WNV is a member of flavivirus genus of flaviviridae family, which is a human pathogen of considerable medical importance. Host response to WNV in vertebrates primarily relies upon type I IFN pathways and virus has evolved to counterpart host defence employing viral non-structural proteins and noncoding RNA (sfRNA). However, we have now found that sfRNA is not the only noncoding RNA generated from the 3’UTR of WNV.
Using RNA-Seq analysis of WNV-infected cells we have identified novel noncoding RNAs encompassing or derived from the 3’-terminal stem loop (3’SL) of the WNV 3’UTR. The production of these RNAs was confirmed by Northern blot analysis. We also showed that these RNAs were produced by different strains of WNV in all tested cell types from mammalian and mosquito hosts. These RNAs were also detected in the brain tissue of WNV-infected mice. The biogenesis of these RNAs was found to be independent of host miRNA/siRNA processing machinery, RNaseL and XRN-1, and required viral RNA replication and/or viral non-structural proteins. It involves two cleavage steps undertaken by different yet unknown nucleases. First, the larger 100nt RNA that includes the 3’SL and the preceeding small hairpin is excised from viral RNA by an RNase that produces 5’OH-end. Subsequently, this 100nt RNA is cleaved into two smaller RNAs of 32 and 68nt in length, by another RNase that produces 5’-P-end. Unlike siRNAs and miRNAs, these 3’SL-derived noncoding RNAs did not incorporate into RISC and did not regulate expression of reporter mRNAs containing WNV noncoding RNA-complementary sites in their 3’UTR. Instead, these abundant noncoding RNAs bind to translation elongation factor eEF-1A and might act as a switch between replication and translation/packaging of viral RNA.