Site-directed mutagenesis of the avian retrovirus nucleocapsid protein, pp 12. Mutation which affects RNA binding in vitro blocks viral replication.
Site-directed mutagenesis was used to replace the lysine residues of the avian retrovirus nucleocapsid protein pp 12 that have previously been shown to be important for RNA binding. Single amino acid substitutions at Lys-36, -37, and -39 of the protein were not sufficient to affect virus production as measured by reverse transcriptase activity in virus particles released from transfected cells. However, when Lys-36 and Lys-37 were simultaneously replaced by isoleucine residues, there was a complete block of viral replication. As expected from the single mutant analyses, the wild type phenotype could be restored by reverting either or both of the isoleucine residues to lysine residues. Analysis of a bacterially produced rp 12 protein containing Ile-36 and Ile-37 indicated that the protein has a low affinity binding for RNA, as compared to wild type protein. Unlike wild type, the binding is independent of phosphorylation at Ser-40, the major site of phosphorylation of the protein in vivo. A quail cell line was established that expresses virus particles containing the doubly mutated pp 12. Analysis of these particles indicated that they lack viral RNA. Thus, the binding defect in pp 12 is correlated with the inability to package viral RNA.
Fu
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Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Katz
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Skalka
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Leis
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D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics*; D002213:Capsid / Q000235:genetics*; D036022:Capsid Proteins; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D008239:Lysine; D009154:Mutation; D012367:RNA, Viral / Q000378:metabolism*; D012191:Retroviridae Proteins; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000235:genetics*; D014779:Virus Replication
D036022:Capsid Proteins; D012367:RNA, Viral; D012191:Retroviridae Proteins; D014758:Viral Core Proteins; C054897:nucleocapsid protein, Avian retrovirus; D008239:Lysine