TAGAWA Yuko The University of Tokyo, Institute of Medical Science, Research associate, 医科学研究所, 教務職員 (40178538)
NAGAI Yoshiyuki The University of Tokyo, Institute of Medical Science, Professor, 医科学研究所, 教授 (20022874)
|Budget Amount *help
¥7,800,000 (Direct Cost: ¥7,800,000)
Fiscal Year 1998: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥3,100,000 (Direct Cost: ¥3,100,000)
Sendai virus (SeV) is a member of the recently renamed Respirovirus genus of the Paramyxovirinae. SeV P gene gives use to two accessory proteins, V and C, in addition to the phospho (P) protein. While the P protein has been identified to be a modulator protein essential for viral RNA synthesis, the roles of the V and C proteins in viral replication and pathogenesis have remained unclear. It has not even been established whether these proteins are essential or simply auxiliary for viral replication. These issues are now being clarified by SeV reverse genetics.
While the exact copy of the P gene encodes the P protein, the cotranscriptionally edited mRNA directs the V protein. This editing event involves pseudotemplated insertion of one G residue to the nascent chain at a specific site in the template, which shifts the reading frame register form the P ORF to access the -1 ORF encoding a cysteine-rich protein. By disrupting the editing site or introducing nonsense mutations just downstream
of the editing site in a cDNA plasmid generating a full-length copy of the SeV antigenome, we created mutant viruses totally defective in V gene expression (V(-)) or with truncation of die C-terminal half (VDELTAC). Their characterization led to the conclusions that, although completely dispensable for viral replication in tissue cultured cells, the V protein was essential for maintaining a high viral load in mice to cause pneumonia and that. this "luxury" iii vivo function was encoded primarily by the cysteine-rich C-terminal half. The V protein appeared to be essential for SeV to cope with some early host response(s) recruited to clear the virus.
The Sendai C protein is expressed as a nested set of proteins, C', C, Y1 and Y2, from both the unedited P mRNA and the edited V mRNA using the +1 frame relative to the P ORF and starting at different initiation codons. Among them, C protein is the major species expressed in infected cells at molar ratio several-fold higher than the other three. We first silenced C' and C proteins and found that the recovered C/C'(-) viruses were impaired in RNA synthesis and severely attenuated in replication in tissue cultured cells. More notably, the C/C'(-) viruses were almost totally incapable of growing productively in mice and, hence, nonpathogenic for the mice. Silencing all four C proteins was also possible, giving rise to a still more impaired but viable clone, the 4C(-) virus. Thus, it was concluded that SeV C proteins are categorically nonessential gene products but greatly contribute to full replication capability in vitro and are indispensable for in vivo multiplication and pathogenesis. Less