3C Protease-Mediated Cleavage of Cellular Proteins in Enterovirus 71-Infected Cells

Enterovirus 71 (EV71) infection persistently causes a global threat for children due to its neurological complications. The 3C protease (3Cpro) of EV71 plays an important role in viral proteins processing and has the capability to induce host cell apoptosis, which has been considered to be a good molecular target for antiviral drug development. In this proposal we would like to study the 3C protease-associated functions in EV71-infected cells, especially focusing on what kinds of cellular proteins are the substrates for this viral protease. We would also like to address how and why those cellular proteins are cleaved by 3Cpro. Our preliminary results have demonstrated that EV71 3Cpro may enter the nuclei of virus-infected cells. To investigate what nuclear proteins are the potential substrates for viral 3C protease, we compared the amount changes of cellular proteins in wild-type 3Cpro versus mutant 3Cpro-mut (mutations at the catalytic sites to abolish its proteolytic activity)-treated nuclear extracts. Two-dimensional gels electrophoresis and MALDI-TOF were applied to identify nine cellular proteins significantly decrease in wild-type 3C-treated nuclear extract in comparison with those in 3Cpro-mut -treated extract. Sequence analysis shows 7 out of 9 candidates contain the potential 3Cprocleavage sites. Protein No. 1, Cleavage stimulation factor, 3' pre-RNA, subunit 2, 64kD (CstF-64) has been chosen for further studies. The purified recombinant 3Cpro directly cleaved the recombinant CstF-64 without existence of any other cellular or viral proteins. A time-course study in EV71-infected RD cells also demonstrates that viral 3Cpro expression correlates to the reduction of CstF-64. We will continue the detail mechanism study for the cleavage of CstF-64 by 3Cpro, including establishing the in vitro polyadenylation assay. In the second and third year, we will apply the similar study strategy (as CstF-64) to investigate other substrates, such as nuclear matrix protein, NMP200, and/or nuclear protein CTBP2. We believe the roles of 3Cpro in EV71-infected cells will be revealed more though this research, which would also help for drug development based on 3C protease as the target.

Project ID:PC9902-0468
External Project ID:NSC97-2320-B182-007-MY3