SARS-Coronavirus Replication Is Supported by a Reticulovesicular Network of Modified Endoplasmic Reticulum

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. 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. Little is understood about the formation and architecture of these replication structures and their exact role in viral RNA synthesis. 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. Previously, coronaviruses were reported to induce large numbers of isolated “double-membrane vesicles” (DMVs). However, our present studies reveal an elaborate reticulovesicular network of modified endoplasmic reticulum membranes with which SARS-coronavirus replicative proteins are associated. The lumen of this unique membrane network contains numerous large (diameter 250–300 nm) “inner vesicles,” which were formerly thought to reside in isolated DMVs. Intriguingly, although the interior of these vesicles does not appear to be connected to the cytosol, it labels abundantly for double-stranded RNA, which presumably is present at the site of viral RNA synthesis. The ultrastructural dissection of this elaborate “replication network” shows how coronaviruses extensively reorganize the host cell's membrane infrastructure, to coordinate their replication cycle, and possibly also hide replicating RNA from antiviral defense mechanisms.