| Vaccinia virus is presumed to encode all functions necessary for the timely and correct duplication of its 192 kb genome. However, the mechanism of replication of this duplex DNA genome is not completely understood. Several proposed replicative functions remain as yet unidentified.; A search for a candidate single-strand DNA binding protein (SSB) was carried out, largely based on reports in the literature of an abundant 34 kDa phosphoprotein which was isolated from virosomes early during vaccinia infection and which demonstrated some ssDNA binding activity. Hybrid selection was used to screen for open reading frames (ORFs) throughout the genome which encode abundant early proteins of approximately 34 kDa.; The I3L ORF was chosen for further study. The I3 protein was overexpressed in bacteria using the T7 expression system of Studier and coworkers, and a polyclonal antiserum was raised against this recombinant protein. Immunofluorescence analysis revealed that the protein localizes to sites of viral DNA replication in vivo, and immunoprecipitation demonstrated that the I3 protein is a major phosphoprotein of vaccinia infection.; Metabolically labeled infected-cell extracts were examined for SSB activity using single-stranded DNA-cellulose chromatography. In both ({dollar}sp{lcub}35{rcub}{dollar}S) and ({dollar}sp{lcub}32{rcub}{dollar}P) labeled extracts, I3 exhibited the highest degree of single-strand DNA binding of any viral protein. Immunoblot analysis revealed a single binding profile common to both labeled extracts, suggesting that phosphorylated I3 binds as well as the total pool of I3 represented in the ({dollar}sp{lcub}35{rcub}{dollar}S) labeled extracts. These experiments reveal I3 as the best candidate for the vaccinia SSB. Attempted isolation of a recombinant virus with a null I3 allele were unsuccessful, indicating that a functional I3 gene is essential for viral replication.; Both the recombinant and native proteins were purified to homogeneity and subjected to electrophoretic mobility shift analysis in order to further characterize the DNA binding properties of the protein. I3 binds tightly to single-stranded DNA of lengths greater than 17 nucleotides, while binding to double-stranded DNA of any size is negligible. The protein forms discrete complexes on single-stranded oligonucleotides irrespective of sequence, which allows for an estimate of the binding site size of approximately 10 nucleotides. Cooperativity appears to be low, as saturated complexes are not seen in shifts with sub-saturating concentrations of protein. |
