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Related Experiment Videos

Poxvirus DNA primase.

Frank S De Silva1, Whitney Lewis, Peter Berglund

  • 1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-3210, USA.

Proceedings of the National Academy of Sciences of the United States of America
|November 15, 2007
PubMed
Summary
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The vaccinia virus D5 protein, crucial for DNA replication, acts as an NTPase and primase. Its conserved residues are essential for viral replication and genome synthesis, highlighting its potential as a therapeutic target.

Area of Science:

  • Virology
  • Molecular Biology
  • Biochemistry

Background:

  • Poxviruses are large, enveloped viruses replicating in host cell cytoplasm.
  • Vaccinia virus, a poxvirus, requires at least six viral proteins for its double-stranded DNA genome replication.
  • The D5 protein is a key enzyme with both NTPase and primase activities.

Purpose of the Study:

  • To investigate the role of conserved aspartic acid residues in the D5 protein's primase active site.
  • To characterize the in vitro enzymatic activities of the D5 protein.
  • To explore the evolutionary conservation and potential therapeutic relevance of D5 orthologs.

Main Methods:

  • Site-directed mutagenesis of conserved aspartic acid residues in the D5 protein.
  • In vivo complementation assays for infectious virus formation, genome, and plasmid replication.

Related Experiment Videos

  • In vitro enzymatic assays using purified recombinant D5 protein and bacteriophage DNA templates.
  • Biochemical characterization of D5 protein's oligoribonucleotide synthesis and NTPase activity.

    • Main Results:

    • Specific aspartic acid residues in the D5 primase active site are essential for viral replication and genome synthesis in vivo.
    • Purified recombinant D5 protein synthesizes oligoribonucleotides in vitro, dependent on ATP/GTP and divalent cations.
    • Mutagenesis studies demonstrate independent inactivation of D5's primase and NTPase activities.
    • Conserved D5 orthologs are found across poxviruses and other large DNA viruses.

    Conclusions:

    • The D5 protein's primase activity is critical for poxvirus DNA replication.
    • D5 protein's enzymatic functions are essential for viral propagation.
    • Viral primases like D5 are conserved across large DNA viruses and represent potential therapeutic targets.