The DEAD-box RNA helicase Dhx15 controls glycolysis and arbovirus replication in Aedes aegypti mosquito cells

Abstract

Aedes aegypti mosquitoes are responsible for the transmission of arthropod-borne (arbo)viruses including dengue and chikungunya virus (CHIKV), but in contrast to human hosts, arbovirus infected mosquitoes are able to efficiently control virus replication to sub-pathological levels. Yet, our knowledge about the molecular interactions of arboviruses with their mosquito hosts is largely incomplete. Here, we aimed to identify and characterize novel host genes that control arbovirus replication in Aedes mosquitoes. RNA binding proteins (RBPs) are well known to regulate immune signaling pathways in all kingdoms of life. We therefore performed a knockdown screen targeting 461 genes encoding predicted RBPs in Aedes aegypti Aag2 cells and identified 15 genes with antiviral activity against a Sindbis reporter virus. Amongst these, three DEAD-box RNA helicases, AAEL004419/Dhx15, AAEL008728 and AAEL004859 also acted as antiviral factors in dengue and CHIKV infections. Here, we explore the mechanism of Dhx15 in regulating an antiviral transcriptional response in mosquitoes by silencing Dhx15 in Aag2 cells followed by deep-sequencing of poly-A enriched RNAs. Dhx15 knockdown in uninfected or CHIKV-infected cells resulted in differential expression of 856 and 372 genes, respectively. Interestingly, amongst the consistently downregulated genes, glycolytic process was the most strongly enriched GO term as the expression of all core enzymes of the glycolytic pathway was reduced, suggesting that Dhx15 regulates glycolytic function. A decrease in lactate production supported the observation that Dhx15 silencing functionally impaired glycolysis. Modified rates of glycolytic metabolism have been implicated in controlling the replication of several classes of viruses and strikingly, infection of Aag2 cells with CHIKV by itself also resulted in the decrease of several glycolysis genes. Our data suggests that Dhx15 regulates replication of CHIKV, and possibly other arboviruses, by controlling glycolysis in mosquito cells.

The Hsf1-sHsp cascade has pan-antiviral activity in mosquito cells

“…We observed major transcriptional changes at 8 hpi and 48 hpi, which we termed the early and late responses to CHIKV infection, respectively. The late response was more pronounced, with eight upregulated and 51 downregulated genes (Figure 1A), and KEGG pathway analysis suggested that downregulated DEgenes were mainly involved in glycolysis, which we have reported before 35 . In contrast, the early response was modest, with two downregulated and 19 upregulated genes, which were enriched for genes encoding HSP20-like chaperones (Figure 1A).…”

“…For RNA sequencing in Aag2 cells, control dsRNA (dsLuc) treated cells were infected with CHIKV at a MOI of 5. The double stranded RNA treatment was included in the procedure to allow a better comparison of the 4 hpi and 8 hpi samples to the 48 hpi datasets that was generated in parallel for a previously published study 35 .…”

“…4 hpi and 8 hpi samples to the 48 hpi datasets that was generated in parallel for a previously published study35 .Three to five day-old female mosquitoes were anesthetized on a CO 2 pad, washed once in 70% ethanol and three times with 1× PBS for outer de-contamation. Afterwards, crop, fat body, leg, Malpighian tubules, midgut, ovary, salivary glands, and wings were dissected.…”

Arbovirus-vector protein interactomics identifies Loquacious as a co-factor for dengue virus replication in Aedes mosquitoes

“…From the mass spectrometry data, we identified one peptide unique to Loqs-PA in NS1 immunoprecipitations, whereas other peptides were shared between the three isoforms ( [23], and AAEL004859 which was shown to have a strong antiviral effect on DENV [38]. Notably, this analysis confirmed the interaction of Loqs with DENV non-structural proteins NS1, NS3 and NS5, enriched by 4.9-, 6.1-and 10.7-fold, respectively.…”

Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA cluster biogenesis in mosquitoes

“…In other model systems, transcription factors such as A-MYB and Ci are important for piRNA cluster transcription (Goriaux et al ., 2014; Li et al, 2013). We also tested the H3K36me3 writer, Setd2 (Sun et al, 2005), due to the observed readthrough transcription that was marked by rather a strong continuation of H3K36me3 signals (Figures 3A and D); and subsequently, two m 6A writers, Mellt3 and Mellt14, as recent studies suggested H3K36me3 guided the deposition of m 6A at 3’UTRs (Huang et al, 2019a), which may serve as a signal triggering readthrough transcription (C), 16 mRNA 3’end processing genes (D), 18 RNA export genes and confirmation of Nxf1 knockdown (dsNxf1) with another set of dsRNA (lower panel) (E), 26 general RNA processing genes (Halbach et al ., 2020) and 29 RNA helicase genes (Machado et al, 2022) (F).…”