Characterization of putative Japanese encephalitis virus receptor molecules on microglial cells

Abstract

Japanese encephalitis virus (JEV) a mosquito-borne flavivirus is a major cause of viral encephalitis in Asia. While the principle target cells for JEV in the central nervous system are believed to be neurons, microglia are activated in response to JEV and have been proposed to act as a long lasting virus reservoir. Viral attachment to a host cell is the first step of the viral entry process and is a critical mediator of tissue tropism. This study sought to identify molecules associated with JEV entry to microglial cells. Virus overlay protein-binding assay (VOPBA) and liquid chromatography-mass spectrometry (LC/MS/MS) identified the 37/67 kDa high-affinity laminin receptor protein and nucleolin as a potential JEV-binding proteins. These proteins were subsequently investigated for a contribution to JEV entry to mouse microglial BV-2 cells together with other possible candidate receptor molecules including Hsp70, Hsp90, GRP78, CD14, and CD4. In antibody mediated inhibition of infection experiments, both anti-laminin receptor and anti-CD4 antibodies significantly reduced virus entry while anti-Hsp70 and 90 antibodies produced a slight reduction. Significant inhibition of virus entry (up to 80%) was observed in the presence of lipopolysaccharide (LPS) which resulted in a complete down-regulation of CD4 and moderate down-regulation of CD14. These results suggest that multiple receptor proteins may mediate the entry of JEV to microglial cells, with CD4 playing a major role.

GRP78 is a novel receptor initiating a vascular barrier protective response to oxidized phospholipids

“…It was initially identified as a key component of the cellular stress pathway induced by accumulation of unfolded proteins. GRP78 is mainly located in the ER, where it binds denatured or incorrectly folded polypeptides and initiates a cascade of protective reactions helping the cell to survive under conditions of stress induced by protein overload (He et al ., 2011), viral infection (Shi-Chen Ou et al ., 2011; Thongtan et al ., 2012), and so on. Alterations in GRP78 tissue levels or activity have been implicated in cancer, immune regulation, aging, Alzheimer disease, and other diseases associated with ER stress (Misra et al ., 2005; Pfaffenbach and Lee, 2011; Wei et al ., 2012; Soejima et al ., 2013).…”

GRP78 Is an Important Host Factor for Japanese Encephalitis Virus Entry and Replication in Mammalian Cells

“…Though this technique has limitations, it has been widely employed in several studies, most likely because of its technical ease. Thus, proteins that have been suggested to play a role in JEV entry are HSP70, vimentin, laminin receptor, CD4, ␣5␤3 integrin, and DC-SIGN (14)(15)(16)(17)(18).…”

Nucleolin Interacts with the Dengue Virus Capsid Protein and Plays a Role in Formation of Infectious Virus Particles

“…NCL is involved in attachment or entry of several unrelated viruses, including human parainfluenza virus type 3, respiratory syncytial virus, Crimean-Congo hemorrhagic fever virus, Japanese encephalitis virus (JEV), and HIV (41)(42)(43)(44)(45)(46)(47). NCL colocalizes with hepatitis D virus delta antigen and interacts with herpes simplex virus 1 protein US11, cytomegalovirus UL44, white-spot syndrome virus VP15, influenza virus (H3N2) NS1, and the NS5B protein of hepatitis C virus.…”