Zika Virus Production Is Resistant to RNase L Antiviral Activity

Abstract

There is currently no knowledge of how the emerging human pathogen Zika virus (ZIKV) interacts with the antiviral endoribonuclease L (RNase L) pathway during infection. Since activation of RNase L during infection typically limits virus production dramatically, we used CRISPR-Cas9 gene editing technology to knockout (KO) targeted host genes involved in the RNase L pathway to evaluate the effects of RNase L on ZIKV infection in human A549 cells. RNase L was activated in response to ZIKV infection, which degraded ZIKV genomic RNA. Surprisingly, despite viral genome reduction, RNase L activity did not reduce ZIKV infectious titers. In contrast, both the flavivirus dengue virus and the alphavirus Sindbis virus replicated to significantly higher titers in RNase L KO cells compared to wild-type (WT) cells. Using MAVS/RNase L double KO cells, we demonstrated that the absence of increased ZIKV production in RNase L KO cells was not due to compensation by enhanced type I interferon transcripts to thus inhibit virus production. Finally, when synthetic double-stranded RNA was detected by OAS3 to induce RNase L antiviral activity prior to ZIKV infection, we observed reduced ZIKV replication factory formation, as well as a 42-fold reduction in virus yield in WT but not RNase L KO cells. This study proposes that ZIKV evades RNase L antiviral activity by generating a viral genome reservoir protected from RNase L cleavage during early infection, allowing for sufficient virus production before RNase L activation is detectable.IMPORTANCE With the onset of the 2015 ZIKV outbreak, ZIKV pathogenesis has been of extreme global public health interest, and a better understanding of interactions with the host would provide insight into molecular mechanisms driving the severe neurological outcomes of ZIKV disease. Here is the initial report on the relationship between ZIKV and the host oligoadenylate synthetase-RNase L (OAS-RNase L) system, a potent antiviral pathway effective at restricting replication of diverse viruses. Our study elucidated a unique mechanism whereby ZIKV production is impervious to antiviral RNase L activity, through a mechanism of viral RNA protection that is not mimicked during infection with numerous other RNase L-activating viruses, thus identifying a distinct replication strategy potentially important for ZIKV pathogenesis.

Keywords: RNA; RNase L; Sindbis virus; Zika virus; dengue virus; innate immunity; replication factories; viral genome.