Enhancement of Viral Yields of Picornaviruses by Promoting Ires-Dependent Translation( I )

Many viruses, especially the genetically lean RNA viruses, can utilize internal ribosomal entry site (IRES)- driven translation to synthesize viral proteins while the host cap-dependent translation has been disrupted. In the past few years, we have identified several IRES trans-acting factors (ITAFs) that either positively or negatively regulate viral IRES activity. Our idea in the current proposal is to engineer enhanced vaccine cell lines based on manipulating the expressions of these ITAFs in a Vero (African green monkey kidney) cell line licensed for both poliovirus and EVA-71 vaccine productions. We will engineer the cell based on delivering positive ITAFs and inactivating negative ITAF. There are three specific aims in the proposal: (1) To engineer enhanced vaccine cell lines via overexpressing FBP1 and FBP11-371 We have identified far upstream element binding protein 1 (FUBP1; also known as FBP1) as a positive regulator through a specific binding to the linker region of viral IRES. We have also found that the cleavage occurs at the Gly-371 residue of FBP1 during the EV71 infection process, and this generates a functional cleavage product, FBP11-371. Moreover, FBP1 and FBP11-371 were found to act additively to promote IRES-mediated translation and viral yield. Therefore, we plan to overexpress both FBP1 and FBP11-371 in Vero cell (a licensed cell line for vaccine production) to enhance virus production. To achieve these goals, CRISPR (Clustered regularly interspaced short palindromic repeats)/Cas9 system will be introduced to ensure the accuracy and persistently expression of FBP1 and truncated FBP11-371. (2) To engineer enhanced vaccine cell lines via replacing mutant FBP2 which cannot be ubiquitinated Previously we have identified KH-type splicing regulatory protein (KHSRP; also called FBP2) as a novel negative regulator for EVA-71 IRES-driven translation. More recently we found that ubiquitination is essential for FBP2 to play the negative role. When we mutated the ubiquitination sites in FBP2, the negative effect on EVA-71 translation was lost. To enhance the vaccine cell line to produce more viruses, we will mutate the ubiquitination sites in FBP2. By eliminating the negative regulation from FBP2, we expect more viruses will be produced in the engineered cell line. Again, the technology of CRISPR/Cas 9 system will be applied to introduce mutations in FBP2. (3) To evaluate viral yields in the engineered cell line for different picornaviruses Once the FBP1+/FBP2- cell line (a cell line in which FBP1 and FBP11-371 will be overexpressed and the ubiquitination sites in FBP2 will be mutated) was obtained, we will evaluate viral yields for EVA-71, CV-B3, EVD-68 and HRV-C. These picornaviruses are important pathogens that can cause severe complications and even death in humans.

Project ID:PC10603-0003
External Project ID:MOST106-2321-B182-001