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

Abstract

The nonenveloped polyomavirus (PyV) simian virus 40 (SV40) traffics from the cell surface to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol before mobilizing into the nucleus to cause infection. Prior to ER membrane penetration, ER lumenal factors impart structural rearrangements to the virus, generating a translocation-competent virion capable of crossing the ER membrane. Here we identify ERdj5 as an ER enzyme that reduces SV40's disulfide bonds, a reaction important for its ER membrane transport and infection. ERdj5 also mediates human BK PyV infection. This enzyme cooperates with protein disulfide isomerase (PDI), a redox chaperone previously implicated in the unfolding of SV40, to fully stimulate membrane penetration. Negative-stain electron microscopy of ER-localized SV40 suggests that ERdj5 and PDI impart structural rearrangements to the virus. These conformational changes enable SV40 to engage BAP31, an ER membrane protein essential for supporting membrane penetration of the virus. Uncoupling of SV40 from BAP31 traps the virus in ER subdomains called foci, which likely serve as depots from where SV40 gains access to the cytosol. Our study thus pinpoints two ER lumenal factors that coordinately prime SV40 for ER membrane translocation and establishes a functional connection between lumenal and membrane events driving this process. IMPORTANCE PyVs are established etiologic agents of many debilitating human diseases, especially in immunocompromised individuals. To infect cells at the cellular level, this virus family must penetrate the host ER membrane to reach the cytosol, a critical entry step.In this report, we identify two ER lumenal factors that prepare the virus for ER membrane translocation and connect these lumenal events with events on the ER membrane. Pinpointing cellular components necessary for supporting PyV infection should lead to rational therapeutic strategies for preventing and treating PyV-related diseases. Viruses must penetrate host cell membranes to reach their proper intracellular destination where they replicate their genome, producing viral progenies used for the next round of infection. While enveloped viruses breach host cells by fusing their membrane with a target cell membrane, the mechanism by which nonenveloped viruses penetrate the host cell membrane must be distinct from that of enveloped viruses, as they lack a surrounding membrane. Despite being poorly characterized, a series of biochemical experiments provided a general model describing nonenveloped virus membrane penetration (1-5). In this model, the nonenveloped virus first traffics to the proper site for membrane penetration. Here the viral particle undergoes defined conformational changes induced by host environments and factors (e.g., low pH, proteases, reductases, and chaperones) that either expose hydrophobic moieties buried in the native virus or release small lytic peptides hidden in the intact virion (1-11). In the final step, the hydrophobic viral intermediate (o...

EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus

“…Clearly these two possibilities do not have to be mutually exclusive – EMC1 may operate in both manners to shield the presumed exposed charges and to reinforce interactions between the pentamers. In our in vitro analyses, addition of DTT and EGTA also generated VP1 pentamers (Figure 6B), whose formation can be largely suppressed by addition of purified EMC1; DTT likely mimics viral disulfide bond reduction that occurs in the ER lumen (Inoue et al, 2015), whereas EGTA may imitate loss of Ca2+ from the membrane-embedded virus when it is exposed to the low Ca2+ in the cytosol, as previously hypothesized (Schelhaas et al, 2007). …”

“…This was not surprising because the purifications were intentionally performed in the presence of the harsher detergent Triton X-100, which prevented other EMC subunits from co-purification. Previous studies revealed that SV40 is partially destabilized in the ER (Inoue et al, 2015; Magnuson et al, 2005; Walczak and Tsai, 2011; Nelson et al, 2012), an effect that can be mimicked in part by treating purified SV40 with DTT (which reduces the SV40 disulfide bonds) and EGTA (which removes calcium bound to the virus). Indeed, when SV40 was incubated with either GFP-FLAG or WT EMC1-FLAG in the presence or absence of DTT/EGTA, pull down of EMC1 but not GFP precipitated SV40 (Figure 5F, top panel, compare lane 2 to 1), demonstrating a direct EMC1-SV40 interaction.…”

“…Specifically, upon reaching the ER, protein disulfide isomerase (PDI) family members including PDI and ERdj5 isomerize and reduce SV40’s disulfide bonds (Schelhaas et al, 2007; Inoue et al, 2015); for the murine PyV and possibly JC PyV, another non-catalytically active PDI family member ERp29 locally unfolds the VP1 C-terminal arm (Magnuson et al, 2005; Walczak and Tsai, 2011; Nelson et al, 2012). These reactions disrupt the viral architecture, exposing its hydrophobic proteins VP2 and VP3 (Magnuson et al, 2005).…”

SGTA-Dependent Regulation of Hsc70 Promotes Cytosol Entry of Simian Virus 40 from the Endoplasmic Reticulum

“…In the ER, redox enzymes reduce and isomerize the SV40 disulfide bonds (16)(17)(18), while molecular chaperones locally unfold the VP1 C-terminal arms (17,19,20) to expose the internal proteins VP2 and VP3 (21,22). These events generate a hydrophobic virus that integrates into the ER membrane.…”

A bacterial toxin and a nonenveloped virus hijack ER-to-cytosol membrane translocation pathways to cause disease

“…For instance, the ER-resident ERp57 isomerizes a subset of SV40's disulfide bonds, breaking and forming non-native disulfide bonds to initiate partial disassembly of the viral particle (Figure 3C, step 1) (Schelhaas et al, 2007); in this reaction, ERp57 acts on disulfide bonds involved in stabilizing the 12 five-coordinated pentamers. In addition to isomerization, disulfide bonds present in SV40 are also reduced by ERdj5 (Inoue et al, 2015); whether this reaction is imposed on disulfide bonds surrounding the 60 six-coordinated pentamers is unclear. It is interesting to note that ERdj5 functions as a J-protein and not as a reductase during ERAD of CT. Canonical PDI neither isomerizes nor reduces SV40's disulfide bonds, but probably uses its chaperone function to unfold the virus (Schelhaas et al, 2007, Inoue et al, 2015), reminiscent of PDI's role as an unfoldase during CTA1 retro-translocation.…”