Mutational analysis of the Bunyamwera orthobunyavirus nucleocapsid protein gene.
The bunyavirus nucleocapsid protein, N, is a multifunctional protein that encapsidates each of the three negative-sense genome segments to form ribonucleoprotein complexes that are the functional templates for viral transcription and replication. In addition, N protein molecules interact with themselves to form oligomers, with the viral L (RNA polymerase) protein, with the carboxy-terminal regions of either or both of the virion glycoproteins, and probably also with host cell proteins. Bunyamwera virus (BUNV), the prototype bunyavirus, encodes an N protein of 233 amino acids in length. To learn more about the roles of individual amino acids in the different interactions of N, we performed a wide-scale mutagenic analysis of the protein, and 110 single-point mutants were obtained. When the mutants were employed in a minireplicon assay to examine their effects on viral RNA synthesis, a wide range of activities compared to those of wild-type N protein were observed; changes at nine amino acid positions resulted in severely impaired RNA synthesis. Seventy-seven mutant clones were selected for use in the bunyavirus reverse genetics system, and 57 viable recombinant viruses were recovered. The recombinant viruses displayed a range of plaque sizes and titers in cell culture (from approximately 10(3) to 10(8) PFU/ml), and a number of viruses were shown to be temperature sensitive. Different assays were applied to determine why 20 mutant N proteins could not be recovered into infectious virus. Based on these results, a preliminary domain map of the BUNV N protein is proposed.
Eifan
Saleh A
SA
Centre for Biomolecular Sciences, School of Biology, University of St. Andrews, North Haugh, St. Andrews, Fife KY16 9ST, Scotland, United Kingdom.
Elliott
Richard M
RM
D000595:Amino Acid Sequence; D002042:Bunyamwera virus / Q000235:genetics* / Q000378:metabolism; D002044:Bunyaviridae Infections; D008969:Molecular Sequence Data; D016296:Mutagenesis; D019590:Nucleocapsid Proteins / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames; D010641:Phenotype; D017354:Point Mutation; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA; D014779:Virus Replication / Q000235:genetics
C423924:N protein, Bunyamwera bunyavirus; D019590:Nucleocapsid Proteins; D012367:RNA, Viral