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.
Highlights
Hepatitis C virus (HCV) infection is a global health concern and a leading cause of hepatocellular carcinoma and liver transplantation [1,2,3]
We wished to apply this construct to investigate whether D2 expression can independently induce alterations in metabolic flux observed during HCV infection [36,37,38]
Simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (TPF) revealed an increase in lipid droplets (LDs) size and number in cells transfected with the D2-green fluorescent protein (GFP) construct (Figure 1, Figure S1) compared to adjacent non-transfected cells
Summary
Hepatitis C virus (HCV) infection is a global health concern and a leading cause of hepatocellular carcinoma and liver transplantation [1,2,3]. HCV infection is associated with steatosis in over 40% of patients [10] This hepatic lipid accumulation results from virus-induced alterations in host lipid homeostasis [7], [9]. The core protein strongly associates with LDs and is progressively loaded onto LDs over time after HCV infection is established [12]. HCV core protein controls the size, subcellular localization, and movement of LDs on microtubules [13], [16] These core-induced changes to cytoplasmic LDs represent a subset of the changes HCV core induces in host metabolism to create an environment that is advantageous to the virus
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