Arbovirus infection of Aedes aegypti salivary glands (SGs) determines transmission. However, there is a dearth of knowledge on SG immunity. Here, we characterized SG immune response to dengue, Zika and chikungunya viruses using high-throughput transcriptomics. We also describe a transcriptomic response associated to apoptosis, blood-feeding and lipid metabolism. The three viruses differentially regulate components of Toll, Immune deficiency (IMD) and c-Jun N-terminal Kinase (JNK) pathways. However, silencing of the Toll and IMD pathway components showed variable effects on SG infection by each virus. In contrast, regulation of the JNK pathway produced consistent responses in both SGs and midgut. Infection by the three viruses increased with depletion of the activator Kayak and decreased with depletion of the negative regulator Puckered. Virus-induced JNK pathway regulates the complement factor, Thioester containing protein-20 (TEP20), and the apoptosis activator, Dronc, in SGs. Individual and co-silencing of these genes demonstrate their antiviral effects and that both may function together. Co-silencing either TEP20 or Dronc with Puckered annihilates JNK pathway antiviral effect. Upon infection in SGs, TEP20 induces antimicrobial peptides (AMPs), while Dronc is required for apoptosis independently of TEP20. In conclusion, we revealed the broad antiviral function of JNK pathway in SGs and showed that it is mediated by a TEP20 complement and Dronc-induced apoptosis response. These results expand our understanding of the immune arsenal that blocks arbovirus transmission.

RbdA is a positive egulator ofiofilm ispersal of Its cytoplasmic region (cRbdA) comprises a N-terminal PAS domain followed by a diguanylate cyclase (GGDEF) and an EAL domain, whose phosphodiesterase activity is allosterically stimulated by GTP binding to the GGDEF domain. We report crystal structures of cRbdA and of two binary complexes: with GTP/Mg bound to the GGDEF active site and with the EAL domain bound to the c-di-GMP substrate. These structures unveil a 2-fold symmetric dimer, stabilized by a closely packed N-terminal PAS domain and a non-canonical EAL dimer. The auto-inhibitory switch is formed by an alpha helix (S-helix) immediately N-terminal to the GGDEF domain that interacts with the EAL dimerization helix (α) of the other EAL monomer and maintains the protein in a locked conformation. We propose that local conformational changes in cRbdA upon GTP binding lead to a structure with the PAS domain and S-helix shifted away from the GGDEF-EAL domains, as suggested by SAXS experiments. Domain reorientation should be facilitated by the presence of a α-helical lever (H-helix) that tethers the GGDEF and EAL regions, allowing the EAL domain to rearrange into an active dimeric conformation.Biofilm formation by bacterial pathogens increases resistance to antibiotics. RbdA positively regulates biofilm dispersal of The crystal structures of the cytoplasmic region of RbdA protein presented here reveal that two evolutionary-conserved helices play an important role in regulating the activity of RbdA, with implications for other dual GGDEF-EAL domains that are abundant in the proteomes of several bacterial pathogens. Thus, this work could assist the development of small molecules that would promote bacterial biofilm dispersal.

Zika virus (ZIKV) remains a potentially significant public health concern because it can cause teratogenic effects such as microcephaly in newborns and neurological disease like Guillain-Barré syndrome. Together with efforts to develop a vaccine, the discovery of antiviral molecules is important to control ZIKV infections and to prevent its most severe symptoms. Here we report the development of small non-nucleoside inhibitors (NNIs) of ZIKV RNA-dependent RNA polymerase (RdRp) activity. These NNIs target an allosteric pocket (“N-pocket”) located next to a putative hinge region between the thumb and the palm subdomains, that was originally described for dengue virus (DENV) RdRp. We first tested DENV RdRp N-pocket inhibitors against ZIKV RdRp, introduced chemical modifications into these molecules and assessed their potency using both enzymatic and cell-based assays. The most potent compound has an IC50 value of 7.3 μM and inhibits ZIKV replication in a cell-based assay with an EC50 value of 24.3 μM. Importantly we report four high-resolution crystal structures detailing how these NNIs insert into the N-pocket of ZIKV RdRp. Our observations point to subtle differences in the size, shape, chemical environment and hydration of the N-pocket from ZIKV RdRp compared to DENV RdRp, that are crucial for the design of improved antiviral inhibitors against ZIKV. IMPORTANCE Zika virus belongs to the flavivirus family that comprises several important human pathogens. There is currently neither an approved vaccine nor antiviral drugs available to prevent infection by ZIKV. The NS5 polymerase, which is responsible for replicating the viral RNA genome, represents one of the most promising targets for antiviral drug development. Starting from compounds recently developed against dengue virus NS5, we designed and synthetized inhibitors targeting the Zika virus NS5. We showed that these novel compounds inhibit viral replication by targeting the polymerase activity. High-resolution X-ray crystallographic structures of protein-inhibitor complexes demonstrate specific binding to an allosteric site within the polymerase called the N-pocket. This work paves the way for future structure-based design of potent compounds specifically targeting the ZIKV RNA polymerase activity.

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...