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Journal of Virology, December 2000, p. 11557-11565, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Expression of an Altered Ribonucleotide Reductase Activity Associated with the Replication of Murine Cytomegalovirus in Quiescent Fibroblasts

David Lembo,1 Giorgio Gribaudo,1 Anders Hofer,2 Ludovica Riera,1 Maura Cornaglia,3 Alessandra Mondo,1 Alessandra Angeretti,1 Marisa Gariglio,4 Lars Thelander,2 and Santo Landolfo3,*

Department of Public Health and Microbiology, University of Torino,1 Immunogenetics and Experimental Oncology Center, C.N.R.,3 Turin, and Department of Medical Sciences, University of Novara, Novara,4 Italy, and Department of Medical Biosciences, Medical Biochemistry, Umea University, Umea, Sweden2

Received 16 June 2000/Accepted 14 September 2000

Ribonucleotide reductase (RNR) is an essential enzyme for the de novo synthesis of both cellular and viral DNA and catalyzes the conversion of ribonucleoside diphosphates into the corresponding deoxyribonucleoside diphosphates. The enzyme consists of two nonidentical subunits, termed R1 and R2, whose expression is very low in resting cells and maximal in S-phase cells. Here we show that murine cytomegalovirus (MCMV) replication depends on ribonucleotide reduction since it is prevented by the RNR inhibitor hydroxyurea. MCMV infection of quiescent fibroblasts markedly induces both mRNA and protein corresponding to the cellular R2 subunit, whereas expression of the cellular R1 subunit does not appear to be up-regulated. The increase in R2 gene expression is due to an increase in gene transcription, since the activity of a reporter gene driven by the mouse R2 promoter is induced following virus infection. Cotransfection experiments revealed that expression of the viral immediate-early 1 protein was sufficient to mediate the increase in R2 promoter activity. It was found that the viral gene M45, encoding a putative homologue of the R1 subunit, is expressed 24 and 48 h after infection. Meanwhile, we observed an expansion of the deoxyribonucleoside triphosphate pool between 24 and 48 h after infection; however, neither CDP reduction nor viral replication was inhibited by treatment with 10 mM thymidine. These findings indicate the induction of an RNR activity with an altered allosteric regulation compared to the mouse RNR following MCMV infection and suggest that the virus R1 homologue may complex with the induced cellular R2 protein to reconstitute a new RNR activity.

* Corresponding author. Mailing address: Department of Public Health and Microbiology, University of Torino, Via Santena 9, 10126 Turin, Italy. Phone: 39.011.6706604. Fax: 39.011.6636436. E-mail: landolfo{at}molinette.unito.it.

Journal of Virology, December 2000, p. 11557-11565, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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