Regulatory role of TRIM21 in the type-I interferon pathway in Japanese encephalitis virus-infected human microglial cells.
BACKGROUND
Japanese encephalitis virus (JEV) infection leads to Japanese encephalitis (JE) in humans. JEV is transmitted through mosquitoes and maintained in a zoonotic cycle. This cycle involves pigs as the major reservoir, water birds as carriers and mosquitoes as vectors. JEV invasion into the central nervous system (CNS) may occur via antipodal transport of virions or through the vascular endothelial cells. Microglial cells get activated in response to pathogenic insults. JEV infection induces the innate immune response and triggers the production of type I interferons. The signaling pathway of type I interferon production is regulated by a number of molecules. TRIM proteins are known to regulate the expression of interferons; however, the involvement of TRIM genes and their underlying mechanism during JEV infection are not known.


METHODS
Human microglial cells (CHME3) were infected with JEV to understand the role of TRIM21 in JEV infection and its effect on type I interferon (IFN-β) production. Cells were infected in presence and absence of exogenous TRIM21 as well as after knocking down the TRIM21 mRNA. Levels of activated IRF3 expression were measured through Western blot analyses of anti-p-IRF3 antibody, and IFN-β production was measured by using IFN-β real-time PCR and luciferase activity analyses.


RESULTS
JEV infection increased expression of TRIM21 in CHME3 cells. JEV induced an innate immune response by increasing production of IFN-β via IRF3 activation and phosphorylation. Overexpression of TRIM21 resulted in downregulation of p-IRF3 and IFN-β, while silencing led to increased production of p-IRF3 and IFN-β in JEV-infected CHME3 cells.


CONCLUSIONS
This report demonstrates TRIM21 as a negative regulator of interferon-β (IFN-β) production mediated by IRF-3 during JEV infection in human microglial cells.
Gunjan Dhawan
GD
Manocha
Ritu
R
Mishra
Nikhil
N
Sharma
Kanhaiya Lal
KL
Kumawat
Anirban
A
Basu
Laboratory of Neurovirology and Inflammation Biology, CSIR-Centre for Cellular and Molecular Biology (CCMB), New R&D Building-1st Floor, Uppal Road, Hyderabad 500007, India. sunitsingh2000@gmail.com.
Sunit K
SK
Singh
D002461:Cell Line, Transformed; D004664:Encephalitis Virus, Japanese / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology*; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D007370:Interferon Type I / Q000378:metabolism*; D017628:Microglia / Q000378:metabolism* / Q000821:virology; D010766:Phosphorylation; D012333:RNA, Messenger; D012261:Ribonucleoproteins / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology*; D013997:Time Factors; D014162:Transfection; D010948:Viral Plaque Assay; D000099207:SS-A Antigen
D050838:Interferon Regulatory Factor-3; D007370:Interferon Type I; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D000099207:SS-A Antigen; C494232:IRF3 protein, human