Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

Nirmal Mazumder, Rodney K. Lyn, Ragunath Singaravelu, Andrew Ridsdale, Douglas J. Moffatt, Chih Wei Hu, Han Ruei Tsai, John McLauchlan, Albert Stolow, Fu Jen Kao, John Paul Pezacki

Research output: Contribution to journalArticle

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Abstract

Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced - indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.

Original languageEnglish
Article numbere66738
JournalPLoS One
Volume8
Issue number6
DOIs
Publication statusPublished - 24-06-2013

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Hepatitis C virus
NAD (coenzyme)
Optical Imaging
Metabolism
NAD
Fluorescence
image analysis
fluorescence
Imaging techniques
metabolism
Lipids
Viruses
lipids
proteins
Hepacivirus
droplets
coenzymes
Liver
Biosynthesis
Coenzymes

All Science Journal Classification (ASJC) codes

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Mazumder, Nirmal ; Lyn, Rodney K. ; Singaravelu, Ragunath ; Ridsdale, Andrew ; Moffatt, Douglas J. ; Hu, Chih Wei ; Tsai, Han Ruei ; McLauchlan, John ; Stolow, Albert ; Kao, Fu Jen ; Pezacki, John Paul. / Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein. In: PLoS One. 2013 ; Vol. 8, No. 6.
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abstract = "Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced - indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.",
author = "Nirmal Mazumder and Lyn, {Rodney K.} and Ragunath Singaravelu and Andrew Ridsdale and Moffatt, {Douglas J.} and Hu, {Chih Wei} and Tsai, {Han Ruei} and John McLauchlan and Albert Stolow and Kao, {Fu Jen} and Pezacki, {John Paul}",
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Mazumder, N, Lyn, RK, Singaravelu, R, Ridsdale, A, Moffatt, DJ, Hu, CW, Tsai, HR, McLauchlan, J, Stolow, A, Kao, FJ & Pezacki, JP 2013, 'Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein', PLoS One, vol. 8, no. 6, e66738. https://doi.org/10.1371/journal.pone.0066738

Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein. / Mazumder, Nirmal; Lyn, Rodney K.; Singaravelu, Ragunath; Ridsdale, Andrew; Moffatt, Douglas J.; Hu, Chih Wei; Tsai, Han Ruei; McLauchlan, John; Stolow, Albert; Kao, Fu Jen; Pezacki, John Paul.

In: PLoS One, Vol. 8, No. 6, e66738, 24.06.2013.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Fluorescence Lifetime Imaging of Alterations to Cellular Metabolism by Domain 2 of the Hepatitis C Virus Core Protein

AU - Mazumder, Nirmal

AU - Lyn, Rodney K.

AU - Singaravelu, Ragunath

AU - Ridsdale, Andrew

AU - Moffatt, Douglas J.

AU - Hu, Chih Wei

AU - Tsai, Han Ruei

AU - McLauchlan, John

AU - Stolow, Albert

AU - Kao, Fu Jen

AU - Pezacki, John Paul

PY - 2013/6/24

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N2 - Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced - indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.

AB - Hepatitis C virus (HCV) co-opts hepatic lipid pathways to facilitate its pathogenesis. The virus alters cellular lipid biosynthesis and trafficking, and causes an accumulation of lipid droplets (LDs) that gives rise to hepatic steatosis. Little is known about how these changes are controlled at the molecular level, and how they are related to the underlying metabolic states of the infected cell. The HCV core protein has previously been shown to independently induce alterations in hepatic lipid homeostasis. Herein, we demonstrate, using coherent anti-Stokes Raman scattering (CARS) microscopy, that expression of domain 2 of the HCV core protein (D2) fused to GFP is sufficient to induce an accumulation of larger lipid droplets (LDs) in the perinuclear region. Additionally, we performed fluorescence lifetime imaging of endogenous reduced nicotinamide adenine dinucleotides [NAD(P)H], a key coenzyme in cellular metabolic processes, to monitor changes in the cofactor's abundance and conformational state in D2-GFP transfected cells. When expressed in Huh-7 human hepatoma cells, we observed that the D2-GFP induced accumulation of LDs correlated with an increase in total NAD(P)H fluorescence and an increase in the ratio of free to bound NAD(P)H. This is consistent with an approximate 10 fold increase in cellular NAD(P)H levels. Furthermore, the lifetimes of bound and free NAD(P)H were both significantly reduced - indicating viral protein-induced alterations in the cofactors' binding and microenvironment. Interestingly, the D2-expressing cells showed a more diffuse localization of NAD(P)H fluorescence signal, consistent with an accumulation of the co-factor outside the mitochondria. These observations suggest that HCV causes a shift of metabolic control away from the use of the coenzyme in mitochondrial electron transport and towards glycolysis, lipid biosynthesis, and building of new biomass. Overall, our findings demonstrate that HCV induced alterations in hepatic metabolism is tightly linked to alterations in NAD(P)H functional states.

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