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

PLoS Pathogens

Muriel Mari¹  Fulvio Reggiori¹  Frank J. M. van Kuppeveld²  Eddie G. te Lintelo³  Peter J. M. Rottier³  Cornelis A. M. de Haan³  Monique H. Verheije³  Matthijs Raaben³ 

[1] Department of Cell Biology and Institute of Biomembranes, University Medical Centre Utrecht, Utrecht, The Netherlands;Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands;Virology Division, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, The Netherlands

关键词: Small interfering RNAs;    Luciferase;    Viral replication;    Transfection;    Golgi apparatus;    Guanine nucleotide exchange factors;    Immunostaining;    Vesicles;   

DOI  :  10.1371/journal.ppat.1000088

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY