Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

Abstract

Previous findings that the vaccinia virus uracil DNA glycosylase is required for virus DNA replication, coupled with an inability to isolate a mutant with an active site substitution in the glycosylase gene, were surprising, as such enzymes function in DNA repair and bacterial, yeast, and mammalian null mutants are viable. To further study the role of the viral protein, we constructed recombinant vaccinia viruses with single or double mutations (D68N and H181L) in the uracil DNA glycosylase conserved catalytic site by using a complementing cell line that constitutively expresses the viral enzyme. Although these mutations abolished uracil DNA glycosylase activity, they did not prevent viral DNA replication or propagation on a variety of noncomplementing cell lines or human primary skin fibroblasts. In contrast, replication of a uracil DNA glycosylase deletion mutant occurred only in the complementing cell line. Therefore, the uracil DNA glycosylase has an essential role in DNA replication that is independent of its glycosylase activity. Nevertheless, the conservation of the catalytic site in all poxvirus orthologs suggested an important role in vivo. This idea was confirmed by the decreased virulence of catalytic-site mutants when administered by the intranasal route to mice.

Differential regulation of S-region hypermutation and class-switch recombination by noncanonical functions of uracil DNA glycosylase

“…Interestingly, we also observed that WT and loss-of-catalysis UNG mutants can form a dimer in the cell (Fig. 5 A and B), which raises a possibility that the dimeric structure of UNG could favor a more dynamic scaffolding function, as observed in the case of vaccinia virus UNG (23,24). Future study may reveal how the structure of UNG promotes protein-protein interaction during SHM and CSR at the physiological milieu.…”

“…Curiously, this function of UNG is independent of its catalytic activity. Similarly, vaccinia virus UNG protein, but not its catalytic activity, is essential for the viral replicative cycle by the formation of a large complex to support processive DNA synthesis (23,24). UNG is also known to be recruited to DSB foci in association with γH2AX (25).…”

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

“…In contrast, the poxvirus UNG is an obligate component of the replication machinery. Studies performed on D4 (the UNG encoded by vaccinia virus (VACV)) demonstrated that the presence of the protein is crucial for DNA replication as knockout mutants lacking D4 are not viable (14,15), whereas its glycosylase activity is dispensable (16). Later, data obtained from the Traktman group revealed that D4 is a subunit of the VACV DNA polymerase holoenzyme (17).…”

“…Furthermore, sequence alignments of VACV D4 with related UNGs identified conserved active site residues that are predicted to form the uracil recognition pocket (i.e. Tyr-70, Phe-79, and Asn-120) in addition to Asp-68 and His-181 that are needed for glycosyl bond cleavage (16,21). When the crystal structure of D4 is superimposed onto the human enzyme bound to DNA, these residues match perfectly their human counterparts (20).…”

Characterization of the Uracil-DNA Glycosylase Activity of Epstein-Barr Virus BKRF3 and Its Role in Lytic Viral DNA Replication

“…The vaccinia virus D4R gene, which encodes the viral UNG, is essential for replication in tissue culture, although the catalytic activity is dispensable (14), suggesting that vaccinia virus UNG may participate in the formation of multiprotein DNA replication complexes. Indeed, the interaction of vaccinia virus UNG with A20 (a stoichiometric component of the viral processivity factor), along with E9 (viral DNA polymerase), is necessary and sufficient for the processive polymerase holoenzyme (67).…”