Superinfection exclusion is the ability of an established
viral infection to interfere with a second
viral infection. Using West Nile virus (WNV) as a model, we show that replicating replicons in BHK-21 cells suppress subsequent
WNV infection. The WNV replicon also suppresses
superinfections of other flaviviruses but not nonflaviviruses. Mode-of-action analysis indicates that the exclusion of WNV
superinfection occurs at the step of
RNA synthesis. The continuous culturing of WNV in the replicon-containing cells generated variants that could overcome the
superinfection exclusion. The sequencing of the selected viruses revealed mutations in structural (prM S90R or envelope E138K) and nonstructural genes (NS4a K124R and
peptide 2K V9M). Mutagenesis analysis showed that the mutations in structural genes nonselectively enhance
viral infection in both naïve and replicon-containing BHK-21 cells; in contrast, the mutations in nonstructural genes more selectively enhance viral replication in the replicon-containing cells than in the naïve cells. Mechanistic analysis showed that the envelope mutation functions through the enhancement of virion attachment to BHK-21 cells, whereas the 2K mutation (and, to a lesser extent, the NS4a mutation) functions through the enhancement of
viral RNA synthesis. Furthermore, we show that WNV
superinfection exclusion is reversible by the treatment of the replicon cells with a flavivirus inhibitor. The preestablished replication of the replicon could be suppressed by infecting the cells with the 2K mutant WNV but not with the wild-type virus. These results suggest that WNV
superinfection exclusion is a result of competition for intracellular host factors that are required for
viral RNA synthesis.