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Journal of Virology, December 2002, p. 12135-12148, Vol. 76, No. 23
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.23.12135-12148.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Cell Cycle Arrest by Human Cytomegalovirus 86-kDa IE2 Protein Resembles Premature Senescence

Emanuela Noris,^1, Claudia Zannetti,¹ Anna Demurtas,² John Sinclair,³ Marco De Andrea,^1,4 Marisa Gariglio,⁴ and Santo Landolfo^1*

Department of Public Health and Microbiology, University of Turin,¹ Pathology Service, Molinette Hospital, Turin,² Department of Medical Sciences, University of Eastern Piedmont, Novara, Italy,⁴ Department of Medicine, University of Cambridge, Cambridge, United Kingdom³

Received 19 April 2002/ Accepted 5 August 2002

Primary human embryo lung fibroblasts and adult diploid fibroblasts infected by the human cytomegalovirus (HCMV) display ß-galactosidase (ß-Gal) activity at neutral pH (senescence-associated ß-Gal [SA-ß-Gal] activity) and overexpression of the plasminogen activator inhibitor type 1 (PAI-1) gene, two widely recognized markers of the process designated premature cell senescence. This activity is higher when cells are serum starved for 48 h before infection, a process that speeds and facilitates HCMV infection but that is insufficient by itself to induce senescence. Fibroblasts infected by HCMV do not incorporate bromodeoxyuridine, a prerequisite for the formal definition of senescence. At the molecular level, cells infected by HCMV, beside the accumulation of large amounts of the cell cycle regulators p53 and pRb, the latter in its hyperphosphorylated form, display a strong induction of the cyclin-dependent kinase inhibitor (cdki) p16^INK4a, a direct effector of the senescence phenotype in fibroblasts, and a decrease of the cdki p21^CIP1/WAF. Finally, a replicative senescence state in the early phases of infection significantly increased the number of cells permissive to virus infection and enhanced HCMV replication. HCMV infection assays carried out in the presence of phosphonoformic acid, which inhibits the virus DNA polymerase and the expression of downstream genes, indicated that immediate-early and/or early () genes are sufficient for the induction of SA-ß-Gal activity. When baculovirus vectors expressing HCMV IE1-72 or IE2-86 proteins were inoculated into fibroblasts, the increase of p16^INK4a (observed predominantly with IE2-86) was similar to that observed with the whole virus, as was the induction of SA-ß-Gal activity, suggesting that the viral IE2 gene leads infected cells into senescence. Altogether our results demonstrate for the first time that HCMV, after arresting the cell cycle and inhibiting apoptosis, triggers the cellular senescence program, probably through the p16^INK4a and p53 pathways.

Present address: Istituto di Fitovirologia, CNR, Turin, Italy.

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