Mouse Hepatitis Coronavirus RNA Replication Depends on GBF1-Mediated ARF1 Activation

Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepat...

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Published in:PLoS pathogens Vol. 4; no. 6; p. e1000088
Main Authors: Verheije, Monique H., Raaben, Matthijs, Mari, Muriel, te Lintelo, Eddie G., Reggiori, Fulvio, van Kuppeveld, Frank J. M., Rottier, Peter J. M., de Haan, Cornelis A. M.
Format: Journal Article
Language:English
Published: United States Public Library of Science 01.06.2008
Public Library of Science (PLoS)
Subjects:
ADP-Ribosylation Factor 1 - genetics
ADP-Ribosylation Factor 1 - metabolism
Animals
Brefeldin A - pharmacology
Causes of
Cell Line
Coronavirus
Coronavirus Infections - immunology
Coronavirus Infections - virology
Coronaviruses
Dogs
Guanine Nucleotide Exchange Factors - physiology
Health aspects
Hepatitis
Humans
Medical research
Mice
Microscopy
Murine hepatitis virus - physiology
Mutation
Physiological aspects
Proteins
R&D
Research & development
RNA
RNA, Small Interfering - pharmacology
RNA, Viral - biosynthesis
Virology/Viral and Gene Regulation
Viruses
Netherlands
ISSN:1553-7374, 1553-7366, 1553-7374
Online Access:Get full text
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Summary:Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected.
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Conceived and designed the experiments: MV FR FV PR CD. Performed the experiments: MV MR MM ET CD. Analyzed the data: MV MR MM. Contributed reagents/materials/analysis tools: MM FR FV PR CD. Wrote the paper: MV.
ISSN:1553-7374
1553-7366
1553-7374
DOI:10.1371/journal.ppat.1000088