Promoter-like activity on the minus strand supports sfRNA biogenesis in Japanese encephalitis virus.

Arthropod-borne flaviviruses produce subgenomic RNAs (sfRNAs) from the highly conserved 3'-untranslated region (UTR). While most flaviviruses generate sfRNAs by resisting degradation from the host exoribonuclease XRN1, Japanese encephalitis virus (JEV) maintains sfRNA production even when XRN1 is depleted, suggesting an alternative mechanism. Using an in vitro RNA-dependent RNA polymerase (RdRp) assay, we identified a promoter-like element on the antigenome, designated (-)sfP, which exhibits transcriptional activity comparable to the well-characterized 5' stem-loop A (5'-SLA) promoter of the viral genome. In contrast, the complementary strand of 5'-SLA, termed (-)SLA, located at the 3'-terminus of antigenome, displayed only weak promoter activity. Both (-)SLA and (-)sfP RNAs were found to interact with viral RdRp and a similar set of host proteins, suggesting potential roles in regulating RNA synthesis from the antigenomic template. Detection of minus-strand sfRNA by Northern blot supports the existence of replication intermediates generated through RdRp-mediated transcription rather than simple degradation products. Together, these findings reveal a previously unrecognized promoter-like activity on the JEV antigenome that may contribute to sfRNA formation and genome replication.