Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the Hepatitis C virus core protein

Download
  1. Get@NRC: Fluorescence lifetime imaging of alterations to cellular metabolism by domain 2 of the Hepatitis C virus core protein (Opens in a new window)
DOIResolve DOI: http://doi.org/10.1371/journal.pone.0066738
AuthorSearch for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for: ; Search for:
TypeArticle
Journal titlePLoS ONE
ISSN1932-6203
Volume8
Issue6
Article numbere66738
Subjectcore protein; fat droplet; green fluorescent protein; reduced nicotinamide adenine dinucleotide phosphate; virus RNA; article; cell metabolism; coherent anti Stokes Raman scattering microscopy; correlation analysis; enzyme activity; fluorescence; genetic transfection; glycolysis; hepatitis C; Hepatitis C virus; hepatoma cell; human; human cell; lipogenesis; liver metabolism; metabolic regulation; microscopy; mitochondrial energy transfer; molecular imaging; protein conformation; protein expression; protein function; protein localization; Raman spectrometry
AbstractHepatitis 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. © 2013 Mazumder et al.
Publication date
LanguageEnglish
AffiliationNational Research Council Canada (NRC-CNRC)
Peer reviewedYes
NPARC number21270594
Export citationExport as RIS
Report a correctionReport a correction
Record identifierb7d5af0f-1a8f-4937-b1e9-a16abc21a2fd
Record created2014-02-17
Record modified2016-05-09
Bookmark and share
  • Share this page with Facebook (Opens in a new window)
  • Share this page with Twitter (Opens in a new window)
  • Share this page with Google+ (Opens in a new window)
  • Share this page with Delicious (Opens in a new window)