Exploring the molecular mechanism(s) involved in Wolbachia-Aedes aegypti-dengue virus interactions

Abstract

Dengue is one of the most significant health problems that has magnified its impact globally by affecting about 390 million people annually across 110 countries. The causative agent of this life-threating disease is a positive single stranded RNA arbovirus known as dengue virus (DENV), which uses species of Aedes mosquitoes as vectors. To date, there is no effective available vaccine or cure for dengue, and the control options primarily rely on vector control strategies, mostly through the application of pesticides. However, reports of resistance in Aedes mosquitoes against pesticides has limited this option as well. Therefore, there is an urgent need for alternative approaches to control the spread of DENV. One of the novel options involves the use of the endosymbiotic bacterium Wolbachia, that has successfully limited the ability of Aedes aegypti mosquitoes to transmit a number of life-threatening mosquito-borne viruses such as DENV and Zika virus. Despite its effectiveness to inhibit replication of DENV, very little is known about the mechanism(s) that Wolbachia uses to impart this antiviral effect. In this study, we looked into Ae. aegypti host factors that affect DENV replication and their potential manipulations by Wolbachia to find molecular mechanism(s) that Wolbachia utilizes to limit DENV replication. Recently, studies have shed light on the role of chromodomain DNA binding helicases in Human Immunodeficiency virus (HIV) and Influenza A virus. In Chapter 1, we have identified three Ae. aegypti homologs of chromodomain helicase DNA binding proteins (CHD) and determined their modulation in response to Wolbachia and DENV infections. We have found that among the three CHD members, AeCHD7/Kismet levels are significantly decreased in Wolbachia infection. Further investigations demonstrated that AeCHD7 is significantly increased in the case of DENV replication suggesting that it may facilitate DENV replication. Knock down studies of AeCHD7 confirmed this assumption as it resulted in significant reductions in DENV replication and virion production. In this study, we have identified AeCHD7 as an Ae. aegypti pro-DENV host factor that is downregulated by Wolbachia which may contribute in limiting DENV replication. Vago is an insect-specific secretory protein that has been identified in Culex quinquefasciatus to play an important role in the crosstalk between the mosquito’s immune pathways and reduce West Nile virus (WNV) replication. In Chapter 2, by in-silico identification of Vago characteristic SVWC domain and secretory signal, we identified two potential homologs of the Vago protein in Ae. aegypti and looked at their expression pattern in the case of Wolbachia infection to find that AeVago1 is highly induced in Ae. aegypti upon Wolbachia infection. However, we found no induction of AeVago1 expression in Ae. aegypti mosquitoes infected with DENV. Further, AeVago1 knockdown studies demonstrated that there was a significant increase in DENV replication in Wolbachia infected cells in AeVago1 deficient cells. However, there was no effect on Wolbachia density in AeVago1 depleted cells. The outcomes of this study suggest that in the presence of Wolbachia the immune gene AeVago1 is induced, which might also contribute towards inhibition of DENV replication. Pelo has been recently reported as a positive regulator of Drosophila C virus (DCV) replication. However, its role in the case of DENV replication has not been elucidated yet. In Chapter 3, we looked into the possible involvement of pelo in the case of Wolbachia-Ae. aegypti-DENV interactions. We found that the pelo protein levels increase during DENV replication. Silencing of pelo led to severe reduction of DENV virion production, suggesting its important role in DENV replication. However, in the case of Wolbachia infection, specifically in female Ae. aegypti mosquitoes, there was a significant decrease in the transcript levels of pelo. Further experiments confirmed that Wolbachia changes the subcellular localization of the pelo protein, suggesting that it might be a novel Ae. aegypti host factor that is used by Wolbachia to limit DENV replication. In addition, we found that Wolbachia-mediated downregulation of pelo transcripts might be regulated by aae-miR-2940-5p, which is highly induced by Wolbachia. This study has identified a novel molecular mechanism that is used by Wolbachia to limit DENV replication in Ae. aegypti mosquitoes. Nevertheless, this mechanism of inhibition does not seem to be universally seen in Wolbachia-host-virus interactions. Overall, in this study we have identified three novel host genes that play important roles in DENV replication and that regulation of these genes in the presence of Wolbachia may contribute towards virus blocking in Ae. aegypti mosquitoes.