adopt a polarized morphology in lymphoid organs, where cell-to-cell transmission of HIV-1 is likely frequent. However, despite the importance of understanding virus spread in vivo, little is known about the HIV-1 life cycle, particularly its late phase, in polarized T cells. Polarized T cells form two ends, the leading edge at the front and a protrusion called a uropod at the rear. Using multiple uropod markers, we observed that HIV-1 Gag localizes to the uropod in polarized T cells. Infected T cells formed contacts with uninfected target T cells preferentially via HIV-1 Gag-containing uropods compared to leading edges that lack plasma-membrane-associated Gag. Cell contacts enriched in Gag and CD4, which define the virological synapse (VS), are also enriched in uropod markers. These results indicate that Gag-laden uropods participate in the formation and/or structure of the VS, which likely plays a key role in cell-to-cell transmission of HIV-1. Consistent with this notion, a myosin light chain kinase inhibitor, which disrupts uropods, reduced virus particle transfer from infected T cells to target T cells. Mechanistically, we observed that Gag copatches with antibody-crosslinked uropod markers even in non-polarized cells, suggesting an association of Gag with uropod-specific microdomains that carry Gag to uropods. Finally, we determined that localization of Gag to the uropod depends on higher-order clustering driven by its NC domain. Taken together, these results support a model in which NC-dependent Gag accumulation to uropods establishes a preformed platform that later constitutes T-cell-T-cell contacts at which HIV-1 virus transfer occurs.

Viral Determinants of Polarized Assembly for the Murine Leukemia Virus

“…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determinants identified here are distinct from the requirements for Env incorporation into virions (28; Marc Johnson, unpublished data).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).…”

Rho’ing in and out of cells

“…187 At the same time, the nucleocapsid protein of HIV-1 promotes localization of virus structural proteins to uropods of polarized T cells, which may contribute to virus transfer via T-cell-T-cell contacts through so-called virological synapses, a process that depends on cytoskeletal remodeling. 189,190 Nef-PAK2 also affects T lymphocyte migration, as it leads to inactivation of cofilin, a downstream effector of PAK signaling, which results in reduced chemoattractant-induced T cell motility. 168,176 Importantly, the Nef-PAK2 interaction also interferes with T-lymphocyte circulation in vivo.…”

Basic Motifs Target PSGL-1, CD43, and CD44 to Plasma Membrane Sites Where HIV-1 Assembles

“…Indeed, previous studies suggested that most membrane proteins are incorporated into retrovirus particles passively, i.e., with no enrichment relative to the plasma membrane (28). During assembly, Gag has been observed to associate with plasma membrane microdomains, such as lipid rafts (29,30) and tetraspanin-enriched microdomains (TEMs) (31)(32)(33)(34)(35)(36), and reorganize these domains in ways that do not occur in uninfected cells (37)(38)(39). Therefore, at least a subset of membrane proteins (e.g., glycosylphosphatidylinositol-anchored proteins and tetraspanins) may be incorporated into virions actively (as opposed to by passive incorporation, which does not involve enrichment of membrane proteins at assembly sites) without specific interactions, other than association with the plasma membrane microdomains.…”