Genetic and biochemical evidence for an oligomeric structure of the functional L polymerase of the prototypic arenavirus lymphocytic choriomeningitis virus.
The arenavirus L protein has the characteristic sequence motifs conserved among the RNA-dependent RNA polymerase L proteins of negative-strand (NS) RNA viruses. Studies based on the use of reverse-genetics approaches have provided direct experimental evidence of the key role played by the arenavirus L protein in viral-RNA synthesis. Sequence alignment shows six conserved domains among L proteins of NS RNA viruses. The proposed polymerase module of L is located within its domain III, which contains highly conserved amino acids within motifs designated A and C. We have examined the role of these conserved residues in the polymerase activity of the L protein of the prototypic arenavirus, lymphocytic choriomeningitis virus (LCMV), in vivo using a minigenome rescue assay. We show here that the presence of sequence SDD, a characteristic of motif C of segmented NS RNA viruses, as well as the presence of the highly conserved D residue within motif A of L proteins, is strictly required for the polymerase activity of the LCMV L protein. The strong dominant negative phenotype associated with many of the mutants examined and results from coimmunoprecipitation studies provided genetic and biochemical evidence, respectively, for the requirement of the L-L interaction for the polymerase activity of the LCMV L protein.
Sánchez
Ana B
AB
Department of Neuropharmacology IMM-6, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
de la Torre
Juan C
JC
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D006224:Cricetinae; D005814:Genes, Viral; D008217:Lymphocytic choriomeningitis virus / Q000201:enzymology* / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase