Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.
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 (3C(pro)) 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 3C(pro). CstF-64 was cleaved in vitro by 3C(pro) but neither by mutant 3C(pro) (in which the catalytic site was inactivated) nor by another EV71 protease 2A(pro). Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro) 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 3C(pro)-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro) 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.
Weng
Kuo-Feng
KF
Research Center for Emerging Viral Infections, Chang Gung University, Kwei-Shan Tao-Yuan, Taiwan, ROC.
Li
Mei-Ling
ML
Hung
Chuan-Tien
CT
Shih
Shin-Ru
SR
D000086042:3C Viral Proteases; D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D039241:Cleavage Stimulation Factor; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D029821:Enterovirus A, Human / Q000201:enzymology* / Q000472:pathogenicity; D004769:Enterovirus Infections / Q000378:metabolism / Q000821:virology*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011061:Poly A / Q000378:metabolism; D026723:Polyadenylation; D011485:Protein Binding; D025941:Protein Interaction Mapping; D012322:RNA Precursors / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
C474466:CSTF2T protein, human; D039241:Cleavage Stimulation Factor; D012322:RNA Precursors; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D011061:Poly A; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases