Two second-site mutations compensate the engineered mutation of R7A in vesicular stomatitis virus nucleocapsid protein

•Two second-site mutations were introduced into the genome of rVSVR7A via eight passages.•mutation of either T242 P or U7-U8 can compensate replication defect of rVSVR7A.•Two second-site mutations synergistically compensate replication defect of rVSVR7A.

The functional template for the transcription and replication of vesicular stomatitis virus (VSV) is genomic RNA encapsidated by nucleocapsid (N) protein. Previous studies showed that the amino acid R7 in the N-terminal arm of N is involved in N–N interaction in the N-RNA complex. In our study, the recombinant virus with mutation of R7A (rVSVR7A) in N was recovered, and the replication level of passage 1 (P1) of rVSVR7A was 1000 times lower than that of wild-type rVSV at 37 °C. After eight passages, the replication level of P8 of rVSVR7A with two second-site mutations in the genome (T242 P in N protein and U7-U8 in G-L gene junction) was significantly higher than that of P1. Furthermore, we demonstrate that the mutation of either T242 P or U7-U8 can compensate the effect caused by the mutation of R7A on the replication of rVSVR7A. Therefore, we conclude that two second-site mutations both can compensated the engineered mutation of R7A in VSV N protein.