title: Poxvirus DNA primase.
abstract: 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 double-stranded 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. Furthermore, purified recombinant D5 protein synthesized oligoribonucleotides in vitro. Incorporation of label from [alpha-(32)P]CTP or [alpha-(32)P]UTP into a RNase-sensitive and DNase-resistant product was demonstrated by using single-stranded circular bacteriophage DNA templates and depended on ATP or GTP and a divalent cation. Mutagenesis studies showed that the primase and NTPase activities of the recombinant D5 protein could be independently inactivated. Highly conserved orthologs of D5 are present in all poxviruses that have been sequenced, and more diverged orthologs are found in members of all other families of nucleocytoplasmic large DNA viruses. These viral primases may have roles in initiation of DNA replication or lagging-strand synthesis and represent potential therapeutic targets.
authors.affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210, USA.
authors.forename: Frank S
authors.initials: FS
authors.lastname: De Silva
authors.forename: Whitney
authors.initials: W
authors.lastname: Lewis
authors.forename: Peter
authors.initials: P
authors.lastname: Berglund
authors.forename: Eugene V
authors.initials: EV
authors.lastname: Koonin
authors.forename: Bernard
authors.initials: B
authors.lastname: Moss
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D019915:DNA Primase / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors / Q000235:genetics; D016679:Genome, Viral / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011212:Poxviridae / Q000201:enzymology* / Q000235:genetics
chemical_list: D000596:Amino Acids; D009841:Oligonucleotides; D019915:DNA Primase; D017766:Acid Anhydride Hydrolases