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Journal of Virology, April 2002, p. 3697-3708, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3697-3708.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

RNA Replication of Mouse Hepatitis Virus Takes Place at Double-Membrane Vesicles

Rainer Gosert,1 Amornrat Kanjanahaluethai,2,3 Denise Egger,1 Kurt Bienz,1 and Susan C. Baker2*

Institute for Medical Microbiology, University of Basel, Basel, Switzerland,1 Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois,2 Department of Microbiology, Chiang Mai University, Chiang Mai, Thailand3

Received 23 October 2001/ Accepted 22 January 2002

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Loyola University of Chicago, Stritch School of Medicine, 2160 S. First Ave., Bldg. 105, Rm. 3929, Maywood, IL 60153. Phone: (708) 216-6910. Fax: (708) 216-9574. E-mail: sbaker1{at}lumc.edu.

Journal of Virology, April 2002, p. 3697-3708, Vol. 76, No. 8
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.8.3697-3708.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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