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.
Many viruses contain specific proteases that are essential for processing their own viral proteins. For an efficient replication within their hosts, on the other hand, viruses also utilize these proteases to cleave a number of key host proteins and hijack cellular machineries. In this study, host proteins are identified as the substrates for enterovirus 71 viral protease by adopting a proteomic strategy. Enterovirus 71 infection is highly associated with neurological complication and mortality in an era when poliovirus has been controlled by vaccination. Investigating the host substrates for enterovirus 71 protease may shed light on the viral–host interaction, ultimately providing further insight into the pathogenesis of EV71 infection. We found that 3C protease (3C pro) cleaved CstF-64, the latter a cleavage stimulation factor in host polyadenylation machinery. 3C pro is known to enter host nucleus, whereas the replication of the virus occurs in cytoplasm. While its role in nucleus has been thought to inhibit host transcription, we found that 3C pro may inhibit host-cell gene expression at host 3′-end pre-mRNA processing and polyadenylation steps. Consequently, less polyadenylated host mRNA is synthesized and more cellular resources, such as the translation factors, would be available for viral RNA expression.