Alan G. Goodman

Cotranslocational Degradation Protects the Stressed Endoplasmic Reticulum from Protein Overload

Summary The ERs capacity to process proteins is limited, and stress caused by accumulation of unfolded and misfolded proteins (ER stress) contributes to human disease. ER stress elicits the unfolded protein response (UPR), whose components attenuate protein synthesis, increase folding capacity, and enhance misfolded protein degradation. Here, we report that P58 IPK /DNAJC3 , a UPR-responsive gene previously implicated in translational control, encodes a cytosolic cochaperone that associates with the ER protein translocation channel Sec61. P58 IPK recruits HSP70 chaperones to the cytosolic face of Sec61 and can be crosslinked to proteins entering the ER that are delayed at the translocon. Proteasome-mediated cytosolic degradation of translocating proteins delayed at Sec61 is cochaperone dependent. In P58 IPK−/− mice, cells with a high secretory burden are markedly compromised in their ability to cope with ER stress. Thus, P58 IPK is a key mediator of cotranslocational ER protein degradation, and this process likely contributes to ER homeostasis in stressed cells.

Lethal dissemination of H5N1 influenza virus is associated with dysregulation of inflammation and lipoxin signaling in a mouse model of infection

ABSTRACT Periodic outbreaks of highly pathogenic avian H5N1 influenza viruses and the current H1N1 pandemic highlight the need for a more detailed understanding of influenza virus pathogenesis. To investigate the host transcriptional response induced by pathogenic influenza viruses, we used a functional-genomics approach to compare gene expression profiles in lungs from 129S6/SvEv mice infected with either the fully reconstructed H1N1 1918 pandemic virus (1918) or the highly pathogenic avian H5N1 virus Vietnam/1203/04 (VN/1203). Although the viruses reached similar titers in the lung and caused lethal infections, the mean time of death was 6 days for VN/1203-infected animals and 9 days for mice infected with the 1918 virus. VN/1203-infected animals also exhibited an earlier and more potent inflammatory response. This response included induction of genes encoding components of the inflammasome. VN/1203 was also able to disseminate to multiple organs, including the brain, which correlated with changes in the expression of genes associated with hematological functions and lipoxin biogenesis and signaling. Both viruses elicited expression of type I interferon (IFN)-regulated genes in wild-type mice and to a lesser extent in mice lacking the type I IFN receptor, suggesting alternative or redundant pathways for IFN signaling. Our findings suggest that VN/1203 is more pathogenic in mice as a consequence of several factors, including the early and sustained induction of the inflammatory response, the additive or synergistic effects of upregulated components of the immune response, and inhibition of lipoxin-mediated anti-inflammatory responses, which correlated with the ability of VN/1203 to disseminate to extrapulmonary organs.

The cellular protein P58IPK regulates influenza virus mRNA translation and replication through a PKR-mediated mechanism.

ABSTRACT We previously hypothesized that efficient translation of influenza virus mRNA requires the recruitment of P58IPK, the cellular inhibitor of PKR, an interferon-induced kinase that targets the eukaryotic translation initiation factor eIF2α. P58IPK also inhibits PERK, an eIF2α kinase that is localized in the endoplasmic reticulum (ER) and induced during ER stress. The ability of P58IPK to interact with and inhibit multiple eIF2α kinases suggests it is a critical regulator of both cellular and viral mRNA translation. In this study, we sought to definitively define the role of P58IPK during viral infection of mammalian cells. Using mouse embryo fibroblasts from P58IPK−/− mice, we demonstrated that the absence of P58IPK led to an increase in eIF2α phosphorylation and decreased influenza virus mRNA translation. The absence of P58IPK also resulted in decreased vesicular stomatitis virus replication but enhanced reovirus yields. In cells lacking the P58IPK target, PKR, the trends were reversed—eIF2α phosphorylation was decreased, and influenza virus mRNA translation was increased. Although P58IPK also inhibits PERK, the presence or absence of this kinase had little effect on influenza virus mRNA translation, despite reduced levels of eIF2α phosphorylation in cells lacking PERK. Finally, we showed that influenza virus protein synthesis and viral mRNA levels decrease in cells that express a constitutively active, nonphosphorylatable eIF2α. Taken together, our results support a model in which P58IPK regulates influenza virus mRNA translation and infection through a PKR-mediated mechanism which is independent of PERK.

The NS1 Protein of the 1918 Pandemic Influenza Virus Blocks Host Interferon and Lipid Metabolism Pathways

ABSTRACT The “Spanish influenza” of 1918 claimed an unprecedented number of lives, yet the determinants of virulence for this virus are still not fully understood. Here, we used functional genomics and an in vitro human lung epithelial cell infection model to define the global host transcriptional response to the eight-gene 1918 virus. To better understand the role of the 1918 virus NS1 gene, we also evaluated the host response to a reassortant 1918 virus containing the NS1 gene from A/Texas/36/91 (a seasonal isolate of human influenza virus), as well as the host response to a reassortant of A/Texas/36/91 containing the 1918 NS1 gene. Genomic analyses revealed that the 1918 virus blocked the transcription of multiple interferon-stimulated genes and also downregulated a network of genes associated with lipid metabolism. In contrast, the expression of genes encoding chemokines and cytokines, which serve to attract infiltrating immune cells, was upregulated. Viruses containing the NS1 gene from A/Texas/36/91 induced a significant increase in type I interferon signaling but did not repress lipid metabolism. The 1918 NS1 gene may therefore have contributed to the virulence of the 1918 pandemic virus by disrupting the innate immune response, inducing hypercytokinemia, and by blocking the transcription of certain lipid-based proinflammatory mediators that function as part of the host antiviral response.

P58IPK: A Novel “CIHD” Member of the Host Innate Defense Response against Pathogenic Virus Infection

To support their replication, viruses take advantage of numerous cellular factors and processes. Recent large-scale screens have identified hundreds of such factors, yet little is known about how viruses exploit any of these. Influenza virus infection post-translationally activates P58IPK, a cellular inhibitor of the interferon-induced, dsRNA-activated eIF2α kinase, PKR. Here, we report that infection of P58IPK knockout mice with influenza virus resulted in increased lung pathology, immune cell apoptosis, PKR activation, and mortality. Analysis of lung transcriptional profiles, including those induced by the reconstructed 1918 pandemic virus, revealed increased expression of genes associated with the cell death, immune, and inflammatory responses. These experiments represent the first use of a mammalian infection model to demonstrate the role of P58IPK in the antiviral response. Our results suggest that P58IPK represents a new class of molecule, a cellular inhibitor of the host defense (CIHD), as P58IPK is activated during virus infection to inhibit virus-induced apoptosis and inflammation to prolong host survival, even while prolonging viral replication.

Progressive renal injury from transgenic expression of human carbonic anhydrase IV folding mutants is enhanced by deficiency of p58IPK

Mutations in the human carbonic anhydrase IV (hCAIV) have been associated with retinal degeneration in an autosomal-dominant form of retinitis pigmentosa (RP17). Prior in vitro cell culture studies confirmed that all of the RP17-associated hCAIV mutations cause protein misfolding, leading to endoplasmic reticulum (ER) stress–induced apoptosis in cells expressing the mutant proteins. To evaluate the physiological impacts of these folding mutants in other carbonic anhydrase IV–producing tissues, we generated two transgenic mouse lines expressing R219S or R14W hCAIV under control of the endogenous hCAIV promoter. Expression of either of these mutant proteins in kidneys caused progressive renal injury in male transgenic mice as evidenced by an age-dependent increase in the tubule cell apoptosis starting at approximately 20 weeks of age and vacuolization throughout the renal cortex in older mice. Up-regulation of the ER chaperone, BiP, was observed in the cells of the renal cortex of the male transgenic mice, suggesting ER stress as a causal factor for the renal injury. The renal injury inflicted by expression of the folding mutants was markedly enhanced by haploinsufficiency of the ER cochaperone p58IPK. The transgenic mice expressing the hCAIV folding mutants on a p58IPK heterozygous background showed extensive renal tubular apoptosis by approximately 10 weeks of age in both male and female mice. These data indicate that expression of the RP17-associated folding mutants of hCAIV can adversely affect tissues beyond the retina and their in vivo proteotoxicity is sensitive to modulation of the protein folding environment of the ER.