Barrières d'hôtes dans la spécificité des interactions entre moustiques vecteurs et arbovirus – ArboVEC

In Axis 1 of the project, we test panels of empirically selected candidate genes in each mosquito species using a multi-step screening process:

i) Evaluate transcriptional modulation induced by viral infection, whether homologous or heterologous;

ii) Assess the significant effect on Anopheles–ONNV and Aedes–CHIKV interactions — and vice versa — after gene silencing via dsRNA.

Axis 1 will reveal the spectrum of phenotypes associated with candidate genes in Aedes and Anopheles cells, establishing a panel of significant pro- and antiviral factors specific to each virus in each mosquito species.

In Axis 2, the partners of the putative immune complex in Anopheles are identified by transfecting Anopheles cells with tagged plasmids encoding APL1, LRIM4, LRIM10, and TEP12, followed by infection with ONNV-WT and subsequent capture of the complex. Peptide sequencing reveals the subunit composition of this antiviral complex.

In Aedes, the existence of the putative immune complex is demonstrated by silencing the orthologs of APL1 and TEP12 in Aedes cells, followed by infection with CHIKV-WT. The complex subunits are identified using the same approach as in Anopheles. Furthermore, the function of this complex is analyzed during heterologous viral infection to determine whether it acts as a restriction factor.

Other selected candidate genes are studied in the same way, with the difference that genes that are transcriptionally repressed are tested by overexpression through plasmid transfection. Mechanisms and genes studied in mosquito cells are also investigated in whole mosquitoes.

Axis 3 focuses on Rasputin, whose role must be clarified in the infection of Anopheles and Aedes cells by ONNV and CHIKV. The interaction between nsP3 and Rasputin is believed to influence the host specificity of the virus. Physical interactions between nsP3 from ONNV and CHIKV and mosquito Rasputin are examined through pull-down assays coupled with proteomics.

Using nsP3 mutants helps to identify the domains (MD, AUD, or HVD) responsible for the physical interaction. Chimeric ONNV or CHIKV viruses, in which one or two nsP3 domains have been replaced with their counterparts, are used to infect cells that are either depleted of Rasputin or complemented with its ortholog. It tests the dependency of host restriction on Rasputin and compares molecular mechanisms between mosquito species.

Finally, both ONNV and CHIKV inhibit the Toll pathway, and the mammalian ortholog of Rasputin appears to interact with mammalian Toll pathway components. The hypothesis that nsP3, or its interaction with Rasputin, inhibits Toll pathway activity is assessed by activating or repressing the Toll pathway through Cactus or Rel1, respectively.

The use of fluorescent CHIKV revealed that viral dissemination does not occur before 30h post-infection in Aedes. Following RNA-seq of CHIKV-infected Aedes, 92 genes were identified as candidate genes. The most strongly regulated ones were first studied in Aedes cells infected with CHIKV or ONNV, or in cells depleted of Rin (Rin). Ultimately, two genes were tested in Aedes cells, along with one gene identified as overexpressed in Anopheles, AgSGU, which acts as a proviral factor—essential for optimal viral production and indicative of viral manipulation of the host.

In Aedes, two orthologs of AgSGU were identified. Some genes co-regulated with AgSGU had little to no effect on ONNV production, suggesting that AgSGU may be involved in regulating them as part of viral immune manipulation. Like Rin, AgSGU is proviral and exerts its activity on ONNV in a paracrine manner, and it belongs to the same pathway as Rin. Furthermore, Rin has been shown to negatively influence the IMD, JAK-STAT, and RNAi pathways, supporting the idea that AgSGU and Rin, through interaction with nsP3, modulate immune responses in Anopheles during infection.

We also showed that the interaction between Rin and nsP3 alters the protein partners of both nsP3 and Rin individually. NsP3 and Rin interact directly, and the interaction between CHIKV-nsP3 and Rin is stronger with Aedes Rin than with Anopheles Rin, while no notable difference was observed with ONNV-nsP3. These results suggest greater specificity of Aedes Rin for nsP3.

Additionally, while CHIKV genome replication depends on the hypervariable domain (HVD) of CHIKV-nsP3, the influence of this domain is negligible for ONNV. CHIKV replicates well in Aedes cells, whereas ONNV shows very low replication, a pattern not observed in Anopheles cells, where both viral genomes replicate equally well; this suggests that Aedes cells are less permissive than Anopheles cells. This is further supported by viral replication kinetics in both cell types, highlighting the importance of the MD and AUD domains of nsP3 in viral replication.

Finally, we measured the importance of nsP3 domains in infection, dissemination and transmission in Aedes and Anopheles mosquitoes. We showed that CHIKV clones are transmitted by Aedes as early as 3 days post-infection and we found that only wild-type nsP3s of CHIKV and ONNV allow efficient viral transmission at 14 days post-infection.

In Anopheles, only ONNV-WT is well transmitted as early as 3 days post-infection. CHIKV is transmitted only at low levels, and only if it carries at least one ONNV domain in its nsP3. The composition of nsP3 does not affect ONNV infection or dissemination, whereas only CHIKV-WT enables efficient infection and dissemination.

In Anopheles, only CHIKV viruses with the ONNV HVD infect mosquitoes efficiently, while no notable effect on dissemination is observed. For ONNV, both infection and dissemination are positively affected by this same domain.

Perspectives (3000 caractères)

The genes and proteins identified in the context of the response to CHIKV infection in Aedes and ONNV in Anopheles, as well as protein partners of the Rin-nsP3 complex, have highlighted important factors in the regulation of early infection in these mosquitoes. These factors must now be studied in vivo in mosquitoes following RNA depletion by RNAi, in order to assess their impact on viral infection and on previously identified or studied genes of interest, particularly those involved in mosquito immunity.

Furthermore, although protein partners of the Rin-nsP3 complex have been identified in Anopheles via pull-down assays, and while using the Aedes orthologs might initially be informative, it would be preferable to perform the same co-transfection experiment with tagged nsP3 and Rin in Aedes cells to identify the specific protein partners of this complex in Aedes, and to compare them with those found in Anopheles. The direct interaction between Rin and nsP3, mainly attributed to the hypervariable domain (HVD), suggests that the two other domains of nsP3 may also play important roles in this interaction. Therefore, investigating the nature of these interactions with Rin, as well as potential interactions between other non-structural proteins co-transfected with nsP3 and Rin—particularly given that nsP2 interacts with nsP3—should be considered.

Mutational analysis of nsP3 domains should also be carried out to identify the motifs necessary and sufficient for interaction. A better understanding of this interaction, and in particular of the critical motifs involved, could ultimately lead to the development of antiviral strategies, including vaccines that impair viral replication in infected hosts.

As with Anopheles, in Aedes, clarifying the existence of an immune complex should be feasible by building on the work already done in Anopheles, along with data related to mosquito immunity and the genes modulated by infection with the various viral families transmitted by Aedes (Orthoflaviviridae, Togaviridae, Bunyaviridae, etc.).

This study has identified factors that contribute to the transmission of CHIKV and ONNV by Aedes and Anopheles mosquitoes. Using the data already obtained on Rin in African mosquito populations may allow the identification—within mosquito genomes of non-confirmed vector species—of risk factors based on Rin sequence similarity. However, these findings must be validated by experimental infections to assess the potential transmission risk in currently virus-free regions.

Les moustiques anophèles sont les vecteurs connus d’un seul arbovirus, O’nyong nyong (ONNV), en émergence en Afrique. Par contre, les aedes transmettent de nombreux arbovirus tels que la dengue, Zika, et le chikungunya (CHIKV), responsables de pandémies. Nous comparerons les mécanismes antiviraux déclenchés par Anopheles coluzzii et Aedes aegypti contre ONNV et CHIKV. Les résultats permettront de déterminer les facteurs de restriction de l'hôte, de mesurer le risque qu’un arbovirus puisse changer d’hôtes vecteurs, et d’identifier de nouveaux outils pour interrompre la transmission des arbovirus Objectif 1, réaliser un criblage fonctionnel de gènes candidats en vue d’identifier les facteurs qui sous-tendent la fonction antivirale chez Anopheles et Aedes. Objectif 2, déterminer et caractériser les réponses immunitaires anti-virales chez Anopheles et Aedes. Objectif 3, définir le rôle d’un facteur de restriction Rasputin dans l’infection virale chez Anopheles et Aedes..