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Journal of Virology, November 2007, p. 12554-12563, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01257-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Genetic Analysis of Murine Hepatitis Virus nsp4 in Virus Replication{triangledown}

Jennifer S. Sparks,1,3 Xiaotao Lu,2,3 and Mark R. Denison1,2,3*

Departments of Microbiology and Immunology,1 Pediatrics,2 the Elizabeth B. Lamb Center for Pediatric Research, Vanderbilt University School of Medicine, Nashville, Tennessee 372323

Received 8 June 2007/ Accepted 30 August 2007

Coronavirus replicase polyproteins are translated from the genomic positive-strand RNA and are proteolytically processed by three viral proteases to yield 16 mature nonstructural proteins (nsp1 to nsp16). nsp4 contains four predicted transmembrane-spanning regions (TM1, -2, -3, and -4), demonstrates characteristics of an integral membrane protein, and is thought to be essential for the formation and function of viral replication complexes on cellular membranes. To determine the requirement of nsp4 for murine hepatitis virus (MHV) infection in culture, engineered deletions and mutations in TMs and intervening soluble regions were analyzed for effects on virus recovery, growth, RNA synthesis, protein expression, and intracellular membrane modifications. In-frame partial or complete deletions of nsp4; deletions of TM1, -2, and -3; and alanine substitutions of multiple conserved, clustered, charged residues in nsp4 resulted in viruses that were nonrecoverable, viruses highly impaired in growth and RNA synthesis, and viruses that were nearly wild type in replication. The results indicate that nsp4 is required for MHV replication and that while putative TM1, -2, and -3 and specific charged residues may be essential for productive virus infection, putative TM4 and the carboxy-terminal amino acids K398 through T492 of nsp4 are dispensable. Together, the experiments identify important residues and regions for studies of nsp4 topology, function, and interactions.

* Corresponding author. Mailing address: Department of Pediatrics, Vanderbilt University Medical Center, D6127 MCN, 1161 21st Ave. S., Nashville, TN 37232-2581. Phone: (615) 343-9881. Fax: (615) 343-9723. E-mail: mark.denison{at}vanderbilt.edu

{triangledown} Published ahead of print on 12 September 2007.

Journal of Virology, November 2007, p. 12554-12563, Vol. 81, No. 22
0022-538X/07/$08.00+0     doi:10.1128/JVI.01257-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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