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 doublestranded 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.

The host antiviral ribonuclease L protein supports Zika virus replication factory formation to enhance infectious virus production

“…We have previously shown that ZIKV activation of the OAS/RNase L pathway can be 127 detected by 24hpi (Whelan et al, 2019). Using host 28S and 18S rRNA degradation as a 128 measurement for RNase L activation, we show that RNase L activation during ZIKV 129 infection is mediated by the OAS3 isoform specifically (Figure 1A, left panel).…”

“…No reuse allowed without permission. that OAS3 is the primary human OAS isoform required for 2-5A synthesis during viral 185 infections (Li et al, 2016, Whelan et al, 2019, and verified that other OAS isoforms did 186 not compensate for loss of OAS3 expression during ZIKV infections. Together these 187 results suggest that OAS3 is the primary producer of 2-5A during ZIKV infection, thereby 188 providing a useful system for investigating RNase L function independent of its catalytic 189 activity during viral infection.…”

“…During infection with dengue or West Nile Kunjin 35 viruses, RNase L decreased virus production, suggesting that RNase L proviral function 36 is specific to ZIKV. and that this evasion strategy required assembly of RFs to protect genome from RNase 104 L cleavage (Whelan et al, 2019). Despite substantial RNase L-mediated cleavage of 105 intracellular ZIKV genome, a portion of uncleaved genome was shielded from activated 106…”

“…These results indicated that RNase L expression was ultimately 109 proviral during ZIKV infection (Figure 1A, right panel). Unlike ZIKV, DENV RFs did not 110 obstruct antiviral RNase L activity, and infectious virus production was instead decreased 111 in the presence of RNase L (Whelan et al, 2019, Lin et al, 2009. As this was the initial 112 report of viral resistance to catalytically active RNase L during infection, we sought to 113 isolate the differences between ZIKV RFs and those constructed by other flaviviruses, to 114 identify factors that enable this ZIKV evasion mechanism.…”

ISG15/USP18/STAT2 is a molecular hub regulating autocrine IFN I-mediated control of Dengue and Zika virus replication

“…We used an A549 cell line lacking ISG15, previously generated using CRISPR/Cas9 in our lab (Dos Santos et al, 2018). A549 cells were chosen due to their ability to support flavivirus replication and also to produce and respond to IFN-I (Gullberg et al, 2018;Wang et al, 2018;Whelan et al, 2019;Zhang et al, 2018).…”