Virus Particle Release from Glycosphingolipid-Enriched Microdomains Is Essential for Dendritic Cell-Mediated Capture and Transfer of HIV-1 and Henipavirus

Abstract

Human immunodeficiency virus type 1 (HIV-1) exploits dendritic cells (DCs) to promote its transmission to T cells. We recently lentivirus particles by MDCs were severely attenuated upon depletion of GSLs from virus particles. These results suggest that GSL incorporation into virions is critical for the interaction of diverse enveloped RNA viruses with DCs and that the GSL-CD169 recognition nexus might be a conserved viral mechanism of parasitization of DC functions for systemic virus dissemination. IMPORTANCEDendritic cells (DCs) can capture HIV-1 particles and transfer captured virus particles to T cells without establishing productive infection in DCs, a mechanism of HIV-1 trans infection. We have recently identified CD169-mediated recognition of GM3, a host-derived glycosphingolipid (GSL) incorporated into the virus particle membrane, as the receptor and ligand for the DC-HIV trans infection pathway. In this study, we have identified the matrix (MA) domain of Gag to be the viral determinant that governs incorporation of GM3 into HIV-1 particles, a previously unappreciated function of the HIV-1 MA. In addition, we demonstrate that the GSL-CD169-dependent trans infection pathway is also utilized as a dissemination mechanism by henipaviruses. GSL incorporation in henipaviruses was also dependent on the viral capsid (M) protein-directed assembly and budding from GSL-enriched lipid microdomains. These findings provide evidence of a conserved mechanism of retrovirus and henipavirus parasitization of cell-to-cell recognition pathways for systemic virus dissemination.

Access of HIV-2 to CD169-dependent dendritic cell-mediated trans infection pathway is attenuated

“…HIV-1 Gag defines the virus particle assembly site and hence determines specificity of incorporation of host-determinants such as GSLs (Akiyama et al, 2014). To determine if HIV-1 and HIV-2 assemble at divergent membrane locations, HEK293T cells were co-transfected with HIV-1 Gag-mCherry and HIV-1 or HIV-2 Gag-eGFP expression plasmids and processed for confocal microscopy.…”

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

“…Viruscontaining cell supernatants were harvested at 2 days posttransfection, passed through 0.45-m filters, and stored at Ϫ80°C until further use. For some experiments, virus particles were concentrated by ultracentrifugation on a 20% sucrose cushion (24,000 rpm at 4°C for 2 h with an SW28 rotor [Beckman Coulter]) (116). The virus pellets were resuspended in phosphate-buffered saline (PBS), aliquoted, and stored at Ϫ80°C until use.…”

“…Quantitative Western blotting. To detect Gag and Env in cell and virus particle lysates, cell lysates (normalized to equivalent amounts of cell-associated Gag) or 100 ng p24 Gag virus equivalents (as determined by a quantitative ELISA) was run through SDS-PAGE gels and transferred onto nitrocellulose membranes by using a semidry transfer apparatus, as previously described (116). Blots were blocked and probed with rabbit anti-gp120 (a gift from Nancy Haigwood) and mouse anti-p24 Gag (clone p24-2; NIH AIDS Research and Reference Reagent Program), followed by goat anti-mouse IgG DyLight 680 (Pierce) and goat anti-rabbit IgG DyLight 800 (Pierce).…”

A Mechanistic Overview of Dendritic Cell-Mediated HIV-1 Trans Infection: The Story So Far

“…Choice of plasma membrane microdomains preferentially accessed for virus assembly is governed by sequences in the matrix (MA) domain of HIV-1 Gag. Hence, mutations in the N-terminal basic region or deletion of the membrane-targeting domain of HIV-1 matrix (MA) that alters virus assembly site to intracellular membranes result in reduced GM3 incorporation in MA-deficient viruses and diminished virus capture by mature moDCs [90]. Interestingly, diverse enveloped viruses including Nipah and Hendra hemorrhagic fever viruses (paramyxoviruses), and murine leukemia virus (MLV; a retrovirus), incorporate sialylated GSLs in the virus particle membranes and are captured by mature moDCs [69,90–91], suggesting that GSL-dependent interactions with mature moDCs might be a conserved mechanism of DC-mediated enveloped virus dissemination [90].…”

“…Importantly, mature moDC-mediated trans infection to CD4 + T cells was attenuated only when capture of HIV-1 by CD169 was blocked by usage of either shRNAs to knock down CD169 expression or when CD169 function was blocked by antibodies [69]. Furthermore, reduction in GM3 content of HIV-1 particles by targeting virus assembly to GM3-deficient intracellular membranes [90] or by pharmacologic manipulation of GSL biosynthesis in virus producer cells [69,70] resulted in reduced virus capture by CD169 and reduced access to CD169-mediated trans infection pathway. These findings suggest that GM3 (GSL)-dependent recognition of HIV-1 particles by CD169 is the critical interaction that drives mature DC-mediated dissemination of HIV-1 infection to CD4 + T cells (see Figure 1 ).…”