A mouse cell-adapted NS4B mutation attenuates West Nile virus RNA synthesis.

An adaptive mutation (E249G) within West Nile virus (WNV) NS4B gene was consistently recovered from replicon RNAs in C3H/He mouse cells. The E249G is located at the C-terminal tail of NS4B predicted to be on the cytoplasmic side of the endoplasmic reticulum membrane. The E249G substitution reduced replicon RNA synthesis. Compared with the wild-type NS4B, the E249G mutant protein exhibited a similar efficiency in evasion of interferon-beta response. Recombinant E249G virus exhibited smaller plaques, slower growth kinetics, and lower RNA synthesis than the wild-type virus in a host-dependent manner, with the greatest difference in rodent cells (C3H/He and BHK-21) and the least difference in mosquito cells (C3/36). Selection of revertants of E249G virus identified a second site mutation at residue 246, which could compensate for the low replication phenotype in cell culture. These results demonstrate that distinct residues within the C-terminal tail of flavivirus NS4B are critical for viral replication.