Biosynthesis of insulin – critical to metabolic homeostasis – begins with folding of the proinsulin precursor, including formation of three evolutionarily conserved intramolecular disulfide bonds. Remarkably, normal pancreatic islets contain a subset of proinsulin molecules bearing at least one free cysteine thiol. In human (or rodent) islets with a perturbed endoplasmic reticulum folding environment, non-native proinsulin enters intermolecular disulfide-linked complexes. In genetically obese mice with otherwise wild-type islets, disulfide-linked complexes of proinsulin are more abundant, and leptin receptor-deficient mice, the further increase of such complexes tracks with the onset of islet insulin deficiency and diabetes. Proinsulin-Cys(B19) and Cys(A20) are necessary and sufficient for the formation of proinsulin disulfide-linked complexes; indeed, proinsulin Cys(B19)-Cys(B19) covalent homodimers resist reductive dissociation, highlighting a structural basis for aberrant proinsulin complex formation. We conclude that increased proinsulin misfolding via disulfide-linked complexes is an early event associated with prediabetes that worsens with ß-cell dysfunction in type two diabetes.

Although the precise mechanism by which nonenveloped viruses penetrate biological membranes is unclear, a more coherent understanding of this process is starting to emerge. To initiate membrane penetration, nonenveloped viruses engage host cell factors that impart conformational changes on the viral particles, resulting in the exposure of a hydrophobic moiety or the release of a lytic factor. The viruses' interactions with the limiting membrane subsequently compromise the bilayer integrity. This reaction presumably perforates the bilayer to enable the virus to cross the membrane and reach the cytosol. Valuable insights into this process can be gleaned from the membrane transport mechanisms of enveloped viruses and bacterial toxins. To identify systematically the cellular components that facilitate nonenveloped virus membrane penetration, sensitive assays that monitor the transport event directly must first be established. Moreover, higher-resolution structures of penetration intermediates, particularly those solved in complex with membranes, would provide important molecular details into this process.

The mechanisms by which receptors guide intracellular virus transport are poorly characterized. The murine polyomavirus (Py) binds to the lipid receptor ganglioside GD1a and traffics to the endoplasmic reticulum (ER) where it enters the cytosol and then the nucleus to initiate infection. How Py reaches the ER is unclear. We show that Py is transported initially to the endolysosome where the low pH imparts a conformational change that enhances its subsequent ER-to-cytosol membrane penetration. GD1a stimulates not viral binding or entry, but rather sorting of Py from late endosomes and/or lysosomes to the ER, suggesting that GD1a binding is responsible for ER targeting. Consistent with this, an artificial particle coated with a GD1a antibody is transported to the ER. Our results provide a rationale for transport of Py through the endolysosome, demonstrate a novel endolysosome-to-ER transport pathway that is regulated by a lipid, and implicate ganglioside binding as a general ER targeting mechanism. To cause infection, viruses must reach appropriate compartments within the cell where they undergo a programmed series of conformational changes that enable the viral genome to be exposed and released. The mechanisms that target viruses to these compartments are often not clear. Here we study the infectious pathway of the murine polyomavirus (Py). Py is transported from the cell surface to the intracellular organelle called the endoplasmic reticulum (ER), where it breaches the ER membrane to reach the nucleus to stimulate infection. How Py is transported from the cell surface to the ER is poorly characterized. Our studies show that Py first enters the endolysosome compartments before reaching the ER. The low pH of the endolysosome imparts a structural change on the virus that facilitates its subsequent ER membrane penetration. Importantly, transport of Py from the endolysosome to the ER is guided by the lipid receptor ganglioside GD1a. We also demonstrate that an artificial bead capable of binding to GD1a is transported to the ER as well. Collectively, our data identify a lipid-dependent mechanism that targets a virus to its appropriate organelle during infection.

To cause infection, a virus enters a host cell, replicates, and assembles, with the resulting new viral progeny typically released into the extracellular environment to initiate a new infection round. Virus entry, replication, and assembly are dynamic and coordinated processes that require precise interactions with host components, often within and surrounding a defined subcellular compartment. Accumulating evidence pinpoints the endoplasmic reticulum (ER) as a crucial organelle supporting viral entry, replication, and assembly. This review focuses on the molecular mechanism by which different viruses co-opt the ER to accomplish these crucial infection steps. Certain bacterial toxins also hijack the ER for entry. An interdisciplinary approach, using rigorous biochemical and cell biological assays coupled with advanced microscopy strategies, will push to the next level our understanding of the virus-ER interaction during infection.

Although flaviviruses co-opt the function of the host endoplasmic reticulum (ER) membrane protein complex (EMC) during infection, a mechanistic explanation for this observation remains unclear. Here, we show that the EMC promotes biogenesis of dengue virus (DENV) and Zika virus (ZIKV) non-structural multi-pass transmembrane proteins NS4A and NS4B, which are necessary for viral replication. The EMC binds to NS4B and colocalizes with the DENV replication organelle. Mapping analysis reveals that the two N-terminal marginally hydrophobic domains of NS4B confer EMC dependency. Furthermore, altering the hydrophobicity of these two marginally hydrophobic domains relieves NS4B's EMC dependency. We demonstrate that NS4B biogenesis, but not its stability, is reduced in EMC-depleted cells. Our data suggest that the EMC acts as a multi-pass transmembrane chaperone required for expression of at least two virally encoded proteins essential for flavivirus infection and point to a shared vulnerability during the viral life cycle that could be exploited for antiviral therapy.