Mutations in the C-terminal loop of the nucleocapsid protein affect vesicular stomatitis virus RNA replication and transcription differentially.
The 2.9-A structure of the vesicular stomatitis virus nucleocapsid (N) protein bound to RNA shows the RNA to be tightly sequestered between the two lobes of the N protein. Domain movement of the lobes of the N protein has been postulated to facilitate polymerase access to the RNA template. We investigated the roles of individual amino acid residues in the C-terminal loop, involved in long-range interactions between N protein monomers, in forming functional ribonucleoprotein (RNP) templates. The effects of specific N protein mutations on its expression, interaction with the phosphoprotein, and formation of RNP templates that supported viral RNA replication and transcription were examined. Mutations introduced into the C-terminal loop, predicted to break contact with other residues in the loop, caused up to 10-fold increases in RNA replication without an equivalent stimulation of transcription. Mutation F348A, predicted to break contact between the C-terminal loop and the N-terminal arm, formed templates that supported wild-type levels of RNA replication but almost no transcription. These data show that mutations in the C-terminal loop of the N protein can disparately affect RNA replication and transcription, indicating that the N protein plays a role in modulating RNP template function beyond its structural role in RNA encapsidation.
Harouaka
Djamila
D
Department of Pathology, University of Virginia, Charlottesville, VA 22908-0904, USA.
Wertz
Gail W
GW
D015153:Blotting, Western; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D047468:Immunoprecipitation; D009154:Mutation / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics*; D012367:RNA, Viral / Q000096:biosynthesis / Q000235:genetics; D048348:Reverse Transcription / Q000235:genetics*; D012260:Ribonucleases / Q000378:metabolism; D018116:Vesiculovirus / Q000235:genetics*; D014779:Virus Replication / Q000235:genetics*
D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D012260:Ribonucleases