Abstract
To examine the possible role of the vaccinia virus glutaredoxin as a cofactor for viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were measured in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus and with mutants of vaccinia that lacked a functional reductase or glutaredoxin. In infections of untreated host cells, the lack of viral ribonucleotide reductase or glutaredoxin had only small effects upon virus growth. When host cells were pretreated with alpha-amanitin, which blocks host RNA polymerase II but not viral transcription, viral DNA synthesis was markedly reduced in infections with either of the mutants when compared with wild type infections. Relative to dNTP levels in wild type infections, pools of dCTP, but not of the other dNTPs, were significantly reduced in infections of amanitin-treated cells with either mutant. The parallel depletion of dCTP in the two mutant suggests that the role of glutaredoxin may be to function as a cofactor for viral ribonucleotide reductase. The data suggest that both viral proteins become essential for DNA replication only when levels of the corresponding host cell proteins are depleted.
Highlights
From the ‡Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, §Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0455, and ¶Department of Genetic Engineering, Korea University, Seoul, 136-701, Korea
The protein is synthesized after DNA replication has begun and may well have other roles in the virus growth cycle, one cannot rule out the possibility that it is acting as a hydrogen donor for ribonucleotide reductase
To assess the role of the viral glutaredoxin in relation to the activity of the viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were compared in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus, and with two mutant viruses, M1, with an inactive ribonucleotide reductase R1 subunit, and vGRXϪ(gpt), a glutaredoxin mutant
Summary
From the ‡Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331-7305, §Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892-0455, and ¶Department of Genetic Engineering, Korea University, Seoul, 136-701, Korea. To examine the possible role of the vaccinia virus glutaredoxin as a cofactor for viral ribonucleotide reductase, viral growth, DNA synthesis, and dNTP pools were measured in infections of B-SC-40 monkey kidney cells with wild type vaccinia virus and with mutants of vaccinia that lacked a functional reductase or glutaredoxin. M1, which has been shown to have no ribonucleotide reductase activity as measured by conversion of tritiated CDP to dCDP, has been reported to replicate at wild type levels, as measured by plaque-forming ability at 24 and 48 h postinfection [4] This would suggest that in the absence of functional viral RNR, viral replication could be sustained by a supply of dNTPs produced by the host cell’s ribonucleotide reductase. We show that in the absence of host cell transcription, the synthesis of dNTPs, as well as of viral DNA, is greatly affected in mutant viruses for either ribonucleotide reductase or glutaredoxin
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