Vaccinia Virus–Encoded Ribonucleotide Reductase Subunits Are Differentially Required for Replication and Pathogenesis

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

doi:10.1371/journal.ppat.1000984

Don B. Gammon, David H. Evans + Show 5 more

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https://doi.org/10.1371/journal.ppat.1000984

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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. Co-immunoprecipitation 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.

Highlights

Critical for the replication of all organisms and DNA viruses is the conversion of ribonucleotides to deoxynucleotides to serve as building blocks for genome synthesis and repair
We report here that the R2, but not the R1, subunit of vaccinia virus (VACV) Ribonucleotide reductases (RRs) is required for efficient replication and virulence
We provide evidence that several poxvirus R2 proteins form novel complexes with host R1 subunits and this interaction is required for efficient VACV replication in primate cells

Summary

Introduction

Critical for the replication of all organisms and DNA viruses is the conversion of ribonucleotides to deoxynucleotides to serve as building blocks for genome synthesis and repair. Class I RR enzymes are assembled from both large (R1; 80– 100 kDa) and small (R2; 37–44 kDa) protein subunits, which associate to form enzymatically-active R12R22 tetrameric complexes [1]. These complexes require oxygen to generate a tyrosyl radical found within R2 subunits [1,4], which is transferred to R1 subunits to generate a thiyl radical used in rNDP reduction. Mutant proteins containing amino acid substitutions at either the tyrosine involved in radical formation [9] or any of the proposed transfer pathway residues [4,6,8,10,11] form inactive RR complexes, indicating that both radical formation and transfer are required for catalysis

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