Inhibition of Membrane Cholesterol of Astroglial Cells Leads to Reduced Epstein-Barr Virus Latent Gene Expression: Implications for Virus-Associated Neurological Diseases

The enigmatic association of the Epstein-Barr virus (EBV) with neuropathologies is in constant heed over the last few decades. To better understand the role of membrane cholesterol in EBV infection and the pathogenesis of astroglial cells, here we have used its inhibitor, methyl-β-cyclodextrin (MβCD). The astroglial cells treated with MβCD showed a consistent decrease in ebna1 transcripts compared to untreated EBV-infected cells. Notably, lmp1 and lmp2a were significantly downregulated upon MβCD treatment. Molecules involved in EBV-mediated downstream signalling namely STAT3 and RIP, exhibited a decrease in protein levels after exposure to MβCD while NF-kB levels were found to increase. The levels of TNF-α were decreased in MβCD+EBV at 1, 2 and 4 hrs compared to EBV infection alone. Systemic changes in biomolecular fingerprints were analyzed using Raman microspectroscopy (RS). The intensity of the Raman spectra revealed an increase in triglycerides and fatty acids in the periphery of EBV-infected LN-229 cells compared to MβCD+EBV. The full width at half maximum (FWHM) which corresponds to the peak width of cholesterol, lipid and fatty acids was found to be reduced in EBV-infected samples. This indicates an increase in unique cholesterol moieties. The shift of Raman peak at 1 hr of periphery manifested a change i.e., protein to triglycerides. An opposite pattern was observed in the nucleus. The ingenuity pathway analysis revealed novel protein molecules such as UCP-2, CYP11A1, USP-22, DBH, ADM, and SMPD1 that bridge an attachment with an altered profile of cholesterol, fatty acids and triglycerides with infection-related CNS disorders. Taken together, our results underline the important role of membrane cholesterol in EBV entry/pathogenesis in astroglial cells which might further trigger/exacerbate virus-associated neuropathologies.