West Nile Virus Exploits miR-155-5p-Mediated Regulation of TRIM32-IκBα Axis to Evade NF-κB-Mediated Innate Immune Responses in Human Microglial Cells.

West Nile virus (WNV) is a neurotropic virus, having a single-stranded, positive-sense RNA genome belonging to the Flaviviridae family. WNV infection in the brain is associated with many neurological sequelae. Microglia are brain macrophages that respond to pathogenic breaches and eliminate threats. However, WNV is reported to evade cellular challenges and cause infection in the brain. In this study, we explored an underlying strategy by which WNV suppresses inflammation in microglial cells by increasing cellular microRNA-155-5p and dampening nuclear factor of kappa light-chain enhancer of activated B cells (NF-κB) signaling via tripartite motif-containing protein 32 (TRIM32). NF-κB is a crucial transcription factor involved in the production of pro-inflammatory cytokines and mediating cellular response. We observed increased expression of miR-155-5p in WNV-infected human microglial cells. The miR-155-5p-mediated regulation of TRIM32 and downstream NF-κB signaling was validated by miR-155-5p overexpression and knockdown in HMC3 cells. Increased miR-155-5p expression led to decreased TRIM32 expression. The decrease in TRIM32 sequestered the localization of inhibitor of nuclear factor of kappa light chain enhancer of activated B-cells, alpha (IκBα), and reduced its phosphorylation-mediated degradation. Consequently, resulting in decreased phosphorylation of NF-κB and decreased production of inflammatory cytokines. The protein expression patterns of TRIM32, p-IκBα, IκBα, p-NF-κB, NF-κB, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) were assessed by immunoblotting. This study demonstrates that WNV modulates host cellular machinery by dysregulating miR-155-5p, thereby suppressing the host inflammatory response in human microglial cells.