Abstract
While the cell imposes multiple barriers to virus entry, enveloped viruses are remarkably still able to gain entry to their cellular hosts by hitchhiking and remodeling the endomembrane system to traffic within, and eventually escape from, endosomal organelles for their genome release. Elucidating viral entry mechanisms and their interaction with the host trafficking network is necessary for antiviral therapy. Here, we focus on the use of host autophagy molecular factors during the entry of prototypic negative-stranded RNA viruses, and highlight recent progress in our understanding of the role of one such factor, UVRAG, in both viral and cellular endocytic membrane trafficking and fusion events.
Endocytic and autophagic trafficking are 2 tightly regulated processes evolved for the transport and clearance of extracellular and intracellular cargoes, respectively, and which are both implicated in viral infection and pathogenesis. Most enveloped viruses exploit the endosomal machinery to route themselves to specific compartments for membrane fusion and delivery of their genetic material, while autophagy acts as an essential part of the host antiviral defense mechanism. UVRAG (UV radiation resistance-associated) is well known for its crucial role in both pathways: activating autophagy by pairing with BECN1 and accelerating late endocytic transport by pairing with the class C Vps (hereafter referred to as C/Vps) complex. Yet, despite its importance, the functionality of UVRAG in viral infection has not been addressed. Our findings suggest that overexpression of UVRAG confers efficient infection of vesicular stomatitis virus (VSV) and influenza A virus (IAV), both negative-strand RNA viruses; in contrast, silencing of UVRAG results in a substantial reduction of viral replication. This proviral activity of UVRAG is counterintuitive and hard to reconcile with the role of UVRAG in autophagy activation that is largely antiviral. In fact, even in autophagy-deficient Atg5 null cells, removal of UVRAG renders cells less susceptible to VSV infection, suggesting that an alternative mechanism beyond autophagy may be associated with UVRAG. In line with this, expression of UVRAG does not affect overall type I-interferon (IFN) production, and UVRAG-induced viral infection is not due to an altered IFN response––the first line of defense against infection. On the basis of these data, we argue that UVRAG may directly regulate the viral life cycle.
To determine the step(s) in the replication cycle that are regulated by UVRAG, we tracked the movement of VSV labeled with self-quenching dye in living cells, and found that UVRAG does not affect the initial uptake of the virus into cells, nor does it alter the route of delivery of the endocytosed viral particles to early endosomes. However, knockdown of UVRAG results in a significant delay in viral access to late endosomes and in virus-endosome fusion for cytoplasmic delivery of nucleocapsids. Encouraged by these results, we conducted a single-cycle entry assay using a Moloney murine leukemia virus (MLV)-based pseudo-retroviral system carrying different viral envelopes as entry factors, and found that knockdown of UVRAG considerably inhibited infection mediated by the glycoprotein of VSV; it also blocked the entry of multiple highly pathogenic strains of IAVs. In contrast, suppression of UVRAG did not prevent arenavirus infection that enters cells via a different endocytic pathway. Our data suggest a functional specificity and essential role of UVRAG during virus entry.
How might UVRAG regulate the virus entry process? UVRAG appears to have close association with the endosome machinery. In addition to C/Vps, interactions between UVRAG and late endosomal SNARE proteins have been detected in our study. The region of UVRAG that is responsible for this interaction is mapped to the coiled-coil domain (CCD) in UVRAG, the region that had previously been shown to confer BECN1-binding and autophagy activation. Although BECN1 has been implicated in the antiviral response and mutations in BECN1 are associated with pathogenesis, its function in virus entry is unclear. In fact, our findings suggest that BECN1 is a minor participant in UVRAG-mediated viral entry. Genetic deletion or siRNA-mediated depletion of BECN1 has minimal effect on viral infection, and it does not affect UVRAG-SNAREs interactions. Instead, our observations strongly support an important role for the UVRAG-C/Vps-SNAREs complex in promoting virus entry. First, deletion of the C2 or CCD domain of UVRAG that disrupts the interaction of UVRAG with C/Vps and SNAREs, respectively, severely impairs the ability of UVRAG to promote cells’ susceptibility to VSV and IAV infection. Second, the siRNA-mediated depletion of the individual C/Vps complex subunits and SNARE proteins potently suppresses virus infection. Finally, the overexpression of wild-type UVRAG, but not the C/Vps- or SNAREs-binding defective UVRAG mutant, promotes the assembly of fusogenic trans-SNAREs at endosomes, a decisive step in driving membrane fusion.
The formation of fusogenic trans-SNARE complexes confers specificity to the membrane fusion process in cells. We observed that the UVRAG-associated C/Vps is able to mediate the pairing of Q-SNAREs, including STX7 (syntaxin 7), STX8 (syntaxin 8), and VTI1B, with both the late endosome-related R-SNARE VAMP8 and the lysosome-related R-SNARE VAMP7 in normal conditions. However, an altered interaction pattern of endosomal SNAREs is observed shortly after viral infection. Concomitant with a robust increase in the pairing of Q-SNAREs with VAMP8, we observed a significant decrease in their pairing with VAMP7. In support of this, suppression of VAMP7 that is required for lysosome-related membrane fusion, does not inhibit virus entry; rather, it results in a slight increase in viral infection. Moreover, unlike VAMP8 that is recruited to the virus-carrying vacuoles, VAMP7 is largely excluded, and this process requires UVRAG and its interaction with C/Vps. These findings suggest that UVRAG-mediated SNARE assembly is not a random event hitchhiked by the virus; instead, it may represent a mechanism of viral evasion of lysosome fusion that is designed for pathogen destruction and immune detection.
In summary, our study clearly reveals a previously unknown function of UVRAG in regulating virus entry through multiple interactions with the membrane fusion machinery of cells, independent of autophagy. To the best of our knowledge, this is the first time the cellular outcome of UVRAG in viral infection has been investigated, which also poses interesting questions. First, what is the molecular mechanism that allows the virus to interact with and remodel host cell fusion machinery? Are there any viral entry factors also involved in this process? Furthermore, are different endocytic cargoes (viral and nonviral) differentially regulated by UVRAG? How does UVRAG coordinate its distinct membrane-associated (autophagosome and endosome) trafficking activities in the context of viral infection? Although it remains to be tested whether this regulation of virus entry we have discovered with VSV and IAV might also be seen with other viruses, the existence of intensive interactions between viral particles and the host endomembrane network suggests that remodeling host cell fusion machinery may be a shared strategy for productive entry, and that technologies that interfere with the viral entry step and/or their interaction with the host endomembrane system could have considerable promise in treating some virally associated diseases.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.