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.

Poxviruses are large enveloped viruses that replicate in the cytoplasm of vertebrate or invertebrate cells. At least six virus-encoded proteins are required for synthesis and processing of the doublestranded DNA genome of vaccinia virus, the prototype member of the family. One of these proteins, D5, is an NTPase that contains an N-terminal archaeoeukaryotic primase domain and a C-terminal superfamily III helicase domain. Here we report that individual conserved aspartic acid residues in the predicted primase active site were required for in vivo complementation of infectious virus formation as well as genome and plasmid replication. T he poxviruses comprise a large family of DNA viruses that include the causal agent of smallpox (1). Unlike most other DNA viruses, poxviruses replicate entirely in the cytoplasm. To accommodate this unique lifestyle, they encode enzymes and factors needed for genome replication and transcription, which are potential targets for antivirals (2). Poxvirus genomes are 130,000-300,000 bp long and consist of two complementary strands of DNA that are covalently linked to form hairpins at each end. A transcription system is packaged in infectious virus particles allowing early mRNAs to be synthesized soon after cell entry. Early proteins are used for host defense, genome replication, and transcription of intermediate stage genes. Intermediate proteins include late-stage transcription factors, whereas late proteins are mostly involved in virus assembly and dissemination.Most laboratory studies of poxviruses are carried out by using vaccinia virus (VACV). Studies with conditional lethal mutants indicate that five VACV early proteins are required for DNA replication, namely, E9 DNA polymerase, D4 uracil DNA glycosylase, A20 processivity factor, B1 protein kinase, and D5 NTPase (reviewed in ref.3). The polymerase catalyzes primerand template-dependent DNA synthesis and possesses 3Ј to 5Ј exonucleolytic activity (4, 5). The essential role of D4 in DNA replication (6) is independent of its uracil DNA glycosylase activity (7), which presumably has a facultative repair function. The A20 and D4 proteins physically interact (8, 9) and together provide processivity for the DNA polymerase (10). The B1 kinase phosphorylates a cellular DNA-binding protein called BAF and prevents the latter from blocking VACV DNA replication (11). The fast stop DNA replication phenotype of conditional lethal D5 mutants suggests a function at the replication fork (12). D5 also interacts with A20 (8, 9) and forms multimers (13). Extensive protein sequence analyses have indicated that the C-terminal region of the 90-kDa D5 protein belongs to the helicase superfamily III within the AAAϩ class of NTPases, which includes the replicative helicases of numerous other DNA and RNA viruses (14,15). Furthermore, the N-terminal domain of D5 has sequence and structural features that are common to the archaeoeukaryotic primase superfamily, the members of which have diverse roles in DNA replication and repair (16). Nevertheless, the onl...

Background: Replication of the vaccinia virus genome occurs in cytoplasmic factory areas and is dependent on the virus-encoded DNA polymerase and at least four additional viral proteins. DNA synthesis appears to start near the ends of the genome, but specific origin sequences have not been defined. Surprisingly, transfected circular DNA lacking specific viral sequences is also replicated in poxvirus-infected cells. Origin-independent plasmid replication depends on the viral DNA polymerase, but neither the number of additional viral proteins nor the site of replication has been determined.