Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

Abstract

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

DEAD-Box Helicases: Sensors, Regulators, and Effectors for Antiviral Defense

“…Bunyaviruses snatch mRNA caps from host transcripts in P bodies, and thus cellular factors such as DDX6, which promote the decapping of host mRNAs and thereby deplete the pool of capped RNAs in P bodies, attenuate bunyaviral infection including La Crosse virus (LACV) and RVFV [40]. In mosquitoes, DDX6 is antiviral against the flaviviruses West Nile virus and Zika virus and is counteracted by the noncoding sfRNA derived from the flavivirus 3 UTR, which binds to DDX6 and sequesters it [67]. Human DDX6 is similarly antiviral against ZIKV and is sequestered by sfRNA [68].…”

“…IBV [81] dsRNA sensing (with DDX21 and DHX36) [21] Transmissible gastroenteritis virus [18] NF-κB signaling [19] DDX3X Arenavirus [38] JEV [47] HIV [74,84] IFN-β induction (with TBK1) [26] Innate immune signaling [27,28] HBV [36,93] Myxoma virus [88] VACV K7 [34] HBV polymerase [35] HCV 3 UTR [37] DDX5 JEV [47] HIV [84] Myxoma virus [88] DDX6 Negative regulation of ISG induction [66] RVFV, LACV [40] IVB [65] Flaviviruses: sequestered by sfRNA [67,68] DDX10 HIV [89] DDX17 HIV [84,94] RVFV [41] Cofactor for ZAP [43] Tombusviruses [45] DDX18…”

Differential small RNA responses against co-infecting insect-specific viruses inAedes albopictusmosquitoes

“…We speculate that these viral small RNAs from hotspots could derive from secondary structure of the virus rather than replication intermediates: DCR2 could cleave the hairpin structure of viral RNAs and the loop structure adjacent to the cleavage site might result in 26-30 nt long small RNAs as by-product of vsiRNAs production. Interestingly, previous studies suggested that the subgenomic RNA of flaviviruses, which corresponds to the 3’ UTR, is capable of supressing the RNAi machinery, possibly as an RNA decoy or by inhibiting the loading of vsiRNA into the RISC [21, 23, 24]. The high amount of siRNAs from the the 3’ UTR of AEFV could be a strategy from the virus to escape from the siRNA pathway.…”

“…The siRNA duplexes are loaded into the RNA-induced silencing complex (RISC) containing Argonaute2 (Ago2) that cleaves the target viral RNA under the guide of siRNA. Some mosquito viruses have evolved avoidance mechanisms to the siRNA pathway, demonstrating that virus-derived siRNAs (vsiRNAs) play an important antiviral function in mosquito vectors [19-24].…”

Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

“…sfRNAs have been associated with pathogenic outcomes and cytopathicity during infection (Funk et al 2010;Chang et al 2013;Walker et al 2013;Donald et al 2016;Filomatori et al 2017;Junglen et al 2017). Although their full set of functions remains an active area of investigation (Slonchak and Khromykh 2018) sfRNAs have been shown to bind a number of host proteins (Bidet et al 2014;Schnettler et al 2014;Manokaran et al 2015;Moon et al 2015;Goertz et al 2019;Michalski et al 2019) suggesting they affect several processes during infection including inhibiting the interferon response (Schuessler et al 2012) and suppressing RNAi pathways in insects (Schnettler et al 2012;Schnettler et al 2014;Moon et al 2015). Furthermore, there is evidence that sfRNAs are involved in switching between insect vectors and mammalian hosts (Filomatori et al 2017;de Borba et al 2019), enhancing transmission by mosquitoes (Pompon et al 2017;Yeh and Pompon 2018;Goertz et al 2019;Slonchak et al 2020), and altering the cell's mRNA decay program (Moon et al 2012;Michalski et al 2019).…”