Disruption of Zika virus xrRNA1-dependent sfRNA1 production results in tissue-specific attenuated viral replication

Zika virus (ZIKV), like other flaviviruses, produces several species of sub-genomic RNAs (sfRNAs) during infection, corresponding to noncoding RNA fragments of different lengths derived from the viral 3’ untranslated region (UTR). Over the course of infection, these sfRNAs accumulate in the cell as a result of incomplete viral genome degradation of the 3’UTR by host 5’ to 3’ exoribonuclease (Xrn1). The halting of Xrn1 in the 3’UTR is due to two RNA pseudoknot structures in the 3’UTR termed exoribonuclease-resistant RNA1 and 2 (xrRNA1&2). Studies with related flaviviruses have shown that sfRNAs are important for pathogenicity and inhibiting both mosquito and mammalian host defense mechanisms. However, these investigations have not included ZIKV and there is very limited data addressing how sfRNAs impact infection in a whole animal model or specific tissues. In this study, we rescued a sfRNA1-deficient ZIKV (X1) by targeted mutation in the xrRNA1 3’ UTR structure. We found that virus which lacks the production of the largest ZIKV sfRNA species, sfRNA1. Using the X1 virus to infect adult IFNAR1 -/- mice, we found that while the lack of sfRNA1 does not alter ZIKV replication in the spleen, there is a significant reduction of ZIKV genome replication in the brain and placenta compared to WT ZIKV infection. Despite thee attenuated phenotype of the X1 ZIKV, mice develop a robust neutralizing antibody response. We conclude that targeted disruption of xrRNA1 results in tissue-specific attenuation while still supporting robust neutralizing antibody responses. Future studies will need to investigate the tissue-specific mechanisms by which ZIKV sfRNAs influence infection and may utilize targeted xrRNA mutations to develop novel attenuated flavivirus vaccine approaches.