Association of toll-like receptor 3 polymorphism with chronic hepatitis B

Abstract

Background: Previous studies in an experimental model of rabies showed neuronal process degeneration in association with severe clinical disease. Cultured adult rat dorsal root ganglion (DRG) neurons infected with CVS-11 strain of rabies virus (RABV) showed axonal swellings and immunostaining for 4-hydroxy2-nonenal (4-HNE) indicating evidence of lipid peroxidation associated with oxidative stress and reduced axonal growth versus mock-infection. We have demonstrated that RABV induces alterations in a variety of mitochondrial parameters, including an increase in reactive oxygen species production after addition of substrates or inhibitors and increased activity of Complex I of the respiratory chain vs. mock infection. We have hypothesized that a RABVprotein targets themitochondria and triggers its dysfunction. Methods & Materials: We used immunocytochemistry, Western immunoblotting, immunoprecipation andproteomicsmethods to studyRABV-andmock-infectionofmouseneuroblastoma(MNA) cells. Results: Immunocytochemistry of CVS-infected cells after 72h post-infection showed strong colocalization of mitochondria with the RABV phosphoprotein (P). Extracts of MNA cells from the mitochondrial subcellular fractionation were analyzed with a proteomics approach. We identified peptides belonging to the RABV P, glycoprotein (G), and nucleocapsid protein (N) with 67% of protein coverage at 95% confidence for P vs. N (8%) andG (6%). Our data showthat themajorityof viral peptides (79%)belong toPvs.N (13%) or G (8%), indicating that the extract was highly enriched with P. P was detected by immunoblotting in RABV-infected purified mitochondrial extracts and in Complex I immunoprecipitates from the extracts, but not in mock-infected extracts. A plasmid expressing P in cells produced an increase in Complex I activity,whereas expression of other RABV proteins did not. We have analyzed a variety of recombinant plasmids encoding various segments of the P gene. Expression of a peptide from amino acid 139 to 172 produced an increase in Complex I activity similar to expression of the entire P protein, whereasmost peptides that did not contain this region did not produce increased activity of Complex I. Conclusion: The RABV phosphoprotein is present in mitochondria and may interact with Complex I causing mitochondrial dysfunction, oxidative stress, neuronal process degeneration, and severe clinical disease in experimental rabies.