Mutational analysis of a predicted zinc-binding motif in the 26-kilodalton protein encoded by the vaccinia virus A2L gene: correlation of zinc binding with late transcriptional transactivation activity.
Transient transfection assays indicated that A2L is one of three vaccinia virus intermediate genes that are required for the transcriptional transactivation of viral late genes. We have expressed the A2L open reading frame in Escherichia coli and shown by blotting experiments that the 26-kDa protein binds zinc, a property predicted by the presence of a CX2CX13CX2C zinc finger motif. The specificity for zinc binding was demonstrated by competition with other metals. The role of the sequence motif in zinc binding was established by analysis of a series of mutations, including truncations and conservative single amino acid substitutions. Mutations that reduced zinc binding in vitro prevented the ability of A2L to transactivate late genes in vivo.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J G
JG
Keck
F
F
Feigenbaum
B
B
Moss
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002460:Cell Line; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis; D015870:Gene Expression; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015032:Zinc / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics*
C081260:A2L protein, Vaccinia virus; D003062:Codon; D011994:Recombinant Proteins; D015534:Trans-Activators; D014764:Viral Proteins; D015032:Zinc