HIV-1 Vpu Promotes Release and Prevents Endocytosis of Nascent Retrovirus Particles from the Plasma Membrane

The human immunodeficiency virus (HIV) type-1 viral protein U (Vpu) protein enhances the release of diverse retroviruses from human, but not monkey, cells and is thought to do so by ablating a dominant restriction to particle release. Here, we determined how Vpu expression affects the subcellular distribution of HIV-1 and murine leukemia virus (MLV) Gag proteins in human cells where Vpu is, or is not, required for efficient particle release. In HeLa cells, where Vpu enhances HIV-1 and MLV release approximately 10-fold, concentrations of HIV-1 Gag and MLV Gag fused to cyan fluorescent protein (CFP) were initially detected at the plasma membrane, but then accumulated over time in early and late endosomes. Endosomal accumulation of Gag-CFP was prevented by Vpu expression and, importantly, inhibition of plasma membrane to early endosome transport by dominant negative mutants of Rab5a, dynamin, and EPS-15. Additionally, accumulation of both HIV and MLV Gag in endosomes required a functional late-budding domain. In human HOS cells, where HIV-1 and MLV release was efficient even in the absence of Vpu, Gag proteins were localized predominantly at the plasma membrane, irrespective of Vpu expression or manipulation of endocytic transport. While these data indicated that Vpu inhibits nascent virion endocytosis, Vpu did not affect transferrin endocytosis. Moreover, inhibition of endocytosis did not restore Vpu-defective HIV-1 release in HeLa cells, but instead resulted in accumulation of mature virions that could be released from the cell surface by protease treatment. Thus, these findings suggest that a specific activity that is present in HeLa cells, but not in HOS cells, and is counteracted by Vpu, traps assembled retrovirus particles at the cell surface. This entrapment leads to subsequent endocytosis by a Rab5a- and clathrin-dependent mechanism and intracellular sequestration of virions in endosomes

Plasma Membrane Is the Site of Productive HIV-1 Particle Assembly

Recently proposed models that have gained wide acceptance posit that HIV-1 virion morphogenesis is initiated by targeting the major structural protein (Gag) to late endosomal membranes. Thereafter, late endosome-based secretory pathways are thought to deliver Gag or assembled virions to the plasma membrane (PM) and extracellular milieu. We present several findings that are inconsistent with this model. Specifically, we demonstrate that HIV-1 Gag is delivered to the PM, and virions are efficiently released into the extracellular medium, when late endosome motility is abolished. Furthermore, we show that HIV-1 virions are efficiently released when assembly is rationally targeted to the PM, but not when targeted to late endosomes. Recently synthesized Gag first accumulates and assembles at the PM, but a proportion is subsequently internalized via endocytosis or phagocytosis, thus accounting for observations of endosomal localization. We conclude that HIV-1 assembly is initiated and completed at the PM, and not at endosomal membranes

African swine fever virus : transport perinuclear assembly sites to the plasma membrane

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

African Swine Fever Virus: Transport From Perinuclear Assembly Sites to The Plasma Membrane.

African swine fever virus (ASFV) is a large DNA virus that assembles in viral factories located close to the microtubule organising centre. This study has investigated the mechanisms by which ASFV reaches the cell surface from these perinuclear assembly sites. The first step in this transport is dependent on intact microtubules and involves the motor protein conventional kinesin. This plus-end motor is recruited to virus factories and to individual cytoplasmic virions. Consistent with a role for conventional kinesin during ASFV egress, over-expression of the cargo-binding domain of the kinesin light chain, which acts as a dominant negative protein, severely inhibits movement of viruses to the plasma membrane. Preliminary binding studies show that p73, the main capsid protein, may bind the cargo binding domain of kinesin light chain in vitro. p73 may therefore recruit conventional kinesin to ASFV particles. Once delivered to the periphery of the cell, virions were found associated with microfilaments, possibly using cortical actin fibres as tracks. Once at the plasma membrane, particles induce the formation of long actin projections that morphologically and structurally resemble filopodia. A second generation of particles was capable of moving within these filopodia-like structures in a bidirectional manner. Altogether, these data suggest that ASFV transport from assembly sites to the plasma membrane involves at least four mechanisms: microtubule-based motility, transport along cortical actin filaments, actin polymerisation from the plasma membrane and bi-directional movement along pre-existing projections

African Swine Fever Virus: Transport From Perinuclear Assembly Sites to The Plasma Membrane.

African swine fever virus (ASFV) is a large DNA virus that assembles in viral factories located close to the microtubule organising centre. This study has investigated the mechanisms by which ASFV reaches the cell surface from these perinuclear assembly sites. The first step in this transport is dependent on intact microtubules and involves the motor protein conventional kinesin. This plus-end motor is recruited to virus factories and to individual cytoplasmic virions. Consistent with a role for conventional kinesin during ASFV egress, over-expression of the cargo-binding domain of the kinesin light chain, which acts as a dominant negative protein, severely inhibits movement of viruses to the plasma membrane. Preliminary binding studies show that p73, the main capsid protein, may bind the cargo binding domain of kinesin light chain in vitro. p73 may therefore recruit conventional kinesin to ASFV particles. Once delivered to the periphery of the cell, virions were found associated with microfilaments, possibly using cortical actin fibres as tracks. Once at the plasma membrane, particles induce the formation of long actin projections that morphologically and structurally resemble filopodia. A second generation of particles was capable of moving within these filopodia-like structures in a bidirectional manner. Altogether, these data suggest that ASFV transport from assembly sites to the plasma membrane involves at least four mechanisms: microtubule-based motility, transport along cortical actin filaments, actin polymerisation from the plasma membrane and bi-directional movement along pre-existing projections

Stimulation of Innate Immunity by Host and Viral RNAs

International audienceThe interferon (IFN) response, a major vertebrate defense mechanism against viral infections, is initiated by RIG-I-like receptor (RLR)-mediated recognition of viral replicative intermediates in the cytosol. RLR purification methods coupled to RNA sequencing have recently led to the characterization of viral nucleic acid features recognized by RLRs in infected cells. This work revealed that some cellular RNAs can bind to RLRs and stimulate the IFN response. We provide an overview of self and non-self RNAs that activate innate immunity, and discuss the cellular dysregulation that allows recognition of cellular RNAs by RLRs, including RNA mislocalization and downregulation of RNA-shielding proteins. These discussions are relevant because manipulating RLR activation presents opportunities for treating viral infections and autoimmune disorders

Clash of the titans: interferons and SARS-CoV-2

International audienceInterferons are our first line of defense against invading viruses. However, viruses encode effector proteins that can modulate human interferon responses. In this forum article, we highlight important discoveries and discuss outstanding questions that will enable us to better understand the nuances of this evolutionary battle between interferons and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)

Editorial: Balanced and Unbalanced Immune Response to Dengue Virus in Disease Protection and Pathogenesis

International audienceThe ongoing COVID-19 pandemic has highlighted a central principle of the host immune response to RNA virus infections: while most infected patients remain asymptomatic or mild symptomatic, a portion of patients experience severe clinical symptoms, suggesting the existence of host immune factors that determine susceptibility to symptomatic disease..