Cis-acting sequences and secondary structures in untranslated regions of duck Tembusu virus RNA are important for cap-independent translation and viral proliferation.

Duck Tembusu virus (DTMUV; genus Flavivirus) is a causative agent of duck egg drop syndrome and has zoonotic potential. The positive strand RNA genomes of flaviviruses are commonly translated in a cap-dependent manner. However, Dengue and Zika viruses also exhibit cap-independent translation. In this study, we show that RNAs containing 5' and 3' untranslated regions (UTRs) of DTMUV, mosquito-borne Tembusu virus (TMUV) and Japanese encephalitis virus can be translated in a cap-independent manner in mammalian, avian and mosquito cells. The ability of the 5'UTRs of flaviviruses to direct the translation of a second open reading frame in bicistronic RNAs was much lower than that observed for internal ribosome entry site (IRES) encephalomyocarditis virus, indicating a lack of substantial IRES activity. Instead, cap-independent translation of DTMUV RNA was dependent on the presence of a 3'UTR, RNA secondary structures located in both UTRs and specific RNA sequences. Mutations inhibiting cap-independent translation decreased DTMUV proliferation in vitro and delayed, but did not prevent, the death of infected duck embryos. Thus, the 5' and 3'UTRs of DTMUV enable this virus to use a cap- and IRES-independent RNA genome translation strategy that is important for its propagation and virulence.IMPORTANCE Genus Flavivirus includes major human pathogens as well as animal-infecting viruses with zoonotic potential. In order to counteract the threats these viruses represent, it is important to understand their basic biology, to develop universal attenuation strategies. Here we demonstrated that five different flaviviruses use cap-independent translation, indicating that this phenomenon is probably general for all members of the genus. The mechanism used for flavivirus cap-independent translation was found to be different from that of IRES mediated translation and dependent both on 5' and 3'UTRs that act in cis. As cap-independent translation was also observed in mosquito cells, its role in flavivirus infection is unlikely limited to the evasion of consequences of the shutoff of host translation. We found that the inhibition of cap-independent translation results in decrease viral proliferation, indicating that this strategy could be applied to produce attenuated variants of flaviviruses as potential vaccine candidates.