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Journal of Virology, August 1999, p. 6862-6871, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Putative Helicase of the Coronavirus Mouse
Hepatitis Virus Is Processed from the Replicase Gene Polyprotein
and Localizes in Complexes That Are Active in Viral RNA
Synthesis
Mark R.
Denison,1,*
Willy J. M.
Spaan,2
Yvonne
van der
Meer,2
C. Anne
Gibson,1
Amy C.
Sims,1
Erik
Prentice,1 and
Xiao
Tao
Lu1
Department of Pediatrics, Department of
Microbiology and Immunology, and The Elizabeth B. Lamb Center for
Pediatric Research, Vanderbilt University, Nashville,
Tennessee,1 and Department of Virology,
Leiden University Medical Center, Leiden, The
Netherlands2
Received 29 January 1999/Accepted 20 April 1999
The coronavirus mouse hepatitis virus (MHV) translates its
replicase gene (gene 1) into two co-amino-terminal polyproteins, polyprotein 1a and polyprotein 1ab. The gene 1 polyproteins are processed by viral proteinases to yield at least 15 mature products, including a putative RNA helicase from polyprotein 1ab that is presumed
to be involved in viral RNA synthesis. Antibodies directed against
polypeptides encoded by open reading frame 1b were used to characterize
the expression and processing of the MHV helicase and to define the
relationship of helicase to the viral nucleocapsid protein (N) and to
sites of viral RNA synthesis in MHV-infected cells. The antihelicase
antibodies detected a 67-kDa protein in MHV-infected cells that was
translated and processed throughout the virus life cycle. Processing of
the 67-kDa helicase from polyprotein 1ab was abolished by E64d, a known
inhibitor of the MHV 3C-like proteinase. When infected cells were
probed for helicase by immunofluorescence laser confocal microscopy,
the protein was detected in patterns that varied from punctate
perinuclear complexes to large structures that occupied much of the
cell cytoplasm. Dual-labeling studies of infected cells for helicase
and bromo-UTP-labeled RNA demonstrated that the vast majority of
helicase-containing complexes were active in viral RNA synthesis.
Dual-labeling studies for helicase and the MHV N protein showed that
the two proteins almost completely colocalized, indicating that N was
associated with the helicase-containing complexes. This study
demonstrates that the putative RNA helicase is closely associated with
MHV RNA synthesis and suggests that complexes containing helicase, N,
and new viral RNA are the viral replication complexes.
*
Corresponding author. Mailing address: Department of
Pediatrics, Vanderbilt University Medical Center, D7235 MCN, Nashville, TN 37232-2581. Phone: (615) 343-9881. Fax: (615) 343-9723. E-mail: mark.denison{at}mcmail.vanderbilt.edu.
Journal of Virology, August 1999, p. 6862-6871, Vol. 73, No. 8
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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