Genetic analysis of Murine hepatitis virus nsp4 in virus replication.
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 K(398) through T(492) of nsp4 are dispensable. Together, the experiments identify important residues and regions for studies of nsp4 topology, function, and interactions.
Sparks
Jennifer S
JS
Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232-2581, USA.
Lu
Xiaotao
X
Denison
Mark R
MR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D019295:Computational Biology; D006224:Cricetinae; D004252:DNA Mutational Analysis; D017353:Gene Deletion; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D009154:Mutation; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000235:genetics*
D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase