JVI Accepts, published online ahead of print on 4 November 2009

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J. Virol. doi:10.1128/JVI.01826-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Integrity of the early secretory pathway promotes, but is not required for SARS-coronavirus RNA synthesis and virus-induced remodeling of ER membranes

Kèvin Knoops, Cindy Swett-Tapia, Sjoerd H.E. van den Worm, Aartjan J.W. te Velthuis, Abraham J. Koster, A. Mieke Mommaas, Eric J. Snijder^*, and Marjolein Kikkert^*

Molecular Virology Laboratory, Department of Medical Microbiology, and Section Electron Microscopy, Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands

^* To whom correspondence should be addressed. Email: e.j.snijder{at}lumc.nl. m.kikkert{at}lumc.nl.

To accommodate its RNA synthesis in the infected cell, SARS-coronavirus (SARS-CoV) induces a cytoplasmic reticulovesicular network (RVN) that is derived from endoplasmic reticulum (ER) membranes. We set out to investigate how the early secretory pathway interacts with the RVN and the viral replication/transcription complex (RTC) that is anchored to it. When the secretory pathway was disrupted by Brefeldin A (BFA) treatment at the start of infection, RVN formation and viral RTC activity were not blocked and continued up to 11 h post infection, although RNA synthesis was reduced by about 80%. In vitro RTC assays, using membrane fractions from infected cells, demonstrated that BFA does not directly interfere with the activity of the viral RNA-synthesizing enzymes. Confocal microscopy studies showed that early secretory pathway components are not associated with SARS-CoV-induced replication sites, although our studies revealed that infection induces a remarkable redistribution of the translocon subunit Sec61. Ultrastructural studies, including electron tomography, revealed that the formation of the RVN and all its previously documented features can occur in the presence of BFA, despite differences in volume and morphology of the network. We therefore conclude that early secretory pathway proteins do not play a direct role in RVN morphogenesis or functionality of the SARS-CoV RTC. The BFA-induced disruption of ER integrity and functionality probably affects the overall quality of the membrane scaffold that is needed to support the viral RTC and/or the availability of specific host factors, which in turn compromises viral RNA synthesis.