Importance of hydrogen bond contacts between the N protein and RNA genome of vesicular stomatitis virus in encapsidation and RNA synthesis.
Vesicular stomatitis virus (VSV) genomic RNA encapsidated by the nucleocapsid (N) protein is the template for transcription and replication by the viral polymerase. We analyzed the 2.9-A structure of the VSV N protein bound to RNA (T. J. Green, X. Zhang, G. W. Wertz, and M. Luo, Science 313:357-360, 2006) and identified amino acid residues with the potential to interact with RNA via hydrogen bonds. The contributions of these interactions to N protein function were investigated by individually substituting the residues with alanine and assaying the effect of these mutations on N protein expression, on the ability of the N protein to interact with the phosphoprotein (P), and on its ability to encapsidate RNA and generate templates that can support transcription and RNA replication. These studies identified individual amino acids critical for N protein function. Nine nucleotides are associated with each N monomer and contorted into two quasi-helices within the N protein RNA binding cavity. We found that N protein residues that formed hydrogen bond contacts with the nucleotides in quasi-helix 2 were critical to the encapsidation of RNA and the production of templates that can support RNA synthesis. Individual hydrogen bond interactions between the N protein and the nucleotides of quasi-helix 1 were not essential for ribonucleoprotein (RNP) template function. Residue R143 forms a hydrogen bond with nucleotide 9, the nucleotide that extends between N monomers. R143A mutant N protein failed to encapsidate RNA and to support RNA synthesis and suppressed wild-type N protein function. These studies show a direct correlation between viral RNA synthesis and N protein residues structurally positioned to interact with RNA.
Rainsford
Edward W
EW
Department of Pathology, University of Virginia, 415 Lane Road, Rm. 3051, MR5 building, P.O. Box 800904, Charlottesville, VA 22908-0904, USA.
Harouaka
Djamila
D
Wertz
Gail W
GW
D019943:Amino Acid Substitution; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D016679:Genome, Viral; D006860:Hydrogen Bonding; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology*; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011487:Protein Conformation; D012367:RNA, Viral / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D014162:Transfection; D018116:Vesiculovirus / Q000235:genetics* / Q000502:physiology*; D015678:Viral Structural Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D019065:Virus Assembly
D017931:DNA Primers; D046911:Macromolecular Substances; D019590:Nucleocapsid Proteins; C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012367:RNA, Viral; D015678:Viral Structural Proteins