Abstract
Although mosquitoes serve as vectors of many pathogens of public health importance, their response to viral infection is poorly understood. It also remains to be investigated whether viruses deploy some mechanism to be able to overcome this immune response. Here, we have used an RNA-Seq approach to identify differentially regulated genes in Culex quinquefasciatus cells following West Nile virus (WNV) infection, identifying 265 transcripts from various cellular pathways that were either upregulated or downregulated. Ubiquitin-proteasomal pathway genes, comprising 12% of total differentially regulated genes, were selected for further validation by real time RT-qPCR and functional analysis. It was found that treatment of infected cells with proteasomal inhibitor, MG-132, decreased WNV titers, indicating importance of this pathway during infection process. In infection models, the Culex ortholog of mammalian Cul4A/B (cullin RING ubiquitin ligase) was found to be upregulated in vitro as well as in vivo, especially in midguts of mosquitoes. Gene knockdown using dsRNA and overexpression studies indicated that Culex Cul4 acts as a pro-viral protein by degradation of CxSTAT via ubiquitin-proteasomal pathway. We also show that gene knockdown of Culex Cul4 leads to activation of the Jak-STAT pathway in mosquitoes leading to decrease viral replication in the body as well as saliva. Our results suggest a novel mechanism adopted by WNV to overcome mosquito immune response and increase viral replication.
Highlights
Flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), pose a huge burden on public healthcare system worldwide
We used an unbiased transcriptome sequencing approach to identify genes differentially regulated after WNV infection resulting in 265 transcripts from various cellular pathways
Using in vitro and in vivo infection models, we identified Culex Cul4 to be acting as pro-viral protein, increasing viral titers
Summary
Flaviviruses, such as West Nile virus (WNV) and dengue virus (DENV), pose a huge burden on public healthcare system worldwide. Mosquito immune pathways are less well understood but some recent studies have shown that they may play an important role during infection in the vector [4,5]. Lacking essential components of the mammalian innate and adaptive immune systems, such as interferons, antibodies, B cells, T cells and MHC antigens, mosquitoes have been shown to respond to viral infection by a range of mechanisms including RNA interference (RNAi) and by activation of several evolutionarily conserved signal transduction pathways, include the Toll, Imd/JNK and Jak-STAT [4,5,6,7]. Transcriptome analysis using genome-wide microarrays [8,9,10,11] have revealed complex dynamics of mosquito transcripts during infection and identified changes in expression of genes from diverse cellular processes, including ion binding, transport, metabolic processes and peptidase activity. Gene expression is tissue-specific, with differences reported between midgut and salivary glands [10]
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