RNA interference (RNAi) plays an essential role in mosquito antiviral immunity, but it is not known whether viral small interfering RNA (siRNA) profiles differ between mosquito-borne and mosquito-specific viruses. A pan-Orthoflavivirus analysis in Aedes albopictus cells revealed that viral siRNAs were evenly distributed across the viral genome of most representatives of the Flavivirus genus. In contrast, siRNA production was biased toward the 3' untranslated region (UTR) of the genomes of classical insect-specific flaviviruses (cISF), which was most pronounced for Kamiti River virus (KRV), a virus with a unique, 1.2 kb long 3' UTR. KRV-derived siRNAs were produced in high quantities and almost exclusively mapped to the 3' UTR. We mapped the 5' end of KRV subgenomic flavivirus RNAs (sfRNAs), products of the 5'-3' exoribonuclease XRN1/Pacman stalling on secondary RNA structures in the 3' UTR of the viral genome. We found that KRV produces high copy numbers of a long, 1,017 nt sfRNA1 and a short, 421 nt sfRNA2, corresponding to two predicted XRN1-resistant elements. Expression of both sfRNA1 and sfRNA2 was reduced in Pacman-deficient Aedes albopictus cells; however, this did not correlate with a shift in viral siRNA profiles. We suggest that cISFs, particularly KRV, developed a unique mechanism to produce high amounts of siRNAs as a decoy for the antiviral RNAi response in an sfRNA-independent manner.IMPORTANCEThe Flavivirus genus contains diverse mosquito viruses ranging from insect-specific viruses circulating exclusively in mosquito populations to mosquito-borne viruses that cause disease in humans and animals. Studying the mechanisms of virus replication and antiviral immunity in mosquitoes is important to understand arbovirus transmission and may inform the development of disease control strategies. In insects, RNA interference (RNAi) provides broad antiviral activity and constitutes a major immune response against viruses. Comparing diverse members of the Flavivirus genus, we found that all flaviviruses are targeted by RNAi. However, the insect-specific Kamiti River virus was unique in that small interfering RNAs are highly skewed toward its uniquely long 3' untranslated region. These results suggest that mosquito-specific viruses have evolved unique mechanisms for genome replication and immune evasion.

Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein–protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.

Efficient virus replication in Aedes vector mosquitoes is essential for the transmission of arboviral diseases such as dengue virus (DENV) in human populations. Like in vertebrates, virus-host protein-protein interactions are essential for viral replication and immune evasion in the mosquito vector. Here, 79 mosquito host proteins interacting with DENV non-structural proteins NS1 and NS5 were identified by label-free mass spectrometry, followed by a functional screening. We confirmed interactions with host factors previously observed in mammals, such as the oligosaccharyltransferase complex, and we identified protein-protein interactions that seem to be specific for mosquitoes. Among the interactors, the double-stranded RNA (dsRNA) binding protein Loquacious (Loqs), an RNA interference (RNAi) cofactor, was found to be essential for efficient replication of DENV and Zika virus (ZIKV) in mosquito cells. Loqs did not affect viral RNA stability or translation of a DENV replicon and its proviral activity was independent of its RNAi regulatory activity. Interestingly, Loqs colocalized with DENV dsRNA replication intermediates in infected cells and directly interacted with high affinity with DENV RNA in the 3' untranslated region in vitro (KD = 48-62 nM). Our study provides an interactome for DENV NS1 and NS5 and identifies Loqs as a key proviral host factor in mosquitoes. We propose that DENV hijacks a factor of the RNAi mechanism for replication of its own RNA.

Neurotropic flavivirus Japanese encephalitis virus (JEV) and West Nile virus (WNV) are amongst the leading causes of encephalitis. Using label-free quantitative proteomics, we identified proteins differentially expressed upon JEV (gp-3, RP9) or WNV (IS98) infection of human neuroblastoma cells. Data are available via ProteomeXchange with identifier PXD016805. Both viruses were associated with the up-regulation of immune response (IFIT1/3/5, ISG15, OAS, STAT1, IRF9) and the down-regulation of SSBP2 and PAM, involved in gene expression and in neuropeptide amidation respectively. Proteins associated to membranes, involved in extracellular matrix organization and collagen metabolism represented major clusters down-regulated by JEV and WNV. Moreover, transcription regulation and mRNA processing clusters were also heavily regulated by both viruses. The proteome of neuroblastoma cells infected by JEV or WNV was significantly modulated in the presence of mosquito saliva, but distinct patterns were associated to each virus. Mosquito saliva favored modulation of proteins associated with gene regulation in JEV infected neuroblastoma cells while modulation of proteins associated with protein maturation, signal transduction and ion transporters was found in WNV infected neuroblastoma cells.

Phosphoprotein (P) and matrix protein (M) cooperate to undermine the immune response to rabies virus (RABV) infections. While P is involved in the modulation of the Jak-Stat pathway through the cytoplasmic retention of interferon (IFN)-activated STAT1 (pSTAT1), M interacts with the RelAp43-p105-ABIN2-TPL2 complex, to efficiently inhibit the nuclear factor-κB (NF-κB) pathway. Using transfections, protein-complementation assays, reverse genetics and DNA ChIP, we identified a role of M protein in the control of Jak-Stat signaling pathway, in synergy with the P protein. In unstimulated cells, both M and P proteins were found to interact with JAK1. Upon type-I IFN stimulation, the M switches toward pSTAT1 interaction, which results in an enhanced capacity of P protein to interact with pSTAT1 and restrain it in the cytoplasm. Furthermore, the role for M-protein positions 77, 100, 104 and 110 was also demonstrated in interaction with both JAK1 and pY-STAT1, and confirmed in vivo. Together, these data indicate that M protein cooperates with P protein to restrain in parallel, and sequentially, NF-κB and Jak-Stat pathways.

Rabies virus relies on cellular machinery for its replication while simultaneously evading the host immune response. Despite their importance, little is known about the key host factors required for rabies virus infection. Here, we focused on the human kinome, at the core of many cellular pathways, to unveil a new understanding of the rabies virus infectious cycle and to discover new potential therapeutic targets in a small interfering RNA screening. The mitogen-activated protein kinase pathway and phosphatidylinositol metabolism were identified as prominent factors involved in rabies virus infection, and those findings were further confirmed in human neurons. While bringing a new insight into rabies virus biology, we also provide a new list of host factors involved in rabies virus infection.

To combat DENV transmission, we aim to screen for molecules that target mosquito host cell factors playing a role in the viral replication, consequently inhibiting the replication of virus in the mosquito. Use case scenarios for such compounds include human mass drug administration in endemic populations, application in sugar baits or bed-nets. In order to accomplish this, we have set up high throughput screening (HTS) using viral replication inhibition assays. We describe here the generation of a novel stable Nanoluciferase-reporter based dengue replicon system in U4.4 mosquito cells. The U4.4-DENV1 replicon cell line has been stably and successfully maintained for over 30 passages without significant loss of reporter signal. In order to characterize the cell line further, the cells were subjected to treatment with antiviral compound and viral inhibition was observed with ribavirin (IC50 =1.69 × 10-6M) and siRNA against NS3. For the purpose of HTS, viral inhibition, cytotoxicity and luciferase interference assays were established on a 384-well plate using the cryopreserved U4.4_DENV1 replicon cells. The latter two were used as deselection assays to differentiate between false- and true- positives. To conclude, we have developed a robust screening cascade to identify small molecules that may act as transmission blocking compounds. Screening of a chemical diversity library is ongoing and the results will be presented at the meeting.

1st purification: a-FLAG (A2220; Sigma-Aldrich) 2nd purification: GFP TRAP_A (ChromoTek)

At the crossroad between the NF-κB and the MAPK pathways, the ternary complex composed of p105, ABIN2 and TPL2 is essential for the host cell response to pathogens. The matrix protein (M) of field isolates of rabies virus was previously shown to disturb the signaling induced by RelAp43, a NF-κB protein close to RelA/p65. Here, we investigated how the M protein disturbs the NF-κB pathway in a RelAp43-dependant manner and the potential involvement of the ternary complex in this mechanism. Using a tandem affinity purification coupled with mass spectrometry approach, we show that RelAp43 interacts with the p105-ABIN2-TPL2 complex and we observe a strong perturbation of this complex in presence of M protein. M protein interaction with RelAp43 is associated with a wide disturbance of NF-κB signaling, involving a modulation of IκBα-, IκBβ-, and IκBε-RelAp43 interaction and a favored interaction of RelAp43 with the non-canonical pathway (RelB and p100/p52). Monitoring the interactions between host and viral proteins using protein-fragment complementation assay and bioluminescent resonance energy transfer, we further show that RelAp43 is associated to the p105-ABIN2-TPL2 complex as RelAp43-p105 interaction stabilizes the formation of a complex with ABIN2 and TPL2. Interestingly, the M protein interacts not only with RelAp43 but also with TPL2 and ABIN2. Upon interaction with this complex, M protein promotes the release of ABIN2, which ultimately favors the production of RelAp43-p50 NF-κB dimers. The use of recombinant rabies viruses further indicates that this mechanism leads to the control of IFNβ, TNF and CXCL2 expression during the infection and a high pathogenicity profile in rabies virus infected mice. All together, our results demonstrate the important role of RelAp43 and M protein in the regulation of NF-κB signaling.

This protocol aims to assess: - the interaction between two proteins harboring two parts of the gaussia luciferase (Glu1 and Glu2) - in presence of a third protein co-expressed - in presence of a rabies virus

The matrix (M) protein of wild isolates of rabies virus such as Tha (M-Tha) was previously shown to be able to interact with RelAp43, a protein of the NF-κB family, and to efficiently suppress NF-κB-dependent reporter gene expression, in contrast with the vaccine strain SAD. Here, we analyze the mechanisms involved in RelAp43-M protein interaction. We demonstrate that the central part of M-Tha, and the specific C-terminal region of RelAp43 are required for this interaction. Four differences in the corresponding amino acid sequences of the M-Tha and M-SAD are shown to be crucial for RelAp43 interaction and subsequent modulation of innate immune response. Furthermore, the capacity of M-Tha to interact with RelAp43 was shown to be crucial for the control of the expression of four genes (IFN, TNF, IL8 and CXCL2) during viral infection. These findings reveal that RelAp43 is a potent regulator of transcription of genes involved in innate immune response during rabies virus infection and that the M protein of wild isolates of rabies virus is a viral immune-modulatory factor playing an important role in this RelAp43-mediated host innate immunity response in contrast to M protein of vaccine strains, which have lost this property.

Le rôle central de la voie NFκB dans l’homéostasie cellulaire en fait une cible de choix pour les virus tel que le virus de la rage afin de contrôler la réponse immunitaire et faciliter la propagation chez l’hôte. Dans le cas des isolats de terrain des Lyssavirus, tel que la souche Thailand (Tha), la protéine de matrice (M) interagit avec la protéine RelAp43 de la voie NFκB en comparaison avec des virus de laboratoire telle que la souche vaccinale SAD. Nous montrons que MTha, en interagissant avec le domaine C-terminal de RelAp43, contrôle l’expression de gènes de l’immunité innée et de la réponse inflammatoire. Nous avons ensuite identifié les partenaires protéiques de RelAp43 dont l’interaction est modulée par MTha et notamment le complexe p105-ABIN2-TLP2. L’interaction de RelAp43 avec p105 stabilise la formation d’un complexe avec ABIN2 et TPL2, une MAPK. De plus, nous avons montré que MTha présente une forte interaction avec RelAp43 et ABIN2 en comparaison avec des mutants et exclus ABIN2 du complexe au cours de l’infection par Tha. Finalement, nous avons montré que MTha favorise la formation de dimères NFκB et confirmé la modulation de gènes de la réponse inflammatoire dépendante des voies NFκB et MAPK lors de l’infection de souris par le virus Tha. Afin de mieux comprendre l’importance des voies de signalisation dans le cycle biologique du virus de la rage, nous avons réalisé un criblage avec des pARNi ciblant les kinases et phosphatases au cours d’une infection par le virus Tha. Ainsi, nous confirmons l’importance des MAPKs au cours de l’infection par le virus de la rage et ouvert de nouveaux champs de recherche pouvant mener à une meilleure compréhension de la biologie du virus de la rage