P036 Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs long-chain fatty acid metabolism

• Published in: Cytokine (Philadelphia, Pa.) Vol. 59; no. 3; p. 529
• Main Authors: Kao, Y.-T., Lin, R.-J., Chang, B.-L., Tsai, H.-J., Lin, Y.-L.
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.09.2012
• Subjects: antagonists; cultured cells; Cytokines; dehydrogenase; DNA-directed RNA polymerase; Encephalitis; Energy; Enzymatic activity; enzyme activity; fatty acid metabolism; Fatty acids; Immunoprecipitation; Infection; Interferon; interferons; Japanese encephalitis virus; long chain fatty acids; Metabolism; Methyltransferase; Mitochondria; Nonstructural proteins; Oxygen consumption; Palmitic acid; Plasmids; precipitin tests; Protein turnover; proteins; Reactive oxygen species; RNA-directed RNA polymerase; Transfection
• ISSN: 1043-4666; 1096-0023
• DOI: 10.1016/j.cyto.2012.06.119

Japanese encephalitis virus (JEV) nonstructural protein NS5 has two enzymatic activities, methyltransferase and RNA-dependent RNA polymerase, and has been identified as a strong antagonist of type I interferon signaling. How NS5 executes these diverse functions is largely unknown. We used immunoprecipitation assay to investigate the cellular proteins interacting with JEV NS5. The interaction of NS5 with cellular protein was confirmed by immunoprecipitation-western assay with NS5 expressed by plasmid transfection and by JEV infection. The defect of long-chain fatty acid metabolism was analyzed through measurement of the oxygen consumption rate (OCR) by XF Extracellular Flux Analyzer. Two mitochondrial proteins, hydroxyacyl-CoA dehydrogenase α and β subunits (HADHα and HADHβ) that form mitochondrial trifunctional protein and are involved in long-chain fatty acid β-oxidation, were immunoprecipitated with NS5. Besides the well-known cytoplasmic location, we find that NS5 could also be detected in mitochondria. The deletion constructs of NS5 suggest that the N-terminal methyltransferase domain of NS5 is essential for the interaction with HADHα. Since HADHα and HADHβ are involved in long-chain fatty acid metabolism, we measured OCR in cells supplemented with or without long-chain fatty acid palmitate. An enhanced OCR was noted in JEV-infected cells cultured in normal medium, however when palmitate was the only energy source, the OCR was greatly reduced in JEV-infected cells. These results suggest that JEV infection requires high energy supply, but long-chain fatty acid β-oxidation is impaired in JEV-infected cells. Impaired mitochondria β-oxidation has been implicated in reactive oxygen species production, cytokine induction, and virus-associated encephalitis, thus the role of NS5 interacting with HADHα/HADHβ in blocking long-chain fatty acid metabolism as well as the various functions of NS5 warrants further study.