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

Abstract

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.

Viral security proteins: counteracting host defences

“…The ability of rhinovirus 2A pro to cleave mitochondrial antiviral-signalling protein (mAVS; also known as VISA, CARDIF and IPS1), an intermediate in the interferon generation pathway, might contribute to the insensitivity of these viruses to interferons 71 . 2A pro also cleaves the catalytic subunit of DNA-dependent protein kinase, which, among other activities, is involved in the induction of pro-inflammatory cytokines 72 . Cardiovirus L also suppresses interferon production by affecting the activation of interferon regulatory factor 3, but the exact mechanism of this interference has not yet been identified 32,50,73,74 .…”

Viable Polioviruses That Encode 2A Proteins with Fluorescent Protein Tags

“…All of these 2A-induced events serve to enhance viral protein synthesis. In addition, poliovirus infection or 2A expression in HeLa cells causes alterations in the nuclear pore structure resulting from cleavage of specific components of the nuclear pore complex (8,15,33). These alterations produce bidirectional increases in permeability of the nuclear envelope, which permit redistribution of cellular proteins that normally reside in the nucleus to the cytoplasm, where they are available for viral translation or other replication reactions.…”

Sources of autoantigens in systemic lupus erythematosus

“…These include histone H3, a substrate for a foot-and-mouth disease virus protease [36], as well as the La antigen, which is cleaved by the poliovirus 3C protease [37]. Recently, we have shown that DNA-PK CS is cleaved by a picornavirus 2A protease [38], indicating that viral proteases may contribute to the generation of novel autoantigenic epitopes. Morecomprehensive screens may identify additional substrates for viral proteases.…”