Impaired heterologous protein-protein interaction is an essential cause for non-viability of WNV/DENV recombinants

Flavivirus RNA replication starts at 3'-end, where it folds into a highly conserved stem-loop structure. We attempted to identify the viral non-structural proteins (NSPs) that might specifically interact with the 3'-stemloop (3'SL) through a genetic approach. WNV/DENV2 chimeric recombinants that contain Dengue2 (DENV2) gene(s) in West Nile virus (WNV) backbone were tested for replication competence. Three of seven recombinant viruses, containing the DENV2 NS1, NS2A, or NS4B gene and terminated with a mutated 3'SL (MutC 3'SL), were viable. Of these three, only those bearing the DENV2 NS1 and NS2A substitutions remained infectious when the MutC 3'SL was replaced by the wildtype WNV 3'SL. However, none of the seven chimeric recombinants bearing the DENV2 3'SL were viable. We then investigated the causes for failed replication of WNV/DENV2 chimeric recombinants. Proteolytic cleavage of NS polyproteins was defective by heterologous protease NS2B/3, but was efficient by homologous DENV2 NS2B/3 protease. Whereas, the heterologous polyproteins that contained DENV2 homologous protease were found to produce abnormal vesicles. WNV/DENV2 recombinants expressing the DENV2 homologous protease did not produce infectious virus either. We examined NS protein-protein interaction (PPI) and found that heterologous PPI (hPPI) between WNV and DENV2 NSPs were impaired to various degrees. Insufficient PPIs occurred mainly between heterologous NS2B and NS3; NS2B and NS4A; NS3 and NS5, correlating to those non-viability of substitution mutants. Our results indicate that impaired PPI may decrease protease activity and affect vesicle formation, and is the essential cause for non-viability of the WNV/DENV2 recombinants.