Requirement for the amino-terminal domain of sindbis virus nsP4 during virus infection

The Sindbis virus RNA-dependent RNA polymerase nsP4 possesses an amino-terminal region that is unique to alphaviruses and is predicted to be disordered. To determine the importance of this region during alphavirus replication, 29 mutations were introduced, and resultant viruses were assessed for growth defects. Three small plaque mutants, D41A, G83L, and the triple mutant GPG((8-10))VAV, had defects in subgenome synthesis, minus-strand synthesis, and overall levels of viral RNA synthesis, respectively. Large plaque viruses were selected following passage in BHK-21 cells, and the genomes of these were sequenced. Suppressor mutations in nsP1, nsP2, and nsP3 that restored viral RNA synthesis were identified. An nsP2 change from M282 to L and an nsP3 change from H99 to N corrected the D41A-induced defect in subgenomic RNA synthesis. Three changes in nsP1, I351 to V, I388 to V, or the previously identified change, N374 to H (C. L. Fata, S. G. Sawicki, and D. L. Sawicki, J. Virol. 76:8641-8649, 2002), suppressed the minus-strand synthetic defect. A direct reversion back to G at position 8 reduced the RNA synthesis defect of the GPG((8-10))VAV virus. These results imply that nsP4's amino-terminal domain participates in distinct interactions with other nsPs in the context of differentially functioning RNA synthetic complexes, and flexibility in this domain is important for viral RNA synthesis. Additionally, the inability of the mutant viruses to efficiently inhibit host protein synthesis suggests a role for nsP4 in the regulation of host cell gene expression.