Vaccinia virus-encoded ribonucleotide reductase subunits are differentially required for replication and pathogenesis

Don B. Gammon, Branawan Gowrishankar, Sophie Duraffour, Graciela Andrei, Chris Upton, David H. Evans

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during Dntp synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R12R22 heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Coimmunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.

Original languageEnglish (US)
Article numbere1000984
Pages (from-to)1-20
Number of pages20
JournalPLoS Pathogens
Volume6
Issue number7
DOIs
StatePublished - Jul 1 2010

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Poxviridae
Ribonucleotide Reductases
Vaccinia virus
Monkeypox virus
Proteins
Growth
Cowpox virus
Variola virus
Orthopoxvirus
Base Composition
Mutant Proteins
Computational Biology
Catalysis
Point Mutation
Genes
Virulence
Mammals
Nucleotides
Viruses
Phenotype

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

Vaccinia virus-encoded ribonucleotide reductase subunits are differentially required for replication and pathogenesis. / Gammon, Don B.; Gowrishankar, Branawan; Duraffour, Sophie; Andrei, Graciela; Upton, Chris; Evans, David H.

In: PLoS Pathogens, Vol. 6, No. 7, e1000984, 01.07.2010, p. 1-20.

Research output: Contribution to journalArticle

Gammon, Don B. ; Gowrishankar, Branawan ; Duraffour, Sophie ; Andrei, Graciela ; Upton, Chris ; Evans, David H. / Vaccinia virus-encoded ribonucleotide reductase subunits are differentially required for replication and pathogenesis. In: PLoS Pathogens. 2010 ; Vol. 6, No. 7. pp. 1-20.
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