Determinant host differences in ZIKV sfRNA accumulation

Zika virus ( Orthoflavivirus zikaese) is part of the Orthoflavivirus genus that contains several viral species posing major and expanding burdens to public health worldwide. Upon infection, orthoflaviviruses produce viral RNA fragments named sfRNAs (short flaviviral RNA), generated from viral genomic RNA incompletely degraded by Xrn1/Pcm, the major cellular 5’-3’ exonuclease. Several conserved elements in the orthoflavivirus 3’ untranslated region (UTR) are responsible for X rnI-resistance, named xr RNA. The ubiquitous prevalence of xrRNA/sfRNA in flaviviruses indicate an important role of these structures in the viral life cycle, although their function is not completely understood. Here, we used in vitro reconstitution and infection models to examine the role of ZIKV xrRNA2 in mammalian and insect cells. SHAPE RNA structure probing revealed that disruption of ZIKV xrRNA2 does not affect xrRNA1 or dumbbell structure elsewhere in the 3’UTR despite disrupting all sfRNA accumulation in mammalian cells. In vitro RNA degradation assays showed that xrRNA1 efficiently stalled human or mosquito Xrn1 independent of xrRNA2 integrity. Reversion in mammalian cells occurred at early passages in protein kinase R (PKR) ^-/- or IFNα-receptor1 (IFAR1) ^-/- /PKR ^-/- knock out (KO) or double KO mammalian A549 cells, respectively, while wild-type virus outcompeted the xrRNA2 defective virus in RNAi-deficient mosquito cells. Together, our data reveals novel details of the mechanism of sfRNA accumulation in mammalian and insect cells We demonstrate a differential sensitivity to the absence of a functional xrRNA2 structure in mammalian and insect cells, with reversion to an active xrRNA2 occurring more rapidly in mammalian cells. Together, our work reveals new details on the requirements for ZIKV sfRNA accumulation in mammalian and mosquito cells.