Ralf Bartenschlager

Positive-strand RNA viruses subvert the cellular endomembrane system for the generation of distinct compartments termed replication organelles (ROs) that harbor the site where viral RNAs are generated. In this study, we corroborate that the SARS-CoV-2 non-structural proteins 3 and 4 (nsp3 and nsp4) suffice to remodel the endoplasmic reticulum to form double-membrane vesicles similar to ROs observed in viral infection. Cellular membrane alterations induced by nsp3/4 expression were evaluated through electron tomography and confocal microscopy, and nsp3/4-associated host factors were identified using mass spectrometry. The role of these host factors in virus infection was determined using gene silencing, identifying several host proteins involved in the SARS-CoV-2 replication cycle. Combining the gene silencing approach with ultrastructural analysis of nsp3/4-expressing cells, we found that the host dependency factors FAM149B1, CCAR2, and ZC3HAV1 play a role in the formation of double-membrane vesicles in a replication-independent manner. IMPORTANCE Remodeling of the cellular endomembrane system by viruses allows for efficient and coordinated replication of the viral genome in distinct subcellular compartments termed replication organelles. As a critical step in the viral life cycle, replication organelle formation is an attractive target for therapeutic intervention, but factors central to this process are only partially understood. In this study, we corroborate that two viral proteins, nsp3 and nsp4, are the major drivers of membrane remodeling in SARS-CoV-2 infection. We further report a number of host cell factors interacting with these viral proteins and supporting the viral replication cycle, some of them by contributing to the formation of the SARS-CoV-2 replication organelle.

Emerging omicron subtypes with immune escape lead to inadequate vaccine response with breakthrough infections in immunocompromised individuals such as Anti-neutrophil Cytoplasmic Antibody (ANCA)-associated vasculitis (AAV) patients. As AAV is considered an orphan disease, there are still limited data on SARS-CoV-2 vaccination and prospective studies that have focused exclusively on AAV patients are lacking. In addition, there are safety concerns regarding the use of highly immunogenic mRNA vaccines in autoimmune diseases, and further studies investigating reactogenicity are urgently needed. In this prospective observational cohort study, we performed a detailed characterization of neutralizing antibody responses against omicron subtypes and provided a longitudinal assessment of vaccine reactogenicity and AAV disease activity. Different vaccine doses were generally well tolerated and no AAV relapses occurred during follow-up. AAV patients had significantly lower anti-S1 IgG and surrogate-neutralizing antibodies after first, second, and third vaccine doses as compared to healthy controls, respectively. Live-virus neutralization assays against omicron subtypes BA.1 and BA.5 revealed that previous SARS-CoV-2 vaccines result in an inadequate neutralizing immune response in immunocompromised AAV patients. These data demonstrate that new vaccination strategies including adapted mRNA vaccines against epitopes of emerging variants are needed to help protect highly vulnerable individuals such as AAV patients.