Vaccinia virus ribonucleotide reductase : gene sequencing, intracellular localization, and interaction with a DNA-binding protein Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/dv13zz54k

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  • Vaccinia virus infected monkey kidney cells had been previously shown to have an increased ribonucleoside diphosphate reductase (RR) activity. DNA from mutant virus resistant to hydroxyurea were digested with restriction endonucleases and were shown to have substoichiometric amounts of the Hind III F fragment. Additional information from Southern blotting experiments localized the putative small subunit (R2) gene to the left end of the Hind III F fragment of the vaccinia virus genome. The entire open reading frame of the R2 gene and the flanking regions was sequenced and the translated sequence found to be 80% homologous to the mouse R2 polypeptide. A combination of in situ and in vitro experiments addressed the question of macromolecular interactions involving vaccinia ribonucleotide reductase (FIR). Replication of double stranded viral DNA occurs in very discrete loci in infected cells and these DNA factories can be isolated from gently lysed cell in sucrose gradients. RR was detected at low levels (less than 5% of the total R2) with the rapidly sedimenting DNA by using antibodies against FIR. In situ crosslinking experiments were attempted with no specific interaction determined at this time. Immunolocalization experiments have given evidence for localization of large subunit (R1) polypeptide to the viral inclusion bodies. The most conclusive results utilized anti-idiotypic antibodies against the antibodies to R2 protein. lmmunolocalization experiments have shown the putative R2 binding protein to be localized at the sites of viral DNA synthesis. lmmunoprecipitations show a single predominant viral polypeptide which also has proven to be a DNA binding (phospho)protein. Screening a lambda phale expression library of vaccinia with the anti-idiotypic antibody localized the binding site to the carboxy terminal 81 amino acids in open reading frame 1-3 of the vaccinia genome. The open reading frame was cloned into a pET11c expression vector and the partially purified recombinant protein was shown to have specificity for single-stranded DNA as well as stimulate vaccinia RR activity.
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