Investigating the importance of the Zika virus 3′ untranslated region to viral fitness

Abstract

The RNA binding protein Musashi 1 (MSI1) has been shown to bind to the Zika virus (ZIKV) 3′ untranslated region (3′UTR) and enhance viral replication. MSI1 is present in high levels in neural progenitor cells of the foetal brain, and previous work proposed that MSI1 could drive the pathogenesis caused by ZIKV, and therefore, could be linked to the cases of congenital microcephaly observed during the 2015/2016 epidemic. The work presented here utilises reverse genetics to investigate the mechanism of MSI1 enhancement of ZIKV replication by introducing putative MSI1 binding site mutations into the 3′UTR. Using U251 cells ± MSI1, it was shown that one of the site mutations (Site D) induced a MSI1 dependent reduction in viral replication, indicating that this site may be particularly important for MSI1 binding to the ZIKV 3′UTR. Following viral characterisation in Vero cells, several of the site mutants displayed varying levels of viral attenuation, in one case so severe it was not possible to rescue virus. Passage experiments revealed the importance of one site mutation (Site B), whereby the partial reversion of one of the two mutated nucleotides was observed, indicating the importance of a single nucleotide in the 3′UTR to the overall fitness of the virus. The structure of the flavivirus 3′UTR is well conserved, highlighting its importance to the viral life-cycle. To structurally map the 3′UTR of the different site mutants, a novel structural cassette was designed and employed, allowing, for the first time, in vitro selective 2′-hydroxyl acylation analysed by primer extension mapping of the full 3′UTR structure. This was used to study the contribution of specific nucleotides to the 3′UTR structure, revealing a disruption in RNA structure in the Site B mutant, and thus sfRNA production. Additionally, sfRNA production was found to be restored following the single nucleotide reversion. Further investigations linked the hampered sfRNA production to reduced virion production in IFN competent A549 cells, which was not observed in IFN deficient Vero cells, reaffirming the link between sfRNA production and immune evasion. Overall, this thesis highlights the importance of the ZIKV 3′UTR to viral fitness and provides useful tools for future investigations.