A Nucleotide Exchange Factor Promotes Endoplasmic Reticulum-to-Cytosol Membrane Penetration of the Nonenveloped Virus Simian Virus 40

Abstract

The nonenveloped simian polyomavirus (PyV) simian virus 40 (SV40) hijacks the endoplasmic reticulum (ER) quality control machinery to penetrate the ER membrane and reach the cytosol, a critical infection step. During entry, SV40 traffics to the ER, where host-induced conformational changes render the virus hydrophobic. The hydrophobic virus binds and integrates into the ER lipid bilayer to initiate membrane penetration. However, prior to membrane transport, the hydrophobic SV40 recruits the ER-resident Hsp70 BiP, which holds the virus in a transport-competent state until it is ready to cross the ER membrane. Here we probed how BiP disengages from SV40 to enable the virus to penetrate the ER membrane. We found that nucleotide exchange factor (NEF) Grp170 induces nucleotide exchange of BiP and releases SV40 from BiP. Importantly, this reaction promotes SV40 ER-to-cytosol transport and infection. The human BK PyV also relies on Grp170 for successful infection. Interestingly, SV40 mobilizes a pool of Grp170 into discrete puncta in the ER called foci. These foci, postulated to represent the ER membrane penetration site, harbor ER components, including BiP, known to facilitate viral ER-to-cytosol transport. Our results thus identify a nucleotide exchange activity essential for catalyzing the most proximal event before ER membrane penetration of PyVs. IMPORTANCEPyVs are known to cause debilitating human diseases. During entry, this virus family, including monkey SV40 and human BK PyV, hijacks ER protein quality control machinery to breach the ER membrane and access the cytosol, a decisive infection step. In this study, we pinpointed an ER-resident factor that executes a crucial role in promoting ER-to-cytosol membrane penetration of PyVs. Identifying a host factor that facilitates entry of the PyV family thus provides additional therapeutic targets to combat PyV-induced diseases. Pathogens hijack protein quality control pathways of host cells to successfully cause infection. One pathway co-opted by pathogens during entry is endoplasmic reticulum (ER)-associated degradation (ERAD) (1-3). While ERAD is a surveillance system normally dedicated to the removal of misfolded ER proteins to the cytosol for proteasomal destruction, pathogens can co-opt elements of this pathway to gain entry into the host cytosol by disguising themselves as misfolded ER proteins. A clearer picture of the nature of this pathogen-host interaction is slowly emerging.Entry of the nonenveloped polyomavirus (PyV) family, including the simian virus 40 (SV40) and the human BK PyVs, serves as a salient example of pathogens that co-opt the ERAD pathway during infection (4-6). Structurally, SV40 is composed of 360 copies of the VP1 major coat protein arranged as 72 pentamers, with each pentamer engaging either the VP2 or VP3 internal hydrophobic minor coat protein. The pentamers are assembled as a 45-nm-diameter icosahedral particle that in turn encapsulates its viral DNA genome (7,8). To infect cells, SV40 undergoes receptor-mediated endocytosis a...

ERdj5 Reductase Cooperates with Protein Disulfide Isomerase To Promote Simian Virus 40 Endoplasmic Reticulum Membrane Translocation

“…We next asked if the ERdj5-dependent reduction of SV40's disulfide bonds affects the ability of SV40 to bind to the ER-resident Hsp70 BiP (31,33). Affinity purification (AP) of S-tagged BiP (BiP-S) derived from infected cells transfected with either scrambled siRNA or ERdj5 siRNA 1 revealed that knockdown of ERdj5 decreased the SV40-BiP interaction (Fig.…”

“…Our laboratory and others previously demonstrated that SV40 infection triggered selective ER membrane (e.g., BAP31) and soluble proteins to reorganize into these foci, where the virus also accumulates (12,25,31,52); these foci are thought to represent entry sites from where the viral particle penetrates the cytosol. If this is the case, we envision that impairment of SV40 entry into the cytosol should cause the virus to accumulate in the foci.…”

“…SV40 preparation, ER-to-cytosol transport, and infection assays were performed as described previously by Inoue and Tsai (24,31). Approximately 1,000 cells were scored in each experiment, and each experiment was repeated at least three times.…”

“…At 20 h postinfection (p.i. ), cells were fixed, and infection was assessed by immunofluorescence microscopy, as previously described (31). In FLAGpositive cells, the extent of T antigen expression in the nuclei was scored.…”

Chaperone-Assisted Protein Folding Is Critical for Yellow Fever Virus NS3/4A Cleavage and Replication

“…Most viruses therefore need cellular chaperones during their life cycle. Hsp70 proteins, as central components of the cellular chaperone network, are indeed frequently recruited by viruses (6,56,(73)(74)(75).…”