During viral RNA synthesis by the viral RNA-dependent RNA polymerase (vRdRp) of vesicular stomatitis virus, the sequestered RNA genome must be released from the nucleocapsid in order to serve as the template. Unveiling the sequestered RNA by interactions of vRdRp proteins, the large subunit (L) and the phosphoprotein (P), with the nucleocapsid protein (N) must not disrupt the nucleocapsid assembly. We noticed that a flexible structural motif composed of an α-helix and a loop in the N protein may act as the access gate to the sequestered RNA. This suggests that local conformational changes in this structural motif may be induced by interactions with the polymerase to unveil the sequestered RNA, without disrupting the nucleocapsid assembly. Mutations of several residues in this structural motif-Glu169, Phe171, and Leu174-to Ala resulted in loss of viral RNA synthesis in a minigenome assay. After implementing these mutations in the viral genome, mutant viruses were recovered by reverse genetics and serial passages. Sequencing the genomes of the mutant viruses revealed that compensatory mutations in L, P, and N were required to restore the viral viability. Corresponding mutations were introduced in L, P, and N, and their complementarity to the N mutations was confirmed by the minigenome assay. Introduction of the corresponding mutations is also sufficient to rescue the mutant viruses. These results suggested that the interplay of the N structural motif with the L protein may play a role in accessing the nucleotide template without disrupting the overall structure of the nucleocapsid.IMPORTANCE During viral RNA synthesis of a negative-strand RNA virus, the viral RNA-dependent RNA polymerase (vRdRp) must gain access to the sequestered RNA in the nucleocapsid to use it as the template, but at the same time may not disrupt the nucleocapsid assembly. Our structural and mutagenesis studies showed that a flexible structural motif acts as a potential access gate to the sequestered RNA and plays an essential role in viral RNA synthesis. Interactions of this structural motif within the vRdRp may be required for unveiling the sequestered RNA. This mechanism of action allows the sequestered RNA to be released locally without disrupting the overall structure of the nucleocapsid. Since this flexible structural motif is present in the N proteins of many NSVs, release of the sequestered RNA genome by local conformational changes in the N protein may be a general mechanism in NSV viral RNA synthesis.