Coronaviruses induce the formation of double-membrane vesicles (DMVs) to facilitate viral RNA replication and transcription by the replication-transcription complexes (RTCs), comprising non-structural proteins (nsps) 2-16 and nucleocapsid protein. Nsp3 and nsp4 are the minimal components necessary for DMV formation and assemble a molecular pore that spans the DMV double membrane, connecting the DMV interior to the cytosol. However, the recruitment mechanisms of additional RTC components and the roles of other viral proteins in DMV assembly remain poorly understood. To dissect these processes independently of viral replication, we sought to establish a surrogate expression system using severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) polypeptides to recapitulate DMV formation and RTC recruitment. We characterized DMVs formed in cells expressing various nsp combinations and assessed the localization of RTC components using proteinase K (PK) protection assays following cell permeabilization. Compared to nsp3-4, expression of nsp3-8 and nsp3-10 resulted in larger and more morphologically heterogeneous DMVs. Portions of nsp5, nsp7, and nsp8 are associated with DMV-enriched membrane fractions, with nsp5 and nsp8 showing partial resistance to proteinase K digestion, suggesting that these proteins are at least partially localized within the DMV interior or protected by the DMV membrane architecture. Notably, mutations in the membrane-associated element (MAE) of nsp6 impaired nsp5-mediated proteolytic processing, abrogated DMV formation, and induced a cross-linked endoplasmic reticulum (ER) phenotype. These results highlight the essential role of nsp6 in the DMV biogenesis and demonstrate the utility of this surrogate system for mechanistic studies of coronavirus-induced membrane remodeling.IMPORTANCECoronaviruses remodel host membranes through the action of non-structural proteins to generate double-membrane vesicles (DMVs), which serve as platforms for viral replication-transcription complexes (RTCs). Deciphering the molecular mechanisms governing DMV assembly and RTC recruitment is critical for understanding coronavirus replication and identifying novel antiviral targets. Here, we developed a surrogate system that recapitulates DMV formation in the absence of viral replication, enabling genetic manipulation and functional dissection of individual proteins. Using this system, we demonstrate that expression of the SARS-CoV-2 nsp3-10 polyprotein is sufficient to drive DMV formation and reveal a pivotal role for the membrane-associated element (MAE) of nsp6 in this process. These findings establish a tractable model for investigating coronavirus-induced membrane remodeling and underscore the essential contributions of nsp6 to DMV biogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat due to the emergence of variants with increased transmissibility and enhanced escape from immune responses. Like other coronaviruses before, SARS-CoV-2 likely emerged after its transmission from bats. The successful propagation of SARS-CoV-2 in humans might have been facilitated by usurping evolutionarily conserved cellular factors to execute crucial steps in its life cycle, such as the generation of replication organelles-membrane structures where coronaviruses assemble their replication-transcription complex. In this study, we found that RAB5, which is highly conserved across mammals, is a critical host dependency factor for the replication of the SARS-CoV-2 genome. Our results also suggest that SARS-CoV-2 uses RAB5+ membranes to build replication organelles with the aid of COPB1, a component of the COP-I complex, and that the virus protein NSP6 participates in this process. Hence, targeting NSP6 represents a promising approach to interfere with SARS-CoV-2 RNA synthesis and halt its propagation.IMPORTANCEIn this study, we sought to identify the host dependency factors that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses for the generation of replication organelles: cellular membranous structures that SARS-CoV-2 builds in order to support the replication and transcription of its genome. We uncovered that RAB5 is an important dependency factor for SARS-CoV-2 replication and the generation of replication organelles, and that the viral protein NSP6 participates in this process. Hence, NSP6 represents a promising target to halt SARS-CoV-2 replication.

Although the importance of virus-specific cytotoxic T lymphocytes (CTL) in virus clearance is evident in COVID-19, the characteristics of virus-specific CTLs related to disease severity have not been fully explored. Here we show that the phenotype of virus-specific CTLs against immunoprevalent epitopes in COVID-19 convalescents might differ according to the course of the disease. We establish a cellular screening method that uses artificial antigen presenting cells, expressing HLA-A*24:02, the costimulatory molecule 4-1BBL, SARS-CoV-2 structural proteins S, M, and N and non-structural proteins ORF3a and nsp6/ORF1a. The screen implicates SARS-CoV-2 M protein as a frequent target of IFNγ secreting CD8+ T cells, and identifies M198-206 as an immunoprevalent epitope in our cohort of HLA-A*24:02 positive convalescent COVID-19 patients recovering from mild, moderate and severe disease. Further exploration of M198-206-specific CD8+ T cells with single cell RNA sequencing reveals public TCRs in virus-specific CD8+ T cells, and shows an exhausted phenotype with less differentiated status in cells from the severe group compared to cells from the moderate group. In summary, this study describes a method to identify T cell epitopes, indicate that dysfunction of virus-specific CTLs might be an important determinant of clinical outcomes.
© 2022. The Author(s).

SARS-CoV-2, like other coronaviruses, builds a membrane-bound replication organelle to enable RNA replication1. The SARS-CoV-2 replication organelle is composed of double-membrane vesicles (DMVs) that are tethered to the endoplasmic reticulum (ER) by thin membrane connectors2, but the viral proteins and the host factors involved remain unknown. Here we identify the viral non-structural proteins (NSPs) that generate the SARS-CoV-2 replication organelle. NSP3 and NSP4 generate the DMVs, whereas NSP6, through oligomerization and an amphipathic helix, zippers ER membranes and establishes the connectors. The NSP6(ΔSGF) mutant, which arose independently in the Alpha, Beta, Gamma, Eta, Iota and Lambda variants of SARS-CoV-2, behaves as a gain-of-function mutant with a higher ER-zippering activity. We identified three main roles for NSP6: first, to act as a filter in communication between the replication organelle and the ER, by allowing lipid flow but restricting the access of ER luminal proteins to the DMVs; second, to position and organize DMV clusters; and third, to mediate contact with lipid droplets (LDs) through the LD-tethering complex DFCP1-RAB18. NSP6 thus acts as an organizer of DMV clusters and can provide a selective means of refurbishing them with LD-derived lipids. Notably, both properly formed NSP6 connectors and LDs are required for the replication of SARS-CoV-2. Our findings provide insight into the biological activity of NSP6 of SARS-CoV-2 and of other coronaviruses, and have the potential to fuel the search for broad antiviral agents.
© 2022. The Author(s), under exclusive licence to Springer Nature Limited.