HIV-1 Gag, Envelope, and Extracellular Determinants Cooperate To Regulate the Stability and Turnover of Virological Synapses

Abstract

Retroviruses spread more efficiently when infected and uninfected cells form tight, physical interfaces known as virological synapses (VSs). VS formation is initiated by adhesive interactions between viral Envelope (Env) glycoproteins on the infected cell and CD4 receptor molecules on the uninfected cell. How high-avidity Env-CD4 linkages are resolved over time is unknown. We describe here a tractable two-color, long-term (>24 h) live cell imaging strategy to study VS turnover in the context of a large cell population, quantitatively. We show that Env's conserved cytoplasmic tail (CT) can potently signal the recruitment of Gag capsid proteins to the VS, a process also dependent on residues within Gag's N-terminal matrix (MA) domain. Additionally, we demonstrate that Env's CT and Gag's MA domain both regulate the duration of interactions between viral donor and target cells, as well as the stability of this interaction over time (i.e., its capacity to resolve or form a syncytium). Finally, we report the unexpected finding that modulating extracellular fluid viscosity markedly impacts target T cell trafficking and thus affects the duration, stability, and turnover of virus-induced cell-cell contacts. Combined, these results suggest a stepwise model for viral cell-tocell transmission wherein (i) Env-receptor interactions anchor target cells to infected cells, (ii) Env signals Gag's recruitment to the cell-cell contact dependent on an intact Env CT and Gag MA, and (iii) Env CT and Gag MA, in conjunction with extracellular forces, combine to regulate VS stability and infectious outcomes. IMPORTANCE HIV-1 spreads efficiently at physical, cell-cell interfaces known as virological synapses (VSs). The VS provides for spatiotemporal coupling of virus assembly and entry into new host cells and may transmit signals relevant to pathogenesis. Disrupting this mode of transmission may be critical to the goal of abolishing viral persistence in infected individuals.We describe here a long-term live cell imaging strategy for studying virus-induced effects on cell behavior in the context of a large cell population. We demonstrate cooperative roles for viral Gag capsid proteins and Envelope glycoproteins in regulating VS formation and turnover. We also show that modulating fluid viscosity markedly affects T cell trafficking and VS stability. Thus, extracellular factors also play an important role in modulating the nature of infectious cell-cell interactions. In sum, our study provides new tools and insights relevant to exposing vulnerabilities in how HIV-1 and other viruses spread infection among cells, tissues, and people. R etroviruses encode transmembrane Envelope (Env) glycoproteins that regulate virion-receptor binding and mediate the fusion of viral and cellular membranes necessary to deliver viral capsids to the cytoplasm of uninfected target cells (1-4). Human immunodeficiency virus type 1 (HIV-1) Env is translated as a 160-kDa polyprotein (also known as gp160) that self-interacts and is cleaved in the secretory path...

Distinct functions for the membrane-proximal ectodomain region (MPER) of HIV-1 gp41 in cell-free and cell–cell viral transmission and cell–cell fusion

“…The cytoplasmic tail (CT) of gp120 -gp41 plays a key role in signaling the recruitment of Gag to the VS, a process that depends on residues within the matrix protein (MA) domain. The functional interaction between the CT and MA regulates the duration and stability of interactions between infected and uninfected target cells (13). Cell-associated virus can be transferred directly from an infected cell to an uninfected cell via the VS, and this cell-tocell mode of viral spread has been observed for CD4 ϩ T cell-CD4 ϩ T cell (7,14), dendritic cell-CD4 ϩ T cell (15,16), and monocyte-derived macrophage (MDM)-CD4 ϩ T cell (17) conjugates.…”

Where in the Cell Are You? Probing HIV-1 Host Interactions through Advanced Imaging Techniques

“…More recently the importance of Gag in synapse formation was also demonstrated by Gardiner et al using live cell imaging of co-cultured infected COS-7 cells with uninfected Jurkat cells, illustrating that Env recruits large amounts of Gag to virological synapses, modulating synapse duration and stability [29]. However, further elucidation and clarification surrounding Gag and Env involvement in synapse formation is still required [29]. …”

“…Scanning electron microscopy of the co-culture further illustrated ‘buttons’ at the cell surface containing oligomerized Gag protein which was eventually sequestered to form virus-laden internal compartments in target cells, suggesting transfer may be coupled with cellular endocytosis [28]. More recently the importance of Gag in synapse formation was also demonstrated by Gardiner et al using live cell imaging of co-cultured infected COS-7 cells with uninfected Jurkat cells, illustrating that Env recruits large amounts of Gag to virological synapses, modulating synapse duration and stability [29]. However, further elucidation and clarification surrounding Gag and Env involvement in synapse formation is still required [29].…”

Antigen structure affects cellular routing through DC-SIGN

“…We next compared the routing of glycopolymers and fluorescently labeled HIV-1 virus like particles (VLPs) by analyzing their localization with CD81. VLPs resemble native virions and thus can be used to model viral uptake and trafficking from cell-to-cell at viral synapses (38)(39)(40). To circumvent potential effects of CD81-mCherry expression on polymer or HIV-1 uptake and trafficking, we examined antigen colocalization with endogenous CD81 in moDCs and Raji/DC-SIGN cells.…”