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Journal of Virology, October 2009, p. 9709-9719, Vol. 83, No. 19
0022-538X/09/$08.00+0 doi:10.1128/JVI.00600-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Mouse Norovirus Replication Is Associated with Virus-Induced Vesicle Clusters Originating from Membranes Derived from the Secretory Pathway
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Jennifer L. Hyde,1,2
Stanislav V. Sosnovtsev,3
Kim Y. Green,3
Christiane Wobus,4
Herbert W. Virgin,5 and
Jason M. Mackenzie2*
School of Molecular and Microbial Science, University of Queensland, Brisbane, Queensland 4072,1 Department of Microbiology, La Trobe University, Melbourne, Victoria 3086, Australia,2 Norovirus Gastroenteritis Unit, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892,3 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0620,4 Washington University School of Medicine, St. Louis, Missouri 631105
Received 23 March 2009/ Accepted 30 June 2009
Human noroviruses (family Caliciviridae) are the leading cause of nonbacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community, the study of human norovirus has largely been hindered due to the inability to cultivate the viruses ex vivo and the lack of a small-animal model. In 2003, the discovery of a novel murine norovirus (MNV-1) and the identification of the tropism of MNV-1 for cells of a mononuclear origin led to the establishment of the first norovirus tissue culture system. Like other positive-sense RNA viruses, MNV-1 replication is associated with host membranes, which undergo significant rearrangement during infection. We characterize here the subcellular localization of the MNV-1 open reading frame 1 proteins and viral double-stranded RNA (dsRNA). Over the course of infection, dsRNA and the MNV-1 RNA-dependent RNA polymerase (NS7) were observed to proliferate from punctate foci located in the perinuclear region. All of the MNV-1 open reading frame 1 proteins were observed to colocalize with dsRNA during the course of infection. The MNV-1 replication complex was immunolocalized to virus-induced vesicle clusters formed in the cytoplasm of infected cells. Both dsRNA and MNV-1 NS7 were observed to localize to the limiting membrane of the individual clusters by cryo-immunoelectron microscopy. We show that the MNV-1 replication complex initially associates with membranes derived from the endoplasmic reticulum, trans-Golgi apparatus, and endosomes. In addition, we show that MNV-1 replication is insensitive to the fungal metabolite brefeldin A and consistently does not appear to recruit coatomer protein complex I (COPI) or COPII component proteins during replication. These data provide preliminary insights into key aspects of replication of MNV-1, which will potentially further our understanding of the pathogenesis of noroviruses and aid in the identification of potential targets for drug development.
* Corresponding author. Mailing address: Department of Microbiology, La Trobe University, Thomas Cherry Bldg., Kingsbury Dr., Melbourne, Victoria 3086, Australia. Phone: (613) 9479-2225. Fax: (613) 9476-1222. E-mail: j.mackenzie{at}latrobe.edu.au
Published ahead of print on 8 July 2009.
Supplemental material for this article may be found at http://jvi.asm.org/.
Journal of Virology, October 2009, p. 9709-9719, Vol. 83, No. 19
0022-538X/09/$08.00+0 doi:10.1128/JVI.00600-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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