Abstract
Positive-strand RNA viruses, a large group including human pathogens such as SARS-coronavirus (SARS-CoV), replicate in the cytoplasm of infected host cells. Their replication complexes are commonly associated with modified host cell membranes. Membrane structures supporting viral RNA synthesis range from distinct spherular membrane invaginations to more elaborate webs of packed membranes and vesicles. Generally, their ultrastructure, morphogenesis, and exact role in viral replication remain to be defined. Poorly characterized double-membrane vesicles (DMVs) were previously implicated in SARS-CoV RNA synthesis. We have now applied electron tomography of cryofixed infected cells for the three-dimensional imaging of coronavirus-induced membrane alterations at high resolution. Our analysis defines a unique reticulovesicular network of modified endoplasmic reticulum that integrates convoluted membranes, numerous interconnected DMVs (diameter 200–300 nm), and “vesicle packets” apparently arising from DMV merger. The convoluted membranes were most abundantly immunolabeled for viral replicase subunits. However, double-stranded RNA, presumably revealing the site of viral RNA synthesis, mainly localized to the DMV interior. Since we could not discern a connection between DMV interior and cytosol, our analysis raises several questions about the mechanism of DMV formation and the actual site of SARS-CoV RNA synthesis. Our data document the extensive virus-induced reorganization of host cell membranes into a network that is used to organize viral replication and possibly hide replicating RNA from antiviral defense mechanisms. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of this “replication network” will aid to further dissect the early stages of the coronavirus life cycle and its virus-host interactions.
Highlights
Viruses rely on the host cell’s infrastructure and metabolism during essentially all stages of their replication cycle and have adopted strategies to coordinate a variety of molecular interactions in both time and intracellular space
Viruses with a positive-stranded RNA genome replicate in the cytoplasm of infected host cells. Their replication is driven by a membrane-bound viral enzyme complex that is commonly associated with modified intracellular membranes
We used electron microscopy and tomography for the three-dimensional imaging of the membrane alterations induced by severe acute respiratory syndrome (SARS)-coronavirus, a member of the virus group with the largest RNA genome known to date
Summary
Viruses rely on the host cell’s infrastructure and metabolism during essentially all stages of their replication cycle and have adopted strategies to coordinate a variety of molecular interactions in both time and intracellular space. The fact that the replication complexes of positive-strand RNA (þRNA) viruses of eukaryotes are invariably associated with (modified) intracellular membranes appears to be a striking example of such a strategy [1,2,3,4,5,6,7,8]. The morphogenesis, ultrastructure, and function of these complexes, sometimes referred to as ‘‘viral factories,’’ are only beginning to be understood. They may facilitate the concentration of viral macromolecules and provide a membrane-based structural framework for RNA synthesis. Defining the structure–function relationships that govern the membrane-associated replication of þRNA viruses, a large virus cluster including many important pathogens, will enhance our general understanding of their molecular biology and may have important implications for the development of novel antiviral control strategies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
