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Journal of Virology, September 2009, p. 9273-9282, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00768-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Simian Varicella Virus Induces Apoptosis in Monkey Kidney Cells by the Intrinsic Pathway and Involves Downregulation of Bcl-2 Expression{triangledown}

Subbiah Pugazhenthi,1,4 Donald H. Gilden,2,3 Sreekala Nair,1,4 Anne McAdoo,1,4 Mary Wellish,2 Elizabeth Brazeau,2,3 and Ravi Mahalingam2*

Departments of Medicine,1 Neurology,2 Microbiology, University of Colorado Denver School of Medicine, Aurora, Colorado 80045,3 Veterans Administration Medical Center, Denver, Colorado 802204

Received 15 April 2009/ Accepted 1 July 2009

Simian varicella virus (SVV) causes varicella in primates, becomes latent in ganglionic neurons, and reactivates to produce zoster. SVV produces a cytopathic effect in monkey kidney cells in tissue culture. To study the mechanism by which SVV-infected cells die, we examined markers of apoptosis 24 to 64 h postinfection (hpi). Western blot analysis of virus-infected cell lysates revealed a significant increase in the levels of the cleaved active form of caspase-3, accompanied by a parallel increase in caspase-3 activity at 40 to 64 hpi. Caspase-9, a marker for the intrinsic pathway, was activated significantly in SVV-infected cells at all time points, whereas trace levels of the active form of caspase-8, an extrinsic pathway marker, was detected only at 64 hpi. Bcl-2 expression at the mRNA and protein levels was decreased by 50 to 70% throughout the course of virus infection. Release of cytochrome c, an activator of caspase-9, from mitochondria into the cytoplasm was increased by 200% at 64 hpi. Analysis of Vero cells infected with SVV expressing green fluorescent protein (SVV-GFP) at 64 hpi revealed colocalization of the active forms of caspase-3 and caspase-9 and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining with GFP. A significant decrease in the bcl-2 mRNA levels along with an abundance of mRNA specific for SVV genes 63, 40, and 21 was seen in the fraction of Vero cells that were infected with SVV-GFP. Together, these findings indicate that SVV induces apoptosis in cultured Vero cells through the intrinsic pathway in which Bcl-2 is downregulated.

* Corresponding author. Mailing address: Department of Neurology, Mail Stop B182, University of Colorado Denver School of Medicine, 12700 E. 19th Ave., Aurora, CO 80045. Phone: (303) 724-4324. Fax: (303) 724-4329. E-mail: ravi.mahalingam{at}ucdenver.edu

{triangledown} Published ahead of print on 15 July 2009.

Journal of Virology, September 2009, p. 9273-9282, Vol. 83, No. 18
0022-538X/09/$08.00+0     doi:10.1128/JVI.00768-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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