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.

SUMMARY Vaccinia virus (VACV) encodes DNA polymerase and additional proteins that enable cytoplasmic replication. We confirmed the ability of VACV DNA ligase mutants to replicate and tested the hypothesis that cellular ligases compensate for loss of viral gene expression. Knock-down of human DNA ligase I but not other ligases with siRNA or a specific inhibitor severely reduced replication of viral DNA in cells infected with VACV ligase-deficient mutants, indicating that the cellular enzyme plays a complementary role. Replication of ligase-deficient VACV was greatly reduced and delayed in resting primary cells, correlating with initial low levels of ligase I and subsequent viral induction and localization of ligase I in virus factories. These studies indicate that DNA ligation is essential for poxvirus replication and explain the ability of ligase deletion mutants to replicate in dividing cells but exhibit decreased pathogenicity in mice. Encoding of a ligase might allow VACV to “jump-start” DNA synthesis.

Background: Low levels of uracil in DNA result from misincorporation of dUMP or cytosine deamination. Vaccinia virus (VACV), the prototype poxvirus, encodes two enzymes that can potentially reduce the amount of uracil in DNA. Deoxyuridine triphosphatase (dUTPase) hydrolyzes dUTP, generating dUMP for biosynthesis of thymidine nucleotides while decreasing the availability of dUTP for misincorporation; uracil DNA glycosylase (UNG) cleaves uracil N-glycosylic bonds in DNA initiating base excision repair. Studies with actively dividing cells showed that the VACV UNG protein is required for DNA replication but the UNG catalytic site is not, whereas the dUTPase gene can be deleted without impairing virus replication. Recombinant VACV with an UNG catalytic site mutation was attenuated in vivo, while a dUTPase deletion mutant was not. However, the importance of the two enzymes for replication in quiescent cells, their possible synergy and roles in virulence have not been fully assessed.

Although B lymphocytes are a major constituent of lymphoid organs and acquire a significantly altered phenotype and function in HIV-infected individuals, it remains unclear whether CD4-negative B cells are a susceptible host for viral entry and long-term productive infection. We screened a number of Epstein-Barr virus (EBV)-positive and-negative Burkitt's lymphoma (BL) B cell lines as well as subpopulations of normal B cells that include tonsillar naive and germinal center/memory B cells for the expression of HIV-1 receptors CD4, CXCR4, and CCR5. Cell lines and resting or activated normal B cells lacked CD4 and CCR5 but expressed CXCR4. We demonstrate HIV-1 infection of a CD4-negative, EBV-negative (BL) cell line, CA46, which remained productively infected yet noncytopathic for more than 36 months in culture. HIV-1 (HTLV-III(B)) infection of CA46 cells was mediated through CXCR4 in a CD4-independent manner and correlated with upregulation of the expression of B cell activation markers CD23 and CD95 (Fas receptor). Despite Fas receptor expression, HIV-1-infected CA46 cells remained resistant to Fas-mediated cell death. CA46-derived, CD4-independent viral isolates were proficient in infecting and causing syncytium formation in Molt4 T cells. The HIV-1 genomic organization in persistently infected CA46 clones was found to be predominantly unintegrated linear and circular DNA. Importantly, naive and germinal center/memory B cells could also be infected by HIV-1 in a CD4-independent manner. Although these B cell subpopulations expressed moderate to high levels of CXCR4, they required activation through CD40 and interleukin 4 receptor for infection. These findings point to B cells as an additional HIV-1 target and suggest a structural evolution of the HIV-1 genome responsible for CD4-independent and noncytopathic infections.

Vaccinia virus encodes a 90-kDa protein, conserved in all poxviruses, with DNA primase and nucleoside triphosphatase activities. DNA primase products, synthesized with a single stranded ϕX174 DNA template, were resolved as dinucleotides and long RNAs on denaturing polyacrylamide and agarose gels. Following phosphatase treatment, the dinucleotides GpC and ApC in a 4:1 ratio were identified by nearest neighbor analysis in which 32P was transferred from [α-32P]CTP to initiating purine nucleotides. Differences in the nucleotide binding sites for initiation and elongation were suggested by the absence of CpC and UpC dinucleotides as well as the inability of deoxynucleotides to mediate primer synthesis despite their incorporation into mixed RNA/DNA primers. Strong primase activity was detected with an oligo(dC) template. However, there was only weak activity with an oligo(dT) template and none with oligo(dA) or oligo(dG). The absence of stringent template specificity is consistent with a role for the enzyme in priming DNA synthesis at the replication fork.

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