Enterovirus 71 3C Protease Cleaves a Novel Target CstF-64 and Inhibits Cellular Polyadenylation

Abstract

Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell–virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71) 3C protease (3Cpro) cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3′ pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3Cpro. CstF-64 was cleaved in vitro by 3Cpro but neither by mutant 3Cpro (in which the catalytic site was inactivated) nor by another EV71 protease 2Apro. Serial mutagenesis was performed in CstF-64, revealing that the 3Cpro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500). An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3′-end pre-mRNA processing and polyadenylation in 3Cpro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3Cpro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

Enterovirus 71 Inhibits Pyroptosis through Cleavage of Gasdermin D

“…We along with others have reported that 3C can mediate cleavage of host factors and affect their functions (12,35,36). To investigate whether EV71 3C targets GSDMD, we conducted cleavage experiments.…”

The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

“…EV71 3C is a multifunctional protein which is essential for viral replication. In addition to its role in viral protein processing, the 3C protein induces apoptosis and inhibits cellular polyadenylation (27,32,46,50). It is notable that the 3C protein bears proteolytic and RNA binding activities.…”

Picornavirus IRES elements: RNA structure and host protein interactions

“…This is noticed at the level of transcription, mRNA processing, nucleocytoplasmic transport, translation, or RNA granules composition. Among other proteins, proteolysis affects splicing factors, RNAprocessing proteins, RNA helicases, nuclear pore factors, stress granules assembly factors or antiviral response proteins (Almstead and Sarnow, 2007;Barral et al, 2009;Castello et al, 2009;Chen et al, 2013;Lawrence et al, 2012;Mukherjee et al, 2011;Park et al, 2010;Pineiro et al, 2012;Rozovics et al, 2012;Shiroki et al, 1999;Watters and Palmenberg, 2011;Weng et al, 2009;White et al, 2007).…”