HIV-1 Tat C-mediated regulation of tumor necrosis factor receptor-associated factor-3 by microRNA 32 in human microglia

Abstract

BackgroundHIV-1 Tat protein is known to be associated with neuroinflammation, a condition that develops in almost half of patients infected with HIV-1. HIV-1 Tat can alter glial neuroprotective functions, leading to neurotoxicity within the CNS. HIV-1 Tat is known to be secreted from productively infected cells and can affect neighboring uninfected cells by modulating cellular gene expression in a bystander fashion.MethodsWe were interested to study whether exogenous exposure to HIV-1 Tat-C protein perturbs the microRNA (miRNA) expression profile of human microglial cells, leading to altered protein expression. We used protein expression and purification, miRNA overexpression, miRNA knockdown, transfection, site-directed mutagenesis, real-time PCR, luciferase assay and western blotting techniques to perform our study.ResultsHIV-1 Tat-C treatment of human microglial cells resulted in a dose-dependent increase in miR-32 expression. We found that tumor necrosis factor-receptor–associated factor 3 TRAF3) is a direct target for miR-32, and overexpression of miR-32 in CHME3 cells decreased TRAF3 both at the mRNA and the protein level. Recovery of TRAF3 protein expression after transfection of anti-miR-32 and the results of the luciferase reporter assay provided direct evidence of TRAF3 regulation by miR-32. We found that the regulation of interferon regulatory factor 3 (IRF3) and IRF7 is controlled by cellular levels of TRAF3 protein in microglial cells, as after overexpression of miR-32 and application of anti-miR-32, expression levels of IRF3 and IRF7 were inversely regulated by expression levels of TRAF3. Thus, our results suggest a novel miRNA mediated mechanism for regulation of TRAF3 in human microglial cells exposed to HIV-1 Tat C protein. These results may help to elucidate the detrimental neuroinflammatory consequences of HIV-1 Tat C protein in bystander fashion.ConclusionHIV-1 Tat protein can modulate TRAF3 expression through miRNA mediated pathway and can change the downstream expression of IRF3 and IRF7. This study demonstrates a novel mechanism of HIV-1 Tat C protein-mediated perturbation of miRNA, resulting in dysregulation of cellular TRAF3.

Role of TRAF3 in neurological and cardiovascular diseases: an overview of recent studies

“…In viral infection (12,22), increased expression of miR-32 was correlated with the levels of HIV-1 Tat protein (associated with HIV-associated neurological disorder). Interestingly, the target of miR-32 is TRAF3.…”

“…Other conditions in which TRAF3 expression is affected are osteoclastogenesis (21) and viral infection, such as human immunodeficiency virus (HIV) infection (12).…”

“…As TRAF3 controls the innate and adaptive immune responses, its deficiency implies the development of immunodeficiency syndromes (Table 1) (12). TRAF3 degradation plays an important role in activating the canonical and non-canonical NF-κβ pathways leading to the production of IFN.…”

“…The main pathways regulated by TRAF3 are the canonical and non-canonical NF-κβ pathway (12) and the JNK pathway (13).…”

“…TRAF3 is an inhibitor of the non-canonical NF-κβ activation induced by the lymphotoxin-β receptor (LTβR) and the canonical NF-κβ pathway, thus inhibiting the inflammatory response (Table 1) (8,12).…”