Flow cytometric reporter assays provide robust functional analysis of signaling complexes.

Conventional assays to probe signaling protein interactions and function involve measurement of luciferase reporter expression within the bulk cell population, with lack of control over target-protein expression level. To address this issue, we have developed a rapid and robust flow cytometric assay for analysis of signaling protein function. A fluorescent reporter and fluorescent tagging of the target protein enables simultaneous assessment of protein expression and signaling within individual cells. We have applied our technique to the analysis of variants of the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) and its adapter protein MyD88, using a NF-кB-responsive promoter driving mScarlet-I expression. The assay enables exclusion of nontransfected cells and overexpressing cells that signal spontaneously. Additionally, our assay allows the identification of protein variants that fail to express. We found that the assays were highly sensitive, with cells expressing an appropriate level of GFP-MyD88 showing approximately 200-fold induction of mScarlet-I by lipopolysaccharide, and we can detect subtle protein concentration-dependent effects of mutations. Importantly, the assay is adaptable to various signaling pathways.

Understanding the role of microRNAs in the interaction of Aedes aegypti mosquitoes with an insect-specific flavivirus.

The Flavivirus genus contains some of the most prevalent vector-borne viruses, such as the dengue, Zika and yellow fever viruses that cause devastating diseases in humans. However, the insect-specific clade of flaviviruses is restricted to mosquito hosts, albeit they have retained the general features of the genus, such as genome structure and replication. The interactions between insect-specific flaviviruses (ISFs) and their mosquito hosts are largely unknown. Pathogenic flaviviruses are known to modulate host-derived microRNAs (miRNAs), a class of non-coding RNAs that are important in controlling gene expression. Alterations in miRNAs may represent changes in host gene expression and promote understanding of virus-host interactions. The role of miRNAs in ISF-mosquito interactions is largely unknown. A recently discovered Australian ISF, Palm Creek virus (PCV), has the ability to suppress medically relevant flaviviruses. Here, we investigated the potential involvement of miRNAs in PCV infection using the model mosquito Aedes aegypti. By combining small-RNA sequencing and bioinformatics analysis, differentially expressed miRNAs were determined. Our results indicated that PCV infection hardly affects host miRNAs. Out of 101 reported miRNAs of Ae. aegypti, only aae-miR-2940-5p had a significantly altered expression over the course of infection. However, further analysis of aae-miR-2940-5p revealed that this miRNA does not have any direct impact on PCV replication in vitro. Thus, overall the results suggest that PCV infection has a limited effect on the mosquito miRNA profile and therefore miRNAs may not play a significant role in the PCV-Ae. aegypti interaction.

The Involvement of Atlastin in Dengue Virus and Wolbachia Infection in Aedes aegypti and Its Regulation by aae-miR-989.

Endoplasmic reticulum (ER)-shaping atlastin proteins (ATLs) have been demonstrated to play a functional role during flavivirus replication in mammalian cells. For dengue virus (DENV), atlastin is required in the formation of the replication organelles and RNA replication, virion assembly, production of the infectious virus particles, and trafficking or directing the association of vesicle packets with furin. Here, we investigated the involvement of atlastin in DENV replication in the mosquito Aedes aegypti and explored the possibility of its manipulation by the endosymbiotic bacterium Wolbachia to interfere with DENV replication. Results showed the expression of Ae. aegypti atlastin gene (AaATL) was upregulated in DENV-infected Aag2 cells, and its silencing led to reduced DENV replication. Contrary to our assumption that AaATL could be downregulated by Wolbachia, we did not find evidence for that in Wolbachia-infected cell lines, but this was the case in mosquitoes. Further, silencing AaATL did not have any effect on Wolbachia density. Our results also suggest that aae-miR-989 miRNA negatively regulates AaATL. The oversupply of the miRNA mimic led to reduced DENV replication consistent with the positive role of AaATL in DENV replication. Overall, the results favor AaATL's involvement in DENV replication; however, there is no support that the protein is involved in Wolbachia-mediated DENV inhibition. In addition, the results contribute to discerning further possible overlapping functions of ATLs in mosquitoes and mammalian cells. IMPORTANCE Atlastin is a protein associated with the endoplasmic reticulum and has been shown to play a role in replication of flaviviruses in mammalian cells. This study aimed to investigate the role of mosquito Aedes aegypti atlastin (AaATL) in dengue virus replication and maintenance of Wolbachia, an endosymbiotic bacterium, in the mosquito. Our results suggest that AaATL facilitates dengue virus replication in mosquito cells, considering silencing the gene led to reductions in virus replication and virion production. Further, AaATL was found to be regulated by a mosquito microRNA, aae-miR-989. Despite an effect on dengue virus, AaATL silencing did not affect Wolbachia replication and maintenance in mosquito cells. The results shed light on the role of atlastins in mosquito-pathogen interactions and their overlapping roles in mosquito and mammalian cells.