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Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs fatty acid β-oxidation.

Kao YT, Chang BL, Liang JJ, Tsai HJ, Lee YL, Lin RJ, Lin YL - PLoS Pathog. (2015)

Bottom Line: LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells.Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP.We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.

No MeSH data available.


Related in: MedlinePlus

Impaired long-chain fatty acid (LCFA) β-oxidation and induction of reactive oxygen species (ROS)-dependent pro-inflammatory cytokines in cells infected with Japanese encephalitis virus (JEV).(A and B) A549 cells infected with JEV (multiplicity of infection [MOI] = 10) for 5 h were replenished with serum-free medium for 1 h, then treated with 200 μM palmitate conjugated to bovine serum albumin (BSA) (PA-BSA) or BSA control. (A) Real-time oxygen consumption rate (OCR) measured from 6 to 24 h post-infection (hpi). The OCR before PA-BSA or BSA treatment was set to 100%. (B) The area under the curve (AUC) OCR compared to that for mock cells treated with BSA (n = 3 per group). (C and D) A549 cells infected with JEV (MOI = 5 and 0.1) for 5 h were changed to medium without serum (C) or with serum (10% FBS) (D) for 1 h. Cells were then treated with PA-BSA or BSA for 18 h before Western blot analysis of protein levels of JEV NS3 and actin in cell lysates and virus titration in culture supernatants by plaque-forming assay (n = 3). (E and F) A549 cells were treated with N-acetylcysteine (NAC) 1 h before JEV (MOI = 10) infection and after virus adsorption. At 5 hpi, cells were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of the relative mRNA levels of interleukin 6 (IL-6) (E) and tumor necrosis factor α (TNF-α) (F) (n = 3). Data are mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.
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ppat.1004750.g001: Impaired long-chain fatty acid (LCFA) β-oxidation and induction of reactive oxygen species (ROS)-dependent pro-inflammatory cytokines in cells infected with Japanese encephalitis virus (JEV).(A and B) A549 cells infected with JEV (multiplicity of infection [MOI] = 10) for 5 h were replenished with serum-free medium for 1 h, then treated with 200 μM palmitate conjugated to bovine serum albumin (BSA) (PA-BSA) or BSA control. (A) Real-time oxygen consumption rate (OCR) measured from 6 to 24 h post-infection (hpi). The OCR before PA-BSA or BSA treatment was set to 100%. (B) The area under the curve (AUC) OCR compared to that for mock cells treated with BSA (n = 3 per group). (C and D) A549 cells infected with JEV (MOI = 5 and 0.1) for 5 h were changed to medium without serum (C) or with serum (10% FBS) (D) for 1 h. Cells were then treated with PA-BSA or BSA for 18 h before Western blot analysis of protein levels of JEV NS3 and actin in cell lysates and virus titration in culture supernatants by plaque-forming assay (n = 3). (E and F) A549 cells were treated with N-acetylcysteine (NAC) 1 h before JEV (MOI = 10) infection and after virus adsorption. At 5 hpi, cells were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of the relative mRNA levels of interleukin 6 (IL-6) (E) and tumor necrosis factor α (TNF-α) (F) (n = 3). Data are mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.

Mentions: Palmitic acid is the most common fatty acid in animals, so we and others have been using sodium palmitate conjugated bovine serum albumin (PA-BSA) to study fatty acid β-oxidation [35]. During fasting, fatty acid oxidation becomes the major energy source [36,37] and oxygen consumption will be mainly resulted from β-oxidation in cells cultured with PA-BSA under starvation condition (without serum). To address whether JEV infection modulates cellular LCFA β-oxidation, we measured the oxygen consumption rate (OCR) in cells cultured with a noncytotoxic dose of PA-BSA (S1 Fig) or BSA control by using a metabolic XF24 analyzer [35]. In JEV-infected human A549 cells cultured with BSA, OCR values continued to increase from 6 to 24 h post-infection (hpi; Fig. 1A, JEV + BSA). However, in JEV-infected cells cultured with PA-BSA, the OCR values increased in the beginning, then decreased from about 11 hpi until the end of the recording (Fig. 1A, JEV + PA-BSA). Mock-infected cells did not show the distinct OCR patterns with BSA and PA-BSA treatments (Fig. 1A). Changes in OCR values represented by area under the curve (AUC) similarly showed that the AUC OCR was lower in JEV-infected A549 cells cultured with PA-BSA than BSA (Fig. 1B). This phenomenon was not limited to a single cell type and also occurred in JEV-infected human neuroblastoma HTB-11 cells cultured with PA-BSA or BSA (S2A Fig). Thus, the reduced OCR in JEV-infected cells cultured with palmitate indicates that JEV cannot utilize LCFA efficiently, probably because of blocked β-oxidation.


Japanese encephalitis virus nonstructural protein NS5 interacts with mitochondrial trifunctional protein and impairs fatty acid β-oxidation.

Kao YT, Chang BL, Liang JJ, Tsai HJ, Lee YL, Lin RJ, Lin YL - PLoS Pathog. (2015)

Impaired long-chain fatty acid (LCFA) β-oxidation and induction of reactive oxygen species (ROS)-dependent pro-inflammatory cytokines in cells infected with Japanese encephalitis virus (JEV).(A and B) A549 cells infected with JEV (multiplicity of infection [MOI] = 10) for 5 h were replenished with serum-free medium for 1 h, then treated with 200 μM palmitate conjugated to bovine serum albumin (BSA) (PA-BSA) or BSA control. (A) Real-time oxygen consumption rate (OCR) measured from 6 to 24 h post-infection (hpi). The OCR before PA-BSA or BSA treatment was set to 100%. (B) The area under the curve (AUC) OCR compared to that for mock cells treated with BSA (n = 3 per group). (C and D) A549 cells infected with JEV (MOI = 5 and 0.1) for 5 h were changed to medium without serum (C) or with serum (10% FBS) (D) for 1 h. Cells were then treated with PA-BSA or BSA for 18 h before Western blot analysis of protein levels of JEV NS3 and actin in cell lysates and virus titration in culture supernatants by plaque-forming assay (n = 3). (E and F) A549 cells were treated with N-acetylcysteine (NAC) 1 h before JEV (MOI = 10) infection and after virus adsorption. At 5 hpi, cells were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of the relative mRNA levels of interleukin 6 (IL-6) (E) and tumor necrosis factor α (TNF-α) (F) (n = 3). Data are mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.
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ppat.1004750.g001: Impaired long-chain fatty acid (LCFA) β-oxidation and induction of reactive oxygen species (ROS)-dependent pro-inflammatory cytokines in cells infected with Japanese encephalitis virus (JEV).(A and B) A549 cells infected with JEV (multiplicity of infection [MOI] = 10) for 5 h were replenished with serum-free medium for 1 h, then treated with 200 μM palmitate conjugated to bovine serum albumin (BSA) (PA-BSA) or BSA control. (A) Real-time oxygen consumption rate (OCR) measured from 6 to 24 h post-infection (hpi). The OCR before PA-BSA or BSA treatment was set to 100%. (B) The area under the curve (AUC) OCR compared to that for mock cells treated with BSA (n = 3 per group). (C and D) A549 cells infected with JEV (MOI = 5 and 0.1) for 5 h were changed to medium without serum (C) or with serum (10% FBS) (D) for 1 h. Cells were then treated with PA-BSA or BSA for 18 h before Western blot analysis of protein levels of JEV NS3 and actin in cell lysates and virus titration in culture supernatants by plaque-forming assay (n = 3). (E and F) A549 cells were treated with N-acetylcysteine (NAC) 1 h before JEV (MOI = 10) infection and after virus adsorption. At 5 hpi, cells were incubated with serum-free medium for 1 h before treatment with PA-BSA or BSA for 18 h. RT-qPCR analysis of the relative mRNA levels of interleukin 6 (IL-6) (E) and tumor necrosis factor α (TNF-α) (F) (n = 3). Data are mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001 and ns, not significant.
Mentions: Palmitic acid is the most common fatty acid in animals, so we and others have been using sodium palmitate conjugated bovine serum albumin (PA-BSA) to study fatty acid β-oxidation [35]. During fasting, fatty acid oxidation becomes the major energy source [36,37] and oxygen consumption will be mainly resulted from β-oxidation in cells cultured with PA-BSA under starvation condition (without serum). To address whether JEV infection modulates cellular LCFA β-oxidation, we measured the oxygen consumption rate (OCR) in cells cultured with a noncytotoxic dose of PA-BSA (S1 Fig) or BSA control by using a metabolic XF24 analyzer [35]. In JEV-infected human A549 cells cultured with BSA, OCR values continued to increase from 6 to 24 h post-infection (hpi; Fig. 1A, JEV + BSA). However, in JEV-infected cells cultured with PA-BSA, the OCR values increased in the beginning, then decreased from about 11 hpi until the end of the recording (Fig. 1A, JEV + PA-BSA). Mock-infected cells did not show the distinct OCR patterns with BSA and PA-BSA treatments (Fig. 1A). Changes in OCR values represented by area under the curve (AUC) similarly showed that the AUC OCR was lower in JEV-infected A549 cells cultured with PA-BSA than BSA (Fig. 1B). This phenomenon was not limited to a single cell type and also occurred in JEV-infected human neuroblastoma HTB-11 cells cultured with PA-BSA or BSA (S2A Fig). Thus, the reduced OCR in JEV-infected cells cultured with palmitate indicates that JEV cannot utilize LCFA efficiently, probably because of blocked β-oxidation.

Bottom Line: LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells.Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP.We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.

ABSTRACT
Infection with Japanese encephalitis virus (JEV) can induce the expression of pro-inflammatory cytokines and cause acute encephalitis in humans. β-oxidation breaks down fatty acids for ATP production in mitochondria, and impaired β-oxidation can induce pro-inflammatory cytokine expression. To address the role of fatty-acid β-oxidation in JEV infection, we measured the oxygen consumption rate of mock- and JEV-infected cells cultured with or without long chain fatty acid (LCFA) palmitate. Cells with JEV infection showed impaired LCFA β-oxidation and increased interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) expression. JEV nonstructural protein 5 (NS5) interacted with hydroxyacyl-CoA dehydrogenase α and β subunits, two components of the mitochondrial trifunctional protein (MTP) involved in LCFA β-oxidation, and NS5 proteins were detected in mitochondria and co-localized with MTP. LCFA β-oxidation was impaired and higher cytokines were induced in cells overexpressing NS5 protein as compared with control cells. Deletion and mutation studies showed that the N-terminus of NS5 was involved in the MTP association, and a single point mutation of NS5 residue 19 from methionine to alanine (NS5-M19A) reduced its binding ability with MTP. The recombinant JEV with NS5-M19A mutation (JEV-NS5-M19A) was less able to block LCFA β-oxidation and induced lower levels of IL-6 and TNF-α than wild-type JEV. Moreover, mice challenged with JEV-NS5-M19A showed less neurovirulence and neuroinvasiveness. We identified a novel function of JEV NS5 in viral pathogenesis by impairing LCFA β-oxidation and inducing cytokine expression by association with MTP.

No MeSH data available.


Related in: MedlinePlus