JNK pathway restricts DENV, ZIKV and CHIKV infection by activating complement and apoptosis in mosquito salivary glands

Abstract

150 words) 23 Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. 24 However, there is a dearth of knowledge on SG immunity. Here, we characterized SG 25 immune response to dengue, Zika and chikungunya viruses using high-throughput 26 transcriptomics. The three viruses regulate components of Toll, IMD and JNK pathways. 27 However, silencing of Toll and IMD components showed variable effects on SG infection by 28 each virus. In contrast, regulation of JNK pathway produced consistent responses. Virus 29 infection increased with depletion of component Kayak and decreased with depletion of 30 negative regulator Puckered. Virus-induced JNK pathway regulates complement and 31 apoptosis in SGs via TEP20 and Dronc, respectively. Individual and co-silencing of these 32 genes demonstrate their antiviral effects and that both may function together. Co-silencing 33 either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. We 34 identified and characterized the broad antiviral function of JNK pathway in SGs, expanding 35 the immune arsenal that blocks arbovirus transmission. 36 37 38Despite the critical role of SGs in transmission, only three studies have examined DENV-87 responsive differential gene expression in SGs. These studies revealed activation of the Toll 88 5 and IMD pathways (Bonizzoni et al., 2012; Luplertlop et al., 2011; Sim et al., 2012) and 89 identified the anti-DENV functions of Cecropin (Luplertlop et al., 2011), putative Cystatin 90 and ankyrin-repeat proteins (Sim et al., 2012). Here, we characterized the SG immune 91 response to DENV, ZIKV and CHIKV. We performed the first high throughput RNA-92 sequencing (RNA-seq) in infected SGs and observed differentially expressed genes (DEGs) 93 related to immunity, apoptosis, blood-feeding and lipid metabolism. Using gene silencing, we 94 discovered that upregulated components of the Toll and IMD pathways had variable effects 95 against DENV, ZIKV and CHIKV. However, silencing of a JNK pathway upregulated 96 component increased, and silencing of a negative regulator decreased infection by the three 97 viruses in SGs. Further, we show that the JNK pathway is activated by all viruses and triggers 98 a cooperative complement and apoptosis response in SGs. This work identifies and 99 characterizes the JNK antiviral response that reduces DENV, ZIKV and CHIKV in A. aegypti 100 SGs. 101 102 Results 103 Transcriptome regulation by DENV, ZIKV and CHIKV in SGs 104 SGs were collected at 14 days post oral infection (dpi) with DENV and ZIKV, and at seven 105 dpi with CHIKV (Supplementary Figure 1) to account for variability between virus extrinsic 106 incubation periods (EIP) (Mbaika et al., 2016; Salazar et al., 2007). Differentially expressed 107 genes (DEGs) were calculated with DESeq2, edgeR and Cuffdiff 2, and showed little overlap 108 among the algorithms (Supplementary Figure 2). To validate DEGs and select which 109 software to use, we quantified the expression of 10 genes in a biological repeat with RT-1...

PEBP regulates balance between apoptosis and autophagy, enabling coexistence of arbovirus and insect vector

“…Varied with the regulatory pathway involved, arbovirus species and vector tissue, apoptosis may have contrasting effects on arbovirus loading within insect vectors. The antiviral JNK pathway-activated apoptosis restricts the arboviruses Dengue, Zika and Chikungunya in salivary gland of Aedes aegypti , while current study shows that MAPK-dependent activation of apoptosis promoted TYLCV loading in MED whitefly (Chowdhury et al, 2020). Likewise, apoptosis occurred in midgut of Ae.…”

“…The copyright holder for this preprint this version posted January 20, 2021. ; https://doi.org/10.1101/2021.01.19.427364 doi: bioRxiv preprint restricts the arboviruses Dengue, Zika and Chikungunya in salivary gland of Aedes aegypti, while current study shows that MAPK-dependent activation of apoptosis promoted TYLCV loading in MED whitefly (Chowdhury et al, 2020). Likewise, apoptosis occurred in midgut of Ae.…”

Capturing the transcription factor interactome in response to sub-lethal insecticide exposure

“…Of the 44 transcription factors, 3 had previously been linked with insecticide resistance in Anopheles mosquitoes and just 11 had been previously studied in mosquito species in any context (Amenya et al, 2010;Chen et al, 2017;Chowdhury et al, 2020;Fu et al, 2020;Ingham et al, 2018Ingham et al, , 2017Luna et al, 2006;Maliti et al, 2016;Ruiz et al, 2019;Wang et al, 2017;Wülbeck and Simpson, 2002). All but 4 of these transcription factors have a welldefined role in Drosophila.…”

“…A number of these transcription factors have known roles in stress response in Drosophila (Table 1); however, only Maf-S, Met and Dm have previously been linked with insecticide response in mosquitoes (Ingham et al, 2018(Ingham et al, , 2017. Of the transcription factors selected for analysis the following have been studied in mosquitoes: p53 has been shown to respond to arboviral infection (Chen et al, 2017); Rbsn-5 has been shown to be involved in egg shell formation (Amenya et al, 2010); l(1)sc is linked with sensory tissue development (Wülbeck and Simpson, 2002); kayak is involved in salivary gland response to arboviral infection through JNK pathway activation (Chowdhury et al, 2020); Hnf4 is linked to ecdysone and Met mediated lipid metabolism (Wang et al, 2017); Cyc controls the circadian ryhthm (Maliti et al, 2016); REL1 and REL2 are involved in immune response (Luna et al, 2006); Kr-h1 is essential for egg development (Fu et al, 2020) and Pan is linked with chromatin changes upon Plasmodium infection (Ruiz et al, 2019). et al (Rämet et al, 2002; Miotto et al 2006(Miotto et al, 2006 RNA-related Processes…”

Untitled

“…Infection of the salivary glands also changes the content of saliva, affecting blood acquisition and skin infection 5 . DENV ZIKV and CHIKV infections activate the c-jun N terminal kinase (JNK) pathway, activating complement and apoptotic effectors, resulting in a broad antiviral response 6 . The ZIKV pandemic was a "perfect storm" in which a novel American subclade arose from the virus's Asian origin and was introduced into a uniformly susceptible population that had never been exposed to ZIKV before 7 .…”