Nidovirus replication structures : hijacking membranes to support viral RNA synthesis

Abstract

Positive-stranded RNA viruses replicate in the cytoplasm of host cells and their replication complexes are associated with modified cell membranes. We investigated the structure of the nidovirus-induced membrane modifications and found that nidoviruses transform the endoplasmic reticulum into a reticulovesicular network (RVN) that integrates numerous inter-connected double-membrane vesicles (DMVs), and, in the case of coronaviruses, __vesicle packets__ and convoluted membranes (CM). The membranes of DMVs and CM labeled abundantly for replicase proteins, whereas the DMV interior contained the double-stranded RNA and therefore is the presumed site of viral RNA synthesis. Electron tomography, semi-permeabilization and nuclease digestion experiments suggested that the DMV interior is inaccessible from the cytosol, implying that double-stranded RNA is compartmentalized by membranes. Furthermore, brefeldin A treatment showed that early secretory pathway proteins do not play a direct role in RVN morphogenesis or functionality of the SARS-CoV replication complex. Continued translation was not an absolute requirement for viral RNA synthesis, however, was necessary for RVN development thus suggesting a link between accumulation of replicase proteins and RVN formation. Together with biochemical studies of the viral enzyme complex, our ultrastructural description of the RVN aids to further dissect the early stages of the nidovirus infection and virus-host interactions.