Deletion of the varicella-zoster virus large subunit of ribonucleotide reductase impairs growth of virus in vitro.
Cells infected with varicella-zoster virus (VZV) express a viral ribonucleotide reductase which is distinct from that present in uninfected cells. VZV open reading frames 18 and 19 (ORF18 and ORF19) are homologous to the herpes simplex virus type 1 genes encoding the small and large subunits of ribonucleotide reductase, respectively. We generated recombinant VZV by transfecting cultured cells with four overlapping cosmid DNAs. To construct a virus lacking ribonucleotide reductase, we deleted 97% of VZV ORF19 from one of the cosmids. Transfection of this cosmid with the other parental cosmids yielded a VZV mutant with a 2.3-kbp deletion confirmed by Southern blot analysis. Virus-specific ribonucleotide reductase activity was not detected in cells infected with VZV lacking ORF19. Infection of melanoma cells with ORF19-deleted VZV resulted in plaques smaller than those produced by infection with the parental VZV. The mutant virus also exhibited a growth rate slightly slower than that of the parental virus. Chemical inhibition of the VZV ribonucleotide reductase has been shown to potentiate the anti-VZV activity of acyclovir. Similarly, the concentration of acyclovir required to inhibit plaque formation by 50% was threefold lower for the VZV ribonucleotide reductase deletion mutants than for parental virus. We conclude that the VZV ribonucleotide reductase large subunit is not essential for virus infection in vitro; however, deletion of the gene impairs the growth of VZV in cell culture and renders the virus more susceptible to inhibition by acyclovir.
Heineman
T C
TC
Medical Virology Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Cohen
J I
JI
D000212:Acyclovir / Q000494:pharmacology; D001483:Base Sequence; D015139:Blotting, Southern; D003360:Cosmids; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D017353:Gene Deletion; D014645:Herpesvirus 3, Human / Q000187:drug effects / Q000201:enzymology / Q000235:genetics / Q000254:growth & development*; D006801:Humans; D015151:Immunoblotting; D008546:Melanoma, Experimental; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012264:Ribonucleotide Reductases / Q000187:drug effects / Q000235:genetics*; D014407:Tumor Cells, Cultured; D010948:Viral Plaque Assay; D014779:Virus Replication / Q000235:genetics
D004274:DNA, Recombinant; D004279:DNA, Viral; D012264:Ribonucleotide Reductases; D000212:Acyclovir