Abstract
Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions.
Highlights
Flaviviruses are positive-sense single-strandedRNA viruses transmitted by arthropods
Based on our results in A549 cells in which Ras homolog family member V (RhoV) has a more dramatic effect on Zika virus (ZIKV) production (Figures 2 and S2), and the recent study [17] that reported a role in ZIKV replication and neuroinflammation for the host hippo signaling pathway, in which WW domain-containing transcription regulator 1 (WWTR1) is a critical player, we focused on further studying RhoV as a novel proviral factor for ZIKV infection
We identified RhoV and WWTR1 as candidate host proviral factors for ZIKV infection by performing a genome-wide CRISPR activation screen, which has the advantage of upregulating all gene isoforms from their endogenous promoter contexts [11]
Summary
Many of these viruses cause significant morbidities and mortalities in humans, including dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Zika virus (ZIKV) [1]. ZIKV, endemic to Africa, was known to only cause rash, fever, headache, arthralgia, and conjunctivitis. During recent outbreaks in French Polynesia (2013) and Brazil (2014–2016), infected individuals developed neurological diseases such as Guillain–Barré syndrome and meningoencephalitis [2]. Pregnant women infected with ZIKV during their first trimesters were more likely to give birth to children with severe brain abnormalities such as microcephaly, lissencephaly, or cortical calcification [3]. Despite the serious long-term consequences, there is no approved vaccine or therapy to control ZIKV infection [4]
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