Zika virus (ZIKV) is a member of flaviviridae family, which has recently emerged as medically important human pathogen. Upon infection of pregnant mothers ZIKV causes abnormalities in development of fetal brain that result in microcephaly, severe brain malformations, and birth defects. ZIKV and other flaviviruses have been shown to produce abundant noncoding subgenomic RNAs (sfRNAs), which are generated from their 3’UTR. sfRNAs are the product of incomplete digestion of viral genomic RNA by host exoribonuclease XRN-1, which stalls on XRN-1 resistant tertiary RNA structures (xrRNAs). Here we examined the role of sfRNAs in ZIKV infection.
3’UTR of ZIKV contains two xrRNA structures, xrRNA1 and xrRNA2 which are responsible for production of two isoforms of sfRNA – longer sfRNA-1 and shorter sfRNA-2, respectively. Using circular polymerase extension reaction (CPER) we created ZIKV mutants deficient in production of each of sfRNA species. Mutant deficient in production of both sfRNAs was not viable in Vero cells, which indicates the important role of sfRNAs in ZIKV replication. We then examined cytopathic properties of sfRNA-deficient ZIKV mutants. We also assessed their replication efficiency in comparison with WT virus in different mammalian and mosquito cells. In addition, we determined the role of ZIKV sfRNA species in suppression of IFN response in mammalian cells and RNAi pathway in insect cells using cell lines deficient in IFNAR (IFNAR-/- MEF) and Dicer-2 (C6/36 cells), respectively.
The results showed that sfRNA is required for ZIKV-induced cytopathic effect and efficient replication in mammalian cells and that production of sfRNAs was important for ZIKV evasion of IFN response in MEFs. We have also shown that destabilization of xrRNA-2 impaired production of both sfRNA-2 and sfRNA-1 in mammalian cells suggesting potential interactions between xrRNA1 and xrRNA2 structures in ensuring sfRNA-1 production. Overall, our results demonstrate an important role for sfRNAs in ZIKV infection.