CVB3 VP1 interacts with MAT1 to inhibit cell proliferation by interfering with Cdk-activating kinase complex activity in CVB3-induced acute pancreatitis

Abstract

Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus of the family Picornaviridae and can cause acute acinar pancreatitis in adults. However, the molecular mechanisms of pathogenesis underlying CVB3-induced acute pancreatitis have remained unclear. In this study, we discovered that CVB3 capsid protein VP1 inhibited pancreatic cell proliferation and exerted strong cytopathic effects on HPAC cells. Through yeast two-hybrid, co-immunoprecipitation, and confocal microscopy, we show that Menage a trois 1 (MAT1), a subunit of the Cdk-Activating Kinase (CAK) complex involved in cell proliferation and transcription, is a novel interaction protein with CVB3 VP1. Moreover, CVB3 VP1 inhibited MAT1 accumulation and localization, thus interfering with its interaction with CDK7. Furthermore, CVB3 VP1 could suppress CAK complex enzymic phosphorylation activity towards RNA Pol II and CDK4/6, direct substrates of CAK. VP1 also suppresses phosphorylation of retinoblastoma protein (pRb), an indirect CAK substrate, especially at phospho-pRb Ser⁷⁸⁰ and phospho-pRb Ser^807/811 residues, which are associated with cell proliferation. Finally, we present evidence using deletion mutants that the C-terminal domain (VP1-D8, 768-859aa) is the minimal VP1 region required for its interaction with MAT1, and furthermore, VP1-D8 alone was sufficient to arrest cells in G1/S phase as observed during CVB3 infection. Taken together, we demonstrate that CVB3 VP1 can inhibit CAK complex assembly and activity through direct interaction with MAT1, to block MAT1-mediated CAK-CDK4/6-Rb signaling, and ultimately suppress cell proliferation in pancreatic cells. These findings substantially extend our basic understanding of CVB3-mediated pancreatitis, providing strong candidates for strategic therapeutic targeting. Coxsackievirus B3 (CVB3) is one of the pathogens causing acute acinar pancreatitis. We show that CVB3 VP1 could lead to inhibit cell proliferation and exert strong cytopathic effects on pancreatic cells. Our further studies indicate that VP1 through interacting with MAT1 disrupts the assembly and reduces the activity of Cdk-Activating Kinase (CAK) complex, which is involved in cellular proliferation and transcription. The interaction not only decreases the amount of MAT1, but interferes with MAT1 binding to CDK7. In addition, VP1 impairs the functions of a series of proteins associated with cellular proliferation, including RNA Pol II, CDK4/6, and retinoblastoma protein (pRb). The C-terminal domain of VP1 (VP1 D8, 768-859aa) is identified as the minimal VP1 region that can cause the same effect as the full length of VP1. Consequently, we propose that the interaction between CVB3 capsid protein VP1 and MAT1 induces apparently cytopathic effect, blocks MAT1-mediated CAK-CDK4/6-Rb signaling pathway, and thereby inhibits cell proliferation ultimately during CVB3 infection in pancreatic cells. It provides us a new insight for understanding CVB3-mediated pancreatitis and new candidates for therapeutic strategy.