Molecular chaperones: from proteostasis to pathogenesis.

Maintaining protein homeostasis (proteostasis) is essential for a functional proteome. A wide range of extrinsic and intrinsic factors perturb proteostasis, causing protein misfolding, misassembly, and aggregation. This compromises cellular integrity and leads to aging and disease, including neurodegeneration and cancer. At the cellular level, protein aggregation is counteracted by powerful mechanisms comprising of a cascade of enzymes and chaperones that operate in a coordinated multistep manner to sense, prevent, and/or dispose of aberrant proteins. Although these processes are well understood for soluble proteins, there is a major gap in our understanding of how cells handle misfolded or aggregated membrane proteins. This article provides an overview of cellular proteostasis with emphasis on membrane protein substrates and suggests host-virus interaction as a tool to clarify outstanding questions in proteostasis.

Dynein Engages and Disassembles Cytosol-Localized Simian Virus 40 To Promote Infection.

During entry, polyomavirus (PyV) is endocytosed and sorts to the endoplasmic reticulum (ER), where it penetrates the ER membrane to reach the cytosol. From the cytosol, the virus moves to the nucleus to cause infection. How PyV is transported from the cytosol into the nucleus, a crucial infection step, is unclear. We found that upon reaching the cytosol, the archetypal PyV simian virus 40 (SV40) recruits the cytoplasmic dynein motor, which disassembles the viral particle. This reaction enables the resulting disassembled virus to enter the nucleus to promote infection. Our findings reveal how a cytosolic motor can be hijacked to impart conformational changes to a viral particle, a process essential for successful infection.IMPORTANCE How a nonenveloped virus successfully traffics from the cell surface to the nucleus to cause infection remains enigmatic in many instances. In the case of the nonenveloped PyV, the viral particle is sorted from the plasma membrane to the ER and then the cytosol, from which it enters the nucleus to promote infection. The molecular mechanism by which PyV reaches the nucleus from the cytosol is not entirely clear. Here we demonstrate that the prototype PyV SV40 recruits dynein upon reaching the cytosol. Importantly, this cellular motor disassembles the viral particle during cytosol-to-nucleus transport to cause infection.

Exploiting the kinesin-1 molecular motor to generate a virus membrane penetration site.

Viruses exploit cellular machineries to penetrate a host membrane and cause infection, a process that remains enigmatic for non-enveloped viruses. Here we probe how the non-enveloped polyomavirus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a crucial infection step. We find that the microtubule-based motor kinesin-1 is recruited to the ER membrane by binding to the transmembrane J-protein B14. Strikingly, this motor facilitates SV40 ER-to-cytosol transport by constructing a penetration site on the ER membrane called a 'focus'. Neither kinesin-2, kinesin-3 nor kinesin-5 promotes foci formation or infection. The specific use of kinesin-1 is due to its unique ability to select posttranslationally modified microtubules for cargo transport and thereby spatially restrict focus formation to the perinucleus. These findings support the idea of a 'tubulin code' for motor-dependent trafficking and establish a distinct kinesin-1 function in which a motor is exploited to create a viral membrane penetration site.

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

A dedicated network of cellular factors ensures that proteins translocated into the endoplasmic reticulum (ER) are folded correctly before they exit this compartment en route to other cellular destinations or for secretion. When proteins misfold, selective ER-resident enzymes and chaperones are recruited to rectify the protein-misfolding problem in order to maintain cellular proteostasis. However, when a protein becomes terminally misfolded, it is ejected into the cytosol and degraded by the proteasome via a pathway called ER-associated degradation (ERAD). Strikingly, toxins and viruses can hijack elements of the ERAD pathway to access the host cytosol and cause infection. This review focuses on emerging data illuminating the molecular mechanisms by which these toxic agents co-opt the ER-to-cytosol translocation process to cause disease.

A Non-enveloped Virus Hijacks Host Disaggregation Machinery to Translocate across the Endoplasmic Reticulum Membrane.

Mammalian cytosolic Hsp110 family, in concert with the Hsc70:J-protein complex, functions as a disaggregation machinery to rectify protein misfolding problems. Here we uncover a novel role of this machinery in driving membrane translocation during viral entry. The non-enveloped virus SV40 penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol, a critical infection step. Combining biochemical, cell-based, and imaging approaches, we find that the Hsp110 family member Hsp105 associates with the ER membrane J-protein B14. Here Hsp105 cooperates with Hsc70 and extracts the membrane-penetrating SV40 into the cytosol, potentially by disassembling the membrane-embedded virus. Hence the energy provided by the Hsc70-dependent Hsp105 disaggregation machinery can be harnessed to catalyze a membrane translocation event.

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

UNLABELLED: 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.

A cytosolic chaperone complexes with dynamic membrane J-proteins and mobilizes a nonenveloped virus out of the endoplasmic reticulum.

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.