The herpes simplex virus (HSV) virion host shutoff (vhs) RNase destabilizes cellular and viral mRNAs, suppresses host protein synthesis, dampens antiviral responses, and stimulates translation of viral mRNAs. vhs mutants display a host range phenotype: translation of viral true late mRNAs is severely impaired and stress granules accumulate in HeLa cells, while translation proceeds normally in Vero cells. We found that vhs-deficient virus activates the double-stranded RNA-activated protein kinase R (PKR) much more strongly than the wild-type virus does in HeLa cells, while PKR is not activated in Vero cells, raising the possibility that PKR might play roles in stress granule induction and/or inhibiting translation in restrictive cells. We tested this possibility by evaluating the effects of inactivating PKR. Eliminating PKR in HeLa cells abolished stress granule formation but had only minor effects on viral true late protein levels. These results document an essential role for PKR in stress granule formation by a nuclear DNA virus, indicate that induction of stress granules is the consequence rather than the cause of the translational defect, and are consistent with our previous suggestion that vhs promotes translation of viral true late mRNAs by preventing mRNA overload rather than by suppressing eIF2␣ phosphorylation. IMPORTANCEThe herpes simplex virus vhs RNase plays multiple roles during infection, including suppressing PKR activation, inhibiting the formation of stress granules, and promoting translation of viral late mRNAs. A key question is the extent to which these activities are mechanistically connected. Our results demonstrate that PKR is essential for stress granule formation in the absence of vhs, but at best, it plays a secondary role in suppressing translation of viral mRNAs. Thus, the ability of vhs to promote translation of viral mRNAs can be largely uncoupled from PKR suppression, demonstrating that this viral RNase modulates at least two distinct aspects of RNA metabolism. Viruses deploy diverse strategies to shut off host protein synthesis in order to gain access to the cellular translational machinery and blunt host antiviral responses. Alpha-and gammaherpesviruses, influenza A virus, poxviruses, and the severe acute respiratory syndrome (SARS) coronavirus do so in part by producing proteins that globally destabilize host mRNAs (reviewed in references 1 and 2). Among the best characterized of these viral mRNA destabilizers is the virion host shutoff (vhs) protein (encoded by the UL41 gene) of herpes simplex virus (HSV) and other alphaherpesviruses (3, 4) (reviewed in reference 5). vhs is a Fen-1 family endoribonuclease that is packaged into the virion tegument and delivered into the cytoplasm following fusion of the viral envelope with the host plasma membrane. Although purified vhs degrades essentially any RNA in vitro (6-9), only mRNAs are targeted in vivo (10)(11)(12). This selectivity appears to arise through the ability of vhs to bind components of the host mRNA cap-binding compl...

Highlights d Glucose suppresses a cell exocytosis by inhibiting P/Q-type Ca 2+ currents d Patch-seq links maturation, respiration, and receptor expression to a cell function d Dysfunction of a cells associates with a ''b cell-like'' electrophysiological signature d Impaired exocytosis occurs in a cells enriched for lineage and immaturity markers

Host encounters with viruses lead to an innate immune response that must be rapid and broadly targeted but also tightly regulated to avoid the detrimental effects of unregulated interferon expression. Viral stimulation of host negative regulatory mechanisms is an alternate method of suppressing the host innate immune response. We examined three key mediators of the innate immune response: NF-KB, STAT1 and STAT2 during HCV infection in order to investigate the paradoxical induction of an innate immune response by HCV despite a multitude of mechanisms combating the host response. During infection, we find that all three are repressed only in HCV infected cells but not in uninfected bystander cells, both in vivo in chimeric mouse livers and in cultured Huh7.5 cells after IFNα treatment. We show here that HCV and Flaviviruses suppress the innate immune response by upregulation of PDLIM2, independent of the host interferon response. We show PDLIM2 is an E3 ubiquitin ligase that also acts to stimulate nuclear degradation of STAT2. Interferon dependent relocalization of STAT1/2 to the nucleus leads to PDLIM2 ubiquitination of STAT2 but not STAT1 and the proteasome-dependent degradation of STAT2, predominantly within the nucleus. CRISPR/Cas9 knockout of PDLIM2 results in increased levels of STAT2 following IFNα treatment, retention of STAT2 within the nucleus of HCV infected cells after IFNα stimulation, increased interferon response, and increased resistance to infection by several flaviviruses, indicating that PDLIM2 is a global regulator of the interferon response.

Altered function and gene regulation of pancreatic islet beta cells is a hallmark of type 2 diabetes (T2D), but a comprehensive understanding of mechanisms driving T2D is still missing. Here we integrate information from measurements of chromatin activity, gene expression and function in single beta cells with genetic association data to identify disease-causal gene regulatory changes in T2D. Using machine learning on chromatin accessibility data from 34 non-diabetic, pre-T2D and T2D donors, we robustly identify two transcriptionally and functionally distinct beta cell subtypes that undergo an abundance shift in T2D. Subtype-defining active chromatin is enriched for T2D risk variants, suggesting a causal contribution of subtype identity to T2D. Both subtypes exhibit activation of a stress-response transcriptional program and functional impairment in T2D, which is likely induced by the T2D-associated metabolic environment. Our findings demonstrate the power of multimodal single-cell measurements combined with machine learning for identifying mechanisms of complex diseases.

Reoviridae virus family members, such as mammalian orthoreovirus (reovirus), encounter a unique challenge during replication. To hide the dsRNA from host recognition, the genome remains encapsidated in transcriptionally active proteinaceous core capsids that transcribe and release +RNA. De novo +RNAs and core proteins must repeatedly assemble into new progeny cores in order to logarithmically amplify replication. Reoviruses also produce outercapsid (OC) proteins μ1, σ3 and σ1 that assemble onto cores to create highly stable infectious full virions. Current models of reovirus replication position amplification of transcriptionally-active cores and assembly of infectious virions in shared factories, but we hypothesized that since assembly of OC proteins would halt core amplification, OC assembly is somehow regulated. Using kinetic analysis of virus +RNA, core and OC proteins, core assembly and whole virus assembly, assembly of OC proteins was found to be temporally delayed. All viral RNAs and proteins were made simultaneously, eliminating the possibility that delayed OC RNAs or proteins account for delayed OC assembly. High resolution fluorescence and electron microscopy revealed that core amplification occurred early during infection at peripheral core-only factories, while all OC proteins associated with lipid droplets (LDs) that coalesced near the nucleus in a μ1–dependent manner. Core-only factories transitioned towards the nucleus despite cycloheximide-mediated halting of new protein expression, while new core-only factories developed in the periphery. As infection progressed, OC assembly occurred at LD-and nuclear-proximal factories. Silencing of OC μ1 expression with siRNAs led to large factories that remained further from the nucleus, implicating μ1 in the transition to perinuclear factories. Moreover, late during infection, +RNA pools largely contributed to the production of de-novo viral proteins and fully-assembled infectious viruses. Altogether the results suggest an advanced model of reovirus replication with spatiotemporal segregation of core amplification, OC complexes and fully assembled virions.