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      A Cytosolic Chaperone Complexes with Dynamic Membrane J-Proteins and Mobilizes a Nonenveloped Virus out of the Endoplasmic Reticulum

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          Abstract

          Nonenveloped viruses undergo conformational changes that enable them to bind to, disrupt, and penetrate a biological membrane leading to successful infection. We assessed whether cytosolic factors play any role in the endoplasmic reticulum (ER) membrane penetration of the nonenveloped SV40. We find the cytosolic SGTA-Hsc70 complex interacts with the ER transmembrane J-proteins DnaJB14 (B14) and DnaJB12 (B12), two cellular factors previously implicated in SV40 infection. SGTA binds directly to SV40 and completes ER membrane penetration. During ER-to-cytosol transport of SV40, SGTA disengages from B14 and B12. Concomitant with this, SV40 triggers B14 and B12 to reorganize into discrete foci within the ER membrane. B14 must retain its ability to form foci and interact with SGTA-Hsc70 to promote SV40 infection. Our results identify a novel role for a cytosolic chaperone in the membrane penetration of a nonenveloped virus and raise the possibility that the SV40-induced foci represent cytosol entry sites.

          Author Summary

          The nonenveloped simian virus 40 (SV40) is a model member of the Polyomaviridae family of viruses containing several related species that cause diseases in immunocompromised individuals. As with other nonenveloped viruses, the membrane penetration step during SV40 entry is mechanistically obscure. Productive SV40 infection requires trafficking of the viral particle to the endoplasmic reticulum (ER) from where it penetrates the ER membrane to reach the cytosol; further transport of the virus into the nucleus causes infection. How SV40 crosses the ER membrane is an enigmatic step. Here, we identify a cytosolic chaperone protein that physically engages SV40 and facilitates virus ER-to-cytosol transport. This factor called SGTA is hijacked specifically at the site of membrane penetration due to its recruitment by ER membrane proteins B14 and B12 previously implicated in supporting virus infection. Additionally, we observe that B14 and B12 reorganize during SV40 entry into discrete foci on the ER membrane. These virus-induced structures likely represent exit sites for the viral particles and could serve to transiently recruit high concentrations of SGTA to complete membrane penetration. Our data reveal that a cytosolic chaperone can play a direct role in membrane penetration of a nonenveloped virus.

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          Most cited references 45

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          Virus entry by endocytosis.

          Although viruses are simple in structure and composition, their interactions with host cells are complex. Merely to gain entry, animal viruses make use of a repertoire of cellular processes that involve hundreds of cellular proteins. Although some viruses have the capacity to penetrate into the cytosol directly through the plasma membrane, most depend on endocytic uptake, vesicular transport through the cytoplasm, and delivery to endosomes and other intracellular organelles. The internalization may involve clathrin-mediated endocytosis (CME), macropinocytosis, caveolar/lipid raft-mediated endocytosis, or a variety of other still poorly characterized mechanisms. This review focuses on the cell biology of virus entry and the different strategies and endocytic mechanisms used by animal viruses.
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            Modification of intracellular membrane structures for virus replication

            Key Points Plus-stranded RNA viruses induce large membrane structures that might support the replication of their genomes. Similarly, cytoplasmic replication of poxviruses (large DNA viruses) occurs in associated membranes. These membranes originate from the endoplasmic reticulum (ER) or endosomes. Membrane vesicles that support viral replication are induced by a number of RNA viruses. Similarly, the poxvirus replication site is surrounded by a double-membraned cisterna that is derived from the ER. Analogies to autophagy have been proposed since the finding that autophagy cellular processes involve the formation of double-membrane vesicles. However, molecular evidence to support this hypothesis is lacking. Membrane association of the viral replication complex is mediated by the presence of one or more viral proteins that contain sequences which associate with, or integrate into, membranes. Replication-competent membranes might contain viral or cellular proteins that contain amphipathic helices, which could mediate the membrane bending that is required to form spherical vesicles. Whereas poxvirus DNA replication occurs inside the ER-enclosed site, for most RNA viruses the topology of replication is not clear. Preliminary results for some RNA viruses suggest that their replication could also occur inside double-membrane vesicles. We speculate that cytoplasmic replication might occur inside sites that are 'enwrapped' by an ER-derived cisterna, and that these cisternae are open to the cytoplasm. Thus, RNA and DNA viruses could use a common mechanism for replication that involves membrane wrapping by cellular cisternal membranes. We propose that three-dimensional analyses using high-resolution electron-microscopy techniques could be useful for addressing this issue. High-throughput small-interfering-RNA screens should also shed light on molecular requirements for virus-induced membrane modifications.
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              Defining human ERAD networks through an integrative mapping strategy

              SUMMARY Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin and proteasome dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organised into a functional network that coordinates recognition, ubiquitination, and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organisation of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics, and the transcriptional response to ER stress. This analysis supports an adaptive organisation for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                March 2014
                27 March 2014
                : 10
                : 3
                Affiliations
                [1 ]Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America
                [2 ]Cellular and Molecular Biology Graduate Program, Ann Arbor, Michigan, United States of America
                National Cancer Institute, United States of America
                Author notes

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: CPW MSR BT. Performed the experiments: CPW MSR TI. Analyzed the data: CPW MSR TI BT. Contributed reagents/materials/analysis tools: CPW MSR TI BT. Wrote the paper: CPW MSR TI BT.

                Article
                PPATHOGENS-D-13-02587
                10.1371/journal.ppat.1004007
                3968126
                24675744

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Pages: 17
                Funding
                CPW is funded by NIH T-32-GM007315, and BT by the NIH (AI064296 and 083252). This work is also partially funded by the Fast Forward Initiative at the University of Michigan Medical School. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Microbiology
                Virology

                Infectious disease & Microbiology

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