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Journal of Virology, June 2004, p. 6313-6321, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6313-6321.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Proteolytic Cleavage of the Catalytic Subunit of DNA-Dependent Protein Kinase during Poliovirus Infection

Kareem L. Graham,¹ Kurt E. Gustin,^2, Carlos Rivera,³ N. Muge Kuyumcu-Martinez,³ Sunny S. Choe,² Richard E. Lloyd,³ Peter Sarnow,² and Paul J. Utz^1*

Division of Immunology and Rheumatology,¹ Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305,² Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030³

Received 16 August 2003/ Accepted 2 February 2004

DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase that has critical roles in DNA double-strand break repair, as well as B- and T-cell antigen receptor rearrangement. The DNA-PK enzyme consists of the Ku regulatory subunit and a 450-kDa catalytic subunit termed DNA-PK_CS. Both of these subunits are autoantigens associated with connective tissue diseases such as systemic lupus erythematosus (SLE) and scleroderma. In this report, we show that DNA-PK_CS is cleaved during poliovirus infection of HeLa cells. Cleavage was visible as early as 1.5 h postinfection (hpi) and resulted in an approximately 40% reduction in the levels of native protein by 5.5 hpi. Consistent with this observation, the activity of the DNA-PK_CS enzyme was also reduced during viral infection, as determined by immunoprecipitation kinase assays. Although it has previously been shown that DNA-PK_CS is a substrate of caspase-3 in vitro, the protein was still cleaved during poliovirus infection of the caspase-3-deficient MCF-7 cell line. Cleavage was not prevented by infection in the presence of a soluble caspase inhibitor, suggesting that cleavage in vivo was independent of host caspase activation. DNA-PK_CS is directly cleaved by a picornaviral 2A protease in vitro, producing a fragment similar in size to the cleavage product observed in vivo. Taken together, our results indicate that DNA-PK_CS is cleaved by the 2A protease during poliovirus infection. Proteolytic cleavage of DNA-PK_CS during poliovirus infection may contribute to inhibition of host immune responses. Furthermore, cleavage of autoantigens by viral proteases may target these proteins for the autoimmune response by generating novel, or "immunocryptic," protein fragments.

Present address: Department of Microbiology, Molecular Biology and Biochemistry, University of Idaho, Moscow, ID 83844-3052.

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