Noncanonical Role for the Host Vps4 AAA+ ATPase ESCRT Protein in the Formation of Tomato Bushy Stunt Virus Replicase

Abstract

Assembling of the membrane-bound viral replicase complexes (VRCs) consisting of viral- and host-encoded proteins is a key step during the replication of positive-stranded RNA viruses in the infected cells. Previous genome-wide screens with Tomato bushy stunt tombusvirus (TBSV) in a yeast model host have revealed the involvement of eleven cellular ESCRT (endosomal sorting complexes required for transport) proteins in viral replication. The ESCRT proteins are involved in endosomal sorting of cellular membrane proteins by forming multiprotein complexes, deforming membranes away from the cytosol and, ultimately, pinching off vesicles into the lumen of the endosomes. In this paper, we show an unexpected key role for the conserved Vps4p AAA+ ATPase, whose canonical function is to disassemble the ESCRT complexes and recycle them from the membranes back to the cytosol. We find that the tombusvirus p33 replication protein interacts with Vps4p and three ESCRT-III proteins. Interestingly, Vps4p is recruited to become a permanent component of the VRCs as shown by co-purification assays and immuno-EM. Vps4p is co-localized with the viral dsRNA and contacts the viral (+)RNA in the intracellular membrane. Deletion of Vps4p in yeast leads to the formation of crescent-like membrane structures instead of the characteristic spherule and vesicle-like structures. The in vitro assembled tombusvirus replicase based on cell-free extracts (CFE) from vps4Δ yeast is highly nuclease sensitive, in contrast with the nuclease insensitive replicase in wt CFE. These data suggest that the role of Vps4p and the ESCRT machinery is to aid building the membrane-bound VRCs, which become nuclease-insensitive to avoid the recognition by the host antiviral surveillance system and the destruction of the viral RNA. Other (+)RNA viruses of plants and animals might also subvert Vps4p and the ESCRT machinery for formation of VRCs, which require membrane deformation and spherule formation.

Novel Mechanism of Regulation of Tomato Bushy Stunt Virus Replication by Cellular WW-Domain Proteins

“…Instead, only the formation of new VRCs should be inhibited at the late stage of infection. It is also possible that the WW-domain protein cannot access the previously assembled VRCs, because those are closed from cytosolic proteins due to the spherule structure (57) or, alternatively, the p33 and p92 proteins are already oligomerized (via p33-p33 self-interaction or p33-p92 interaction) or bound to proviral host factors. (iii) A regulatory protein is expected to block the interaction between the viral replication proteins and the viral RNA in order to facilitate a nonreplicative use of the viral RNA (e.g., for encapsidation or cell-to-cell movement), instead of keeping the viral RNA trapped in the translation/replication cycle.…”

“…These proteins include the host heat shock protein 70 (Hsp70), the eukaryotic elongation factor 1A (eEF1A), Vps23p ESCRT (endosomal sorting complexes required for transport) protein, Bro1p ESCRT-associated protein, and Vps4p AAAϩ ATPase (39,46,47,(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59). Cdc34p E2 ubiquitin-conjugating enzyme binds to p33, and it functions as a permanent member of the viral VRC, affecting the activity of the VRC (47).…”

Drosophila Vps4 promotes Epidermal growth factor receptor signaling independently of its role in receptor degradation

“…Active Vps4 forms a hexameric complex that disassembles ESCRT-III, allowing recycling of its components, and also plays an active role in scission of the vesicle neck (Adell et al, 2014;Cashikar et al, 2014;Lata et al, 2008;Monroe et al, 2014;Mueller et al, 2012). In addition to their endocytic functions, ESCRT proteins, including Vps4, are required for cytokinesis, viral budding, protecting viral genomes from degradation, exosome secretion, receptor shedding on microvesicles, assembly of nuclear pore complexes, cholesterol transport and plasma membrane wound repair (Barajas et al, 2014;Choudhuri et al, 2014;Du et al, 2013;de Gassart et al, 2004;Jimenez et al, 2014;Morita, 2012;Nabhan et al, 2012;Tang, 2012;Webster et al, 2014).…”

Viral Sensing of the Subcellular Environment Regulates the Assembly of New Viral Replicase Complexes during the Course of Infection

“…TBSV has been shown to coopt several host factors involved in the assembly of the membrane-bound VRCs, such as the heat shock protein 70 (Hsp70), the eukaryotic elongation factor 1A (eEF1A), Vps23p ESCRT (endosomal sorting complexes required for transport) protein, Bro1p ESCRT-associated protein, Vps4p AAAϩ ATPase, and Cdc34p E2 ubiquitin-conjugating enzyme (4,(6)(7)(8)(9). In addition, TBSV channels sterols and phospholipids (phosphatidylethanolamine [PE]) to enrich these lipids at the sites of replication, possibly via the use of cellular VAMP/synaptobrevin-associated protein, called VAP (Scs2p in yeast), and oxysterol-binding proteins (ORPs; Osh6 and Osh7 in yeast) and membrane contact sites between the endoplasmic reticulum (ER) and peroxisomes ( Fig.…”

“…The low level of Ded1p leads to reduced replication and the production of high levels of truncated viral RNAs and recombinant viral RNAs (14). Also, depletion of ESCRT proteins (Vps24 or Vps4p) results in incorrect assembly of tombusvirus VRCs in yeast, which gives rise to spherules with wide openings, instead of the narrow "neck-like" opening in wild-type (wt) yeast (8). These open VRC structures are likely less protective of the viral dsRNA intermediates against the host antiviral machinery.…”