R7A mutation in N protein renders temperature sensitive phenotype of VSV by affecting its replication and transcription in vitro

Viral genomic RNA encapsidated by nucleoprotein (N) forms functional template for the transcription and replication of vesicular stomatitis virus (VSV). The crystal structure of the N-RNA complex shows that RNA is tightly sequestered between the two lobes of the N protein. The residue (R7) in N-terminal arm of N is of great importance to the formation of functional N-RNA template. In our study, we found that single amino acid substitution (R7A) resulted in the loss of CAT expression in vitro minigenome system at 37 °C. But the R7A had little effect on CAT expression at 31 °C. Further analysis showed that R7A had great effects on the RNA synthesis and the formation of cytoplasmic inclusions of VSV only at 37 °C not at 31 °C. For the further investigation of the effect of R7A on virus replication, we checked the dominant-negative effect of N_R7A in minigenome system and the single step curve of recombinant virus with R7A mutation in N protein (rVSV_R7A) under 37 °C and 31 °C separately. Our results showed that the mutation of R7A within the N-terminal arm of N affected both replication and transcription and induced VSV to become temperature sensitive.