Mutational analysis of the Sendai virus V protein: importance of the conserved residues for Zn binding, virus pathogenesis, and efficient RNA editing.
The V protein of Sendai virus (SeV) is nonessential for virus replication in cell culture but indispensable for viral pathogenicity in mice. At the C terminus of the V protein, there are amino acid residues conserved among the members of the Paramyxovinae subfamily that are clustered in three regions: region I, just downstream of the RNA editing site; and regions II and III, cysteine-rich zinc-finger-like regions. In the present study, we introduced mutations into the conserved amino acids and generated nine mutant viruses. All of the viruses had impaired virus replication in mouse lungs and attenuated virulence in mice. Furthermore, the C-terminal polypeptides fused with glutathione-S-transferase with a mutation in region I, II, or III all had impaired Zn binding in a (65)Zn-binding assay in solution. These results demonstrate that the conserved amino acids are important for V protein function, probably via protein conformation dependent on Zn binding. One mutant, SeV V-H(318)N, had inefficient RNA editing, indicating that the nucleotide that is a part of the codon encoding histidine at position 318 is conserved for the RNA editing machinery. In addition, to determine the function of the C-terminal extension of the V protein, which is not translated in recent virulent field isolates, a translational stop codon was introduced to generate the corresponding short V protein. The mutant virus showed similar virus propagation and pathogenicity, indicating that C-terminal extension of the V protein is not relevant to virus pathogenesis.
Fukuhara
Noriko
N
Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Huang
Cheng
C
Kiyotani
Katsuhiro
K
Yoshida
Tetsuya
T
Sakaguchi
Takemasa
T
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D017124:Conserved Sequence; D008168:Lung / Q000821:virology; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009154:Mutation; D017393:RNA Editing; D012333:RNA, Messenger / Q000032:analysis; D029082:Sendai virus / Q000472:pathogenicity*; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D014779:Virus Replication; D015032:Zinc / Q000378:metabolism*
D012333:RNA, Messenger; C111739:V protein, Sendai virus; D014764:Viral Proteins; D015032:Zinc