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Journal of Virology, September 1999, p. 7641-7657, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Localization of Mouse Hepatitis Virus Nonstructural Proteins and RNA Synthesis Indicates a Role for Late Endosomes in Viral Replication

Yvonne van der Meer,1 Eric J. Snijder,1 Jessika C. Dobbe,1 Sibylle Schleich,2 Mark R. Denison,3,4,5 Willy J. M. Spaan,1 and Jacomine Krijnse Locker2,*

Department of Virology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands1; EMBL, 69117 Heidelberg, Germany2; and Department of Pediatrics,3 Department of Microbiology and Immunology,4 and the Elizabeth B. Lamb Center for Pediatric Research,5 Vanderbilt University Medical Center, Nashville, Tennessee 37232

Received 29 March 1999/Accepted 8 June 1999

The aim of the present study was to define the site of replication of the coronavirus mouse hepatitis virus (MHV). Antibodies directed against several proteins derived from the gene 1 polyprotein, including the 3C-like protease (3CLpro), the putative polymerase (POL), helicase, and a recently described protein (p22) derived from the C terminus of the open reading frame 1a protein (CT1a), were used to probe MHV-infected cells by indirect immunofluorescence (IF) and electron microscopy (EM). At early times of infection, all of these proteins showed a distinct punctate labeling by IF. Antibodies to the nucleocapsid protein also displayed a punctate labeling that largely colocalized with the replicase proteins. When infected cells were metabolically labeled with 5-bromouridine 5'-triphosphate (BrUTP), the site of viral RNA synthesis was shown by IF to colocalize with CT1a and the 3CLpro. As shown by EM, CT1a localized to LAMP-1 positive late endosomes/lysosomes while POL accumulated predominantly in multilayered structures with the appearance of endocytic carrier vesicles. These latter structures were also labeled to some extent with both anti-CT1a and LAMP-1 antibodies and could be filled with fluid phase endocytic tracers. When EM was used to determine sites of BrUTP incorporation into viral RNA at early times of infection, the viral RNA localized to late endosomal membranes as well. These results demonstrate that MHV replication occurs on late endosomal membranes and that several nonstructural proteins derived from the gene 1 polyprotein may participate in the formation and function of the viral replication complexes.

* Corresponding author. Mailing address: EMBL, Meyerhofstrasse 1, 69117 Heidelberg, Germany. Phone: 49 6221 387508. Fax: 49 6221 387306 or 387512. E-mail: KRIJNSE{at}EMBL-Heidelberg.DE.

Journal of Virology, September 1999, p. 7641-7657, Vol. 73, No. 9
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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