The 3' end of the human norovirus antigenomic sequence is required for its genomic RNA synthesis by RNA-dependent RNA polymerase

Norovirus genome is a single-stranded positive-strand RNA. To reveal the mechanism underlying the initiation of the norovirus genomic RNA synthesis by its RNA-dependent RNA polymerase (RdRp), we used an in vitro assay to detect the complementary RNA synthesis activity. Results showed that the purified recombinant RdRp synthesized the complementary positive-sense RNA from the 100 nt template corresponding to the 3' end region of the viral antisense genome sequence, but that RdRp did not synthesize the antisense genomic RNA from the 100 nt template, corresponding to the 5' end region of the positive-sense genome sequence. The 31 nt region at the 3' end of the RNA antisense template was then predicted to form the stem-loop structure. Its deletion resulted in the loss of complementary RNA synthesis by RdRp. The connection of the 31 nt to the 3' end of the positive-sense RNA template allowed to be recognized by the RdRp. Similarly, an electrophoretic mobility shift assay further revealed that RdRp bound to the antisense RNA specifically, but the 31 nt deletion at the 3' end lost the binding to RdRp. Therefore, combining this observation with further deletion and mutation analysis, we concluded that the predicted stem-loop structure in the 31 nt and region close to the antisense viral genomic stem sequences are important for initiating the positive-sense human norovirus genomic RNA synthesis by its RdRp.