Structure informed xrRNA2 mutations in Zika virus provide a roadmap for vaccine development

Flaviviruses like Zika virus (ZIKV), contain RNA tertiary structures within the 3 untranslated region (UTR) that halt the 5-to-3 RNA exonuclease, Xrn1. Halting of Xrn1 at the two RNA structures, termed exonuclease-resistantRNA1 and 2 (xrRNA1 and xrRNA2), results in the formation of subgenomic flavivirus RNAs (sfRNA) that support viral pathogenesis. While the role of the flavivirus xrRNA1 in pathogenesis is well characterized, the role of the flavivirus xrRNA2 structure is not well studied. Using xrRNA crystal structure data, we inserted structure-informed mutations in ZIKV xrRNA2 to disrupt tertiary folding independent of significant sequence changes, evaluate sfRNA production, and define pathogenesis in a murine model of ZIKV infection. Compared to our prior work with ZIKV xrRNA1, we found that ZIKV xrRNA2 is under increased selection pressure to maintain sfRNA production resulting in multiple targeted mutations in xrRNA2 junctional region to induce a stable mutant. Using three targeted xrRNA junctional mutations termed ZIKV X2.L1, we found that the resulting ZIKV clone exhibits attenuated cell death in cultures and decreased viral growth in tissue cultures. In a murine model of ZIKV infection, mice inoculated with ZIKV X2.L1 exhibit significantly decreased symptomatic infection, improved survival, decreased end-organ infection in the brain, and continued robust neutralizing antibody responses to ZIKV. Despite attenuation, serum from ZIKV X2.L1-infected mice or mice vaccinated with ZIKV X2.L1, exhibited 100% protection from lethal ZIKV challenge. These studies show that RNA structure-informed mutations provide a robust model for flavivirus attenuation and vaccine design. Additional studies defining the mechanisms of robust neutralizing antibody responses and flavivirus-specific vaccine development are needed to continue the development of this novel vaccine platform approach for medically important flavivirus infections. Author summaryZika virus is a member of the Orthoflavivirus (referred to as flavivirus) genus that exhibit conserved RNA structures in the 3 untranslated region of the viral RNA genome. Two concerned RNA structures, termed exonuclease-resistant RNA 1 and 2, are important to support the ability of the virus to cause disease. While the first RNA structure is well studied, less is known about the role of exonuclease-resistant RNA 2 in the flavivirus infection. Using reverse genetics, we made mutations in the Zika virus exonuclease-resistant RNA 2 structure and studied how this mutant Zika virus was weakened or attenuated. We found that the mutant Zika virus clone exhibits reduced virus replication, reduced ability to kill cells, and decreased virulence in mouse models of Zika virus disease. Using this mutant virus as a potential vaccine candidate, we found that Zika virus with mutations in the exonuclease-resistant RNA 2 structure provide complete protection from lethal Zika virus challenge. These data suggest that targeting the second exonuclease resistant RNA structure in flaviviruses is a viable platform for the development of vaccine candidates for this important group of viruses.