Cullin4 Is Pro-Viral during West Nile Virus Infection of Culex Mosquitoes

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.

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

“…Infection of live mosquitoes with ZIKV and treatment with the IR inhibitor Eight to ten, 5-8 day old female mosquitoes of the Higgs and Brisbane strains (kindly provided by Dr. Ary Hoffmann, University of Melbourne, Australia, and Dr Akbari, University of California, respectively), were starved the day before being challenged with an virus-infected blood meal containing 10 5.6 TCID 50 (50 percent tissue culture infectious dose)/mL, through membrane feeding using chicken blood and skin (Paradkar et al, 2015). Chicken blood and skin were provided by the Small Animal Facility of the Australian Animal Health Laboratory from chicken bred in the laboratory with no sign of arboviral infection.…”

Biochemistry and Molecular Biology of Flaviviruses

“…376 Similar results were obtained in human THP-1 cells infected by an antibody-dependent route, 377 and the proteasome inhibitors, bortezomib and MG132, were shown to reduce infection by DENV, ZIKV, and WNV. 378,379 Notably, other groups have reported that the ubiquitin system is important for early events in flavivirus infections. Wang et al showed that several proteasome inhibitors caused internalized JEV particles to be mislocalized to lysosomes, resulting in loss of infection.…”

Emerging Mechanisms of Insulin-Mediated Antiviral Immunity in Drosophila melanogaster

“…This includes TotM during early stages of infection (24) and vir-1 during later stages (12) (Figure 2D). JAK/STAT has been shown to be involved in the immune response against the insect virus Drosophila C virus but not SINV (24) in Drosophila, and JAK/STAT is antiviral against WNV in both Drosophila and Culex mosquitoes (10,50).…”