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

PLoS Pathogens

Shin-Ru Shih¹  Mei-Ling Li²  Kuo-Feng Weng³  Chuan-Tien Hung³ 

[1] Department of Molecular Genetics, Microbiology and Immunology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey, United States of America;Graduate Institute of Biomedical Sciences, Chang Gung University, Kwei-Shan Tao-Yuan,Taiwan, R.O.C.;Research Center for Emerging Viral Infections, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan, R.O.C.

关键词: Polyadenylation;    Virus effects on host gene expression;    Proteases;    Protein extraction;    Recombinant proteins;    Messenger RNA;    Reverse transcriptase-polymerase chain reaction;    Transcription factors;   

DOI  :  10.1371/journal.ppat.1000593

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY