Characterization of a vaccinia virus-encoded double-stranded RNA-binding protein that may be involved in inhibition of the double-stranded RNA-dependent protein kinase.
The work described in this article identifies a vaccinia virus-encoded protein that may be involved in inhibition of the interferon-induced, double-stranded RNA-dependent protein kinase. Extracts prepared from vaccinia virus (WR strain)-infected cells contain an inhibitor of this kinase. Inhibition was reduced in extracts from which dsRNA-binding proteins had been removed by preadsorption to poly(rI).poly(rC)-Sepharose, suggesting that a dsRNA-binding protein may be involved in kinase inhibition. A single major virus-specific polypeptide of Mr = 25,000 (p25) bound to the poly(rI).poly(rC)-Sepharose. p25 was synthesized in a coupled in vitro transcription/translation system programmed with vaccinia cores, indicating that it is a vaccinia-encoded protein. Synthesis of p25 was detected at early times, by 2 hr post infection, peaked at 5 hours postinfection, and decreased during the late phase of virus replication. In the presence of cytosine arabinoside p25 synthesis did not decrease at late times postinfection. Kinase inhibitory activity accumulated with similar kinetics to p25, both in the presence and absence of cytosine arabinoside. Kinase inhibitory activity copurified with p25, through gel filtration, and Cibacron blue-affinity chromatography. Removal of p25 by precipitation with antiserum to p25 decreased kinase inhibitory activity in extracts prepared from vaccinia virus-infected cells. These results suggest that p25 may be necessary for the specific kinase inhibitory activity detected in vaccinia virus-infected cells.
Department of Microbiology, Arizona State University, Tempe 85287-2701.
J C
JC
Watson
H W
HW
Chang
B L
BL
Jacobs
D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003593:Cytoplasm / Q000378:metabolism; D005814:Genes, Viral; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D007739:L Cells; D051379:Mice; D008970:Molecular Weight; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D019892:eIF-2 Kinase
D000906:Antibodies; D047428:Protein Kinase Inhibitors; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins; D019892:eIF-2 Kinase