The 3′-to-5′ exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination

Don B. Gammon, David H. Evans

Research output: Contribution to journalArticle

43 Citations (Scopus)

Abstract

Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3′-to-5′ proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3′ ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV r) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3′-to-5′ exonuclease domain of the viral polymerase. Viruses encoding a CDVr mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3′ ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3′-to-5′ exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

Original languageEnglish (US)
Pages (from-to)4236-4250
Number of pages15
JournalJournal of Virology
Volume83
Issue number9
DOIs
StatePublished - May 1 2009

Fingerprint

genetic recombination
Exonucleases
Vaccinia virus
DNA-directed DNA polymerase
Genetic Recombination
Viruses
viruses
Poxviridae
DNA-Directed DNA Polymerase
DNA
antiviral agents
mutation
Phosphodiesterase I
enzymes
Mutation
Viral DNA
Enzymes
Antiviral Agents
vaccinia virus DNA polymerase
cidofovir

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

The 3′-to-5′ exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination. / Gammon, Don B.; Evans, David H.

In: Journal of Virology, Vol. 83, No. 9, 01.05.2009, p. 4236-4250.

Research output: Contribution to journalArticle

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