A DNA ligase gene in the copenhagen strain of vaccinia virus is nonessential for viral replication and recombination

Colinas, Robert J.; Goebel, Scott J.; Davis, Stephen W.; Johnson, Gerard P.; Norton, Elizabeth; Paoletti, Enzo

doi:10.1016/0042-6822(90)90295-3

Cited by 52 publications

(35 citation statements)

References 37 publications

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“…These enzymes are not essential for virus growth in culture (4,14), but mutant virus lacking a catalytically active enzyme (like the ⌬L29 strain) exhibit defects in DNA synthesis in many cell types (Fig. 2) (27).…”

Section: Discussionmentioning

confidence: 99%

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Lin¹,

Li²,

Irwin³

et al. 2008

J Virol

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Vaccinia virus replication is inhibited by etoposide and mitoxantrone even though poxviruses do not encode the type II topoisomerases that are the specific targets of these drugs. Furthermore, one can isolate drugresistant virus carrying mutations in the viral DNA ligase and yet the ligase is not known to exhibit sensitivity to these drugs. A yeast two-hybrid screen was used to search for proteins binding to vaccinia ligase, and one of the nine proteins identified comprised a portion (residue 901 to end) of human topoisomerase II␤. One can prevent the interaction by introducing a C 11 -to-Y substitution mutation into the N terminus of the ligase bait protein, which is one of the mutations conferring etoposide and mitoxantrone resistance. Coimmunoprecipitation methods showed that the native ligase and a Flag-tagged recombinant protein form complexes with human topoisomerase II␣/␤ in infected cells and that this interaction can also be disrupted by mutations in the A50R (ligase) gene. Immunofluorescence microscopy showed that both topoisomerase II␣ and II␤ antigens are recruited to cytoplasmic sites of virus replication and that less topoisomerase was recruited to these sites in cells infected with mutant virus than in cells infected with wild-type virus. Immunoelectron microscopy confirmed the presence of topoisomerases II␣/␤ in virosomes, but the enzyme could not be detected in mature virus particles. We propose that the genetics of etoposide and mitoxantrone resistance can be explained by vaccinia ligase binding to cellular topoisomerase II and recruiting this nuclear enzyme to sites of virus biogenesis. Although other nuclear DNA binding proteins have been detected in virosomes, this appears to be the first demonstration of an enzyme being selectively recruited to sites of poxvirus DNA synthesis and assembly.Topoisomerases play a critical role in modulating DNA superhelical density and in unknotting and decatenating the structures formed during replication, recombination, and repair (reviewed in references 2 and 5). Mammalian cells carry several different kinds of topoisomerases, classified as being either type I or type II enzymes. Both classes of enzyme catalyze DNA strand breakage and rejoining, but type I enzymes catalyze reactions involving single-strand breaks, while the type II topoisomerases catalyze double-strand cleavage reactions. The mammalian type IB enzyme (Topo I) is well adapted for removing the superhelical stress created by the replication and transcription machinery. The two closely related type IIA enzymes (Topo II␣ and Topo II␤) may also catalyze these same reactions but probably play a more critical role in processes like chromatin reorganization and chromosome segregation. A third class of type IA enzymes (Topo III␣ and Topo III␤)

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Section: Discussionmentioning

confidence: 99%

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Lin¹,

Li²,

Irwin³

et al. 2008

J Virol

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“…Predicted proteins with homology to enzymes include, DNA ligase, TmpK, guanylate kinase (GmpK), SOD, 3fl-HSD and protein kinases. Enzyme activity has been demonstrated for DNA ligase (Kerr & Smith, 1989;Colinas et al, 1990) and TmpK (S. Hughes, unpublished). SOD, GmpK and one of the proteins homologous to protein kinases (B 12R) (Howard & Smith, 1989) are unlikely to be active.…”

Section: Discussionmentioning

confidence: 99%

“…Enzyme activity has been demonstrated for DNA ligase (Kerr & Smith, 1989;Colinas et al, 1990) and TmpK (S. Hughes, unpublished results). In addition three other genes are predicted to encode proteins related to known enzymes.…”

Section: (I) Genes Potentially Encoding Enzymesmentioning

confidence: 99%

Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat

Smith

Chan

Howard

1991

Journal of General Virology

155

107

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The nucleotide sequence of 42 090 bp of vaccinia virus strain WR is presented. The sequence includes the SalI L, F, G and I fragments and starts near the centre of the HindIII A fragment and extends rightwards towards the genomic terminus, finishing approximately 0-5 kb internal of the inverted terminal repeat (ITR). Translation of this region has identified 65 open reading frames (ORFs) of greater than 65 amino acids in length. Fifty-one of these which do not extensively overlap other larger ORFs have been subjected to further analysis; the other 14 are termed minor ORFs. In the rightmost 28.7 kb, the genes are, with one exception, transcribed towards the genomic terminus, similar to the arrangement of genes at the left end of the virus genome. Internal of this region the genes are expressed off either DNA strand but still predominately rightwards. ORFs are tightly packed with few intergenic non-coding regions of greater than 250 bp. Protein sequence comparisons have established a remarkably high number of homologies with entries in existing protein databases. Of these, DNA ligase, thymidylate kinase, two serine-threonine protein kinases, two serine proteinase inhibitors (serpins), two interleukin-1 receptor homologues and a discontinuous ORF related to tumour necrosis factor receptor have been reported. Other homologies include lectins, profilin, 3fl-hydroxy steroid dehydrogenase, superoxide dismutase, guanylate kinase, ankyrin and complement factor H. In addition, there are a number of polypeptides with predicted properties of membraneassociated, secretory or glyco-proteins. Twelve gene families are described here and elsewhere. There is considerable similarity between genes from the right and left end of the virus genome that may have arisen by terminal transposition events. Several differences from the corresponding region of vaccinia virus strain Copenhagen sequence are noted. Near the right terminus the sequences diverge completely, and internal of this there are multiple examples of deletion of short sequences (eight to 10 nucleotides) that lie within penta-or hexanucleotide direct repeats.

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“…However, further studies are needed, because G5R ts mutants exhibited a block in the initial stage of morphogenesis rather than DNA replication (45). Although VACV encodes a DNA ligase that can substitute for the replicative yeast ligase, the gene is not essential and is not conserved in all poxviruses (46)(47)(48). This result is also perplexing, because there is no evidence for the recruitment of cellular DNA ligase I from the nucleus to cytoplasmic virus factories (48).…”

Section: Discussionmentioning

confidence: 99%

Poxvirus DNA primase

Silva

Lewis²,

Berglund³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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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...

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A DNA ligase gene in the copenhagen strain of vaccinia virus is nonessential for viral replication and recombination

Cited by 52 publications

References 37 publications

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Vaccinia Virus DNA Ligase Recruits Cellular Topoisomerase II to Sites of Viral Replication and Assembly

Nucleotide sequence of 42 kbp of vaccinia virus strain WR from near the right inverted terminal repeat

Poxvirus DNA primase

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