Individual co-variation between viral RNA load and gene expression reveals novel host factors during early dengue virus infection of the Aedes aegypti midgut

Abstract

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.

Tudor-SN promotes early replication of dengue virus in the Aedes aegypti midgut

“…We also measured DENV RNA loads one day after the infectious blood meal and did not observe a decrease of viral loads. However, this is most likely due to the presence of viral RNA in the undigested blood still present in the midgut at this time point, as shown in a previous report (30). These results confirmed the proviral role of TSN during DENV infection in the mosquito midgut four day after the infectious blood meal.…”

“…aegypti mosquitoes remain poorly understood. Using a novel approach of transcriptomic analysis, we previously uncovered four genes that not only responded to DENV infection in the mosquito midgut but also had expression levels that correlated with viral loads in infected mosquitoes (30). Here, we focused on one of these four genes, Tudor-SN , encoding a member of the Tudor protein family.…”

“…Besides, most of previous studies have focused on mosquito antiviral or restriction factors that antagonize DENV, but little is known about mosquito host factors with a proviral function, that is factors enhancing DENV propagation. Several human factors required for DENV infectivity were recently discovered through genome-wide CRISPR screens (24–26), whereas only a handful of DENV host factors have been identified in mosquitoes to date (27–30). Although CRISPR screens cannot be readily carried out in live mosquitoes, transcriptome analysis by high-throughput RNA sequencing is a powerful method to identify DENV host and restriction factors in vivo (31).…”

“…In this study, we exploited a unique transcriptomic dataset that we previously generated by performing RNA sequencing on individual midguts in a field-derived Ae. aegypti population during early DENV-1 infection (30). In addition to a conventional pairwise comparison of gene expression between DENV-infected and uninfected controls, we also used an approach to detect correlations between viral RNA load and gene expression.…”

“…In addition to a conventional pairwise comparison of gene expression between DENV-infected and uninfected controls, we also used an approach to detect correlations between viral RNA load and gene expression. Out of 269 candidate genes identified by either method, only four were differentially expressed upon DENV infection and had expression levels that correlated with viral RNA load in infected mosquitoes (30). Amongst the four candidate genes identified by both methods was a gene encoding a member of the Tudor protein family, Tudor Staphylococcal Nuclease (abbreviated Tudor-SN or TSN), which we selected for further investigation in the present study.…”