Coronaviruses (CoV), including SARS and mouse hepatitis virus (MHV), are enveloped RNA viruses that induce formation of double-membrane vesicles (DMVs) and target their replication and transcription complexes (RTCs) on the DMV-limiting membranes. The DMV biogenesis has been connected with the early secretory pathway. CoV-induced DMVs, however, lack conventional endoplasmic reticulum (ER) or Golgi protein markers, leaving their membrane origins in question. We show that MHV co-opts the host cell machinery for COPII-independent vesicular ER export of a short-living regulator of ER-associated degradation (ERAD), EDEM1, to derive cellular membranes for replication. MHV infection causes accumulation of EDEM1 and OS-9, another short-living ER chaperone, in the DMVs. DMVs are coated with the nonlipidated LC3/Atg8 autophagy marker. Downregulation of LC3, but not inactivation of host cell autophagy, protects cells from CoV infection. Our study identifies the host cellular pathway hijacked for supplying CoV replication membranes and describes an autophagy-independent role for nonlipidated LC3-I.

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Soluble ERAD substrates require the Hrd1 E3 ligase for degradation compared with membrane-anchored peptides that use GP78.

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. Unsuccessful folding attempts are eventually interrupted and most folding-defective polypeptides are dislocated across the ER membrane and degraded by cytosolic proteasomes in a complex series of events collectively defined as ER-associated degradation (ERAD). Uncontrolled ERAD activity might prematurely interrupt ongoing folding programs. At steady state, this is prevented by ERAD tuning, that is, the removal of select ERAD regulators from the ER and their degradation by proteasomes and by endo-lysosomal proteases. In Coronaviruses infected cells, the formation of LC3-I coated vesicles containing ERAD regulators cleared from the ER lumen is co-opted to anchor viral replication and transcription complexes to ER-derived membranes.

Normally, non-native polypeptides are not transported through the secretory pathway. Rather, they are translocated from the endoplasmic reticulum (ER) lumen into the cytosol where they are degraded by proteasomes. Here we characterize the function in ER quality control of two proteins derived from alternative splicing of the OS-9 gene. OS-9.1 and OS-9.2 are ubiquitously expressed in human tissues and are amplified in tumors. They are transcriptionally induced upon activation of the Ire1/Xbp1 ER-stress pathway. OS-9 variants do not associate with folding-competent proteins. Rather, they selectively bind folding-defective ones thereby inhibiting transport of non-native conformers through the secretory pathway. The intralumenal level of OS-9.1 and OS-9.2 inversely correlates with the fraction of a folding-defective glycoprotein, the Null hong kong (NHK) variant of ␣1-antitrypsin that escapes retention-based ER quality control. OS-9 up-regulation does not affect NHK disposal, but reduction of the intralumenal level of OS-9.1 and OS-9.2 substantially delays disposal of this model substrate. OS-9.1 and OS-9.2 also associate transiently with non-glycosylated foldingdefective proteins, but association is unproductive. Finally, OS-9 activity does not require an intact mannose 6-P homology domain. Thus, OS-9.1 and OS-9.2 play a dual role in mammalian ER quality control: first as crucial retention factors for misfolded conformers, and second as promoters of protein disposal from the ER lumen.About 30% of the eukaryotic gene products are synthesized by ribosomes attached at the cytosolic face of the endoplasmic reticulum (ER) 2 (1). The nascent polypeptide chains are translocated into the ER lumen where molecular chaperones and folding enzymes assist their maturation. Native proteins are transported at their intra-or extracellular destination through the secretory pathway. The ER lumen also contains chaperones and enzymes that retain and appropriately tag terminally misfolded proteins for destruction. Due to the facility of manipulating the yeast genome, many aspects and components of ERAD have been discovered in Saccharomyces cerevisiae (2-4). Yos9p is no exception. It was initially reported that deletion of Yos9p from the yeast ER selectively inhibits degradation of glycosylated ERAD substrates (5). Subsequent work revealed that Yos9p is required for disposal of substrates with luminal folding defects, whereas it is dispensable for disposal of proteins with defects in the transmembrane and cytosolic domains (6 -10). Studies on the involvement of the mammalian ortholog OS-9 in ERAD have been hampered by data showing that OS-9 is a cytosolic protein (11). This study was followed by a series of publications in which experimental design and interpretation of the data were based on the assumption that OS-9 is a cytosolic protein (12)(13)(14).Our analysis shows that OS-9 is a N-glycosylated protein expressed in two splice variants in the ER lumen. Transcription of both OS-9 variants is enhanced upon activation of the Ire1/ Xbp1...