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Hepatitis C Virus infection dysregulates mitochondrial Fatty Acid Oxidation

Final Report Summary - HCVFAO (Hepatitis C Virus infection dysregulates mitochondrial Fatty Acid Oxidation)

Lipid -oxidation is a major metabolic pathway to combust lipid to produce adenosine tri-phosphate. For instance, -oxidation of one mole palmitate yields 106 moles of ATP, whereas glycolysis of one mole glucose yields 30 moles of ATP. Recently, hepatitis C virus infection was found to alter metabolic expenditure profile, which was featured by impaired ketogenesis, upregulation of glycolysis and low cellular ATP content. The proposed grant was aimed to reveal the relations between hepatitis C virus (HCV) infection and host mitochondrial lipid -oxidation. Utilizing a tissue culture model of HCV infection, we made the important observation that HCV infection attenuated mitochondrial lipid -oxidation activity from very early phase of infection. This metabolic alteration results in poor lipid combustion and low ATP production and also triggers a shift of energy expenditure toward glycolysis. It will be more important that poor lipid combustion contributes to the development of fatty liver. The study also revealed the molecular mechanism of viral infection on mitochondrial lipid -oxidation. First, we discovered that HCV infection attenuates mRNA transcription of mitochondrial trifunctional protein (MTP) α and  subunits. This attenuation of gene expression can be caused by viral replication, not by individual expression of viral proteins. And also, we found that inflammatory cytokines can worsen the situation by causing additive gene suppression. Second, we identified that the viral NS5A protein interacts with MTP α and  subunits. This interaction was thought to occur at endoplasmic reticulum/mitochondria contact sites giving rise to another dysfunction of mitochondria. Gain and loss of function study of MTP revealed that ectopic expression of MTP could exert a protective effect on HCV replication suggesting that HCV attenuates mitochondrial lipid -oxidation to promote its replication. In the light of these observations, we investigated that how attenuation of mitochondrial lipid -oxidation can impact the type I interferon signalling cascade. The result showed that impaired lipid -oxidation rendered the host cell less responsive to exogenously added interferon α to suppress HCV replication. This suggests that poor metabolic status prevent host cell from supporting positive feedback mechanism for IFNα production and ISG induction. Viral interference with lipid β-oxidation plays a pivotal role for HCV to establish a long-term, persistent infection. Further understanding of this aspect of virus/host interaction may leads to the improvement of current standard therapy.

PO1 Viral protein interaction with mitochondrial trifunctional protein.
An interactome study of non-structural protein 5A (NS5A) of hepatitis C virus (HCV) had revealed that MTPα and β subunits are associated with NS5A. One STrEP-Tag HCV system was used to confirm the protein–protein interaction between NS5A and MTP. In either case of HCV infected cell or subgenomic replicon (SGR) harboring cell, MTP proteins were found associated with affinity-captured NS5A in affinity-tag dependant manner. Protein interactions were further evaluated immunofluorescent microscopy. Endogenous MTPα and β proteins were seen predominantly and exclusively in mitochondria, which are marked with MitoTracker dye. Their signals were seen evenly distributed to cytosolic spaces with slightly concentrated in perinuclear region. They are seen as dots or relatively large puncta, been connected to each other to form overall mitochondria network. In HCV infected cells, these even distributions were disturbed by intruding NS5A-positive membranous structure. Overall signal intensity were down-regulated, in accordant with previous observation by Western blotting and qPCR. Stained MTP puncta were occasionally associated with NS5A-positive membranous structure, which are presumably an ER-derived membranous web structure, as they are seen as yellow dots in merged panels. NS5A’s co-localization with mitochondrial protein was more evident with mitofusin 2 (MFN2) staining. Evenly distributed MFN2 staining were disrupted in HCV-infected cells, in the same way as MTPs. Combined with the observed partially cleaved form of MFN2, HCV infection and NS5A are likely to cause deregulation of mitochondrial function through MAM, as well as core and NS3/4A. Thus through MAM, HCV replicase may be acting on mitochondrial networks.

PO2 The impact of HCV infection on mitochondrial lipid β-oxidation.
Tissue culture infection of HCV leads host cell to the condition of low ATP production. Bodies of evidences show HCV proteins interact with host mitochondria to impact mitochondrial functions. We hypothesized that a major lipid -oxidation pathway, mitochondrial pathway, might be affected by HCV infection, which in turn causes impaired ATP production. To access this possibility, we performed lipid -oxidation assay using H3-palmitate as tracer. Results indicated that HCV infection significantly attenuates lipid -oxidation. The attenuation of lipid -oxidation occurs in time-dependant manner and correlates with the progress of HCV infection in tissue culture, whose inhibition magnitude reaching to the 59.3% inhibition at Day5. The lipid -oxidation was also performed for variations of stable HCV replicon cell lines. To access viral contributions to their effect on lipid -oxidation activity, assay was performed for three pairs of replicon cell line and its counterpart, interferon-cured cell line. In both subgenomic replicon (SGR) cell line of genotype 2a (Y-19) and genotype 1b (R6 FLR-N), SGR replication is causing impairment of lipid -oxidation by approximately 24.5% and 35.6%(compared to the counterpart cured cell), respectively. Whereas full-genomic replicon of genotype 2a inhibited lipid -oxidation more severely by 46.0%, compared to the cured-cell control, implying either of additionally expressed viral protein (core, E1, E2 P7 and NS2) might have caused additional inhibition of lipid -oxidation. Although lipid -oxidation was significantly impaired in tested cases, they are not associated with cell viability deterioration.  
The mechanism of HCV induced suppression of mitochondrial lipid -oxidation.
To investigate how viral interference on lipid -oxidation can happen, we measured protein expression level of MTPα and β. As mentioned above, these mitochondrial proteins were identified as binding partners of NS5A. Whole cell lysate from Huh7.5 SGR cell and HCV-infected cells were analyzed by quantitative infra-red Western blotting. Result showed that both SGR replication and HCV infection could down regulate protein expression level of MTPα and β subunits. Whereas internal control -Actin was not affected by HCV. To see if viral suppression on lipid -oxidation could happen in post-translation processes, mitochondrial fractions were isolated from SGR cells and HCV-infected cells and MTP protein levels were compared side by side with whole cell lysates. MTP proteins and all mitochondrial proteins tested were seen enriched in mitochondrial fractions (Voltage-dependent anion-selective channel protein 1, VDAC and mitofusin 2, MFN2). Both HCV core and NS5A remained detectable in mitochondrial fraction. And the magnitude of MTP protein depletion was found comparable to those seen for whole cell lysate. Altogether, these results suggest that MTP protein depletion is caused at the level of gene transcription and/or mRNA translation. A recent study revealed that HCV infection promotes mitophagy, a selective degradation of mitochondria by autophagy. In the light of this finding, we speculated that HCV infection could cause a decline of mitochondria number per cell. We performed quantitative PCR of mitochondrial DNA. Result showed that neither HCV infection nor SGR replication caused a change of mtDNA number per cell. This implies that attenuation of lipid -oxidation is not caused by a decrease of mitochondrial per cell. Although little is known about how mitochondrial biogenesis can be affected by HCV infection, HCV induced mitophagy might be compensated by up-regulated mitochondrial biogenesis to maintain overall mitochondria number. It is noteworthy that MFN2 were found partially cleaved in either cases of SGR and HCV-infected cells, by an unknown protease. And interestingly, MFN2 is known to control mitochondrial fusion, and selective degradation of MFN2 leads the mitochondria to be more permissive for mitophagy, by expelling mitofusin-depleted mitochondria from mitochondrial networks (or mitochondrial reticulum). The mechanism and effect of HCV-induced MFN2 cleavage are unclear. However, this is a strong implication that HCV proteins directly and aggressively interact with mitochondria to alter its functions.
To further investigate the mechanism of viral invasion on lipid -oxidation, mRNAs of MTPα and β were quantified by real-time quantitative RT-PCR. In SGR cell, MTPα messenger level dropped by 59.3%. And MTPβ messenger decreased by 21.6%. While GAPDH mRNA was not affected by SGR replication. Although it is not perfectly accordant with the observation on protein expression level, where MTPβ down regulation was more evident than MTPα. These data altogether support that virus induced attenuation of lipid -oxidation occurs mainly in the gene transcription level. To further confirm this, Huh7.5 cells were infected with tissue culture generated HCV (J6/JFH1 chimera Jc1, M.O.I=0.05) and then followed for transcriptional changes of mRNA of MTP genes, GAPDH and cellular HCV RNA, for subsequent 7 days. Both MTPα and β mRNAs were down regulated by about 50%, when HCV RNA reached to plateau at Day 5. While HCV infection did not impact on GAPDH transcription throughout this period. To look into the mechanism of how MTP gene transcriptions are controlled by viral infection. We took advantage of promoter/reporter vectors for MTPα and β. Both MTPα and β genes are located in chromosome 2p23 of host genome, where two genes face to the other in head-to-head way. They are encoded in the opposing strand and sharing core promoter regions of 350 bases in their 5’ flanking region. In another words, mRNA transcription of MTPα and β are controlled by a bi-directional promoter region. We have cloned these 350 bases by PCR using Huh7 genomic DNA as template, and then inserted this promoter into the same restriction sites in pNL1.2 multi cloning site, which encodes NanoLuc®. The promoter was cloned in opposing way to construct two different promoter/reporter vector and designated p350-MTPα and p350-MTPβ. These vectors were transfected into HCV-infected cells (Day7, Jc1 infected at M.O.I=0.05). A parental promoter/reporter vector pNL1.2 was also paralleled side by side with MTP promoter vector transfection for normalizing purpose. Results clearly showed that HCV infection can cause down-regulation of MTP genes transcription by half and the magnitude of reduction was in accordant with what were seen for quantitative PCR results. To look further, we tested these promoter/reporter vectors on stable cell lines expressing individual HCV proteins. We have chosen core, NS3 and NS5A because these three are known to interact with host mitochondrion. And core and NS5A are known to interfere with host transcription with their predicted activity of nuclear localization. As a result, HCV core protein was found more capable to impact MTP promoter activity, while NS3 and NS5A did not cause any effect on promoter activity. Contribution of core protein expression is interesting because promoter activity was readily affected by HCV infection rather than SGR replication. And FGR achieved more severe inhibition of lipid β-oxidation than SGR. Furthermore we tested inflammatory cytokines’ effect on promoter/reporter. Both Interleukin-1β and Tumor necrosis factor α caused modest inhibition on promoter activity of MTPα and β. These altogether suggest HCV infection can cause orchestrated impact on mitochondrial lipid -oxidation through transcriptional attenuation of MTP gene expression.

PO3 Relations between mitochondrial lipid -oxidation and hepatitis C virus replication.
We performed a series of “loss of function” and “gain of function” study of MTP proteins, in order to study how their functions relate to HCV replication. First, we constructed lentiviral vector-mediated shRNA expression system to achieve specific knock down of MTPα and β genes. 4 controls targeting non human genes like luciferase (Luc), Green fluorescent protein (GFP) and β-galactosidase(LacZ) and 3 each for MTPα and β were made, and used for subsequent experiments in order to target gene expression. 5 out of 6 shMTPs worked effectively to down-regulate both mRNA and protein expression. Surprisingly, RNA interference triggered “co-knock down” of MTPα and β, where shRNA mediated knock down of either part of subunit caused concurrent down regulation of counterpart subunit. As mentioned above, gene transcriptions of MTPα and β are tightly correlated to each other. And this tight relation seems to be extended to post-transcription process including translation and more, until they form a mature octamer complex, which consists of 4 α-subunits and 4 β-subunits. In the level of mRNA expression, despites of “co-knock down” observed for protein blotting, each shMTP constructs down-regulated target mRNA. All shRNA tested in this study did not impact on GAPDH mRNA expression or protein expression of GAPDH and MFN2. They did neither cause significant changes of cellular mtDNA contents. Under these conditions, MTP depleted cells were tested for HCV replication assay to sudy their effect over HCV infection/replication. A reporter virus construct carrying a novel luciferase gene (NanoLuc®) was utilized. This reporter virus construct, designated Jc1-p7NLuc2A, was constructed in the same architecture as p7-Rluc2A. Reporter virus particles were made and used to infect MTP depleted cells at M.O.I=0.05. As a result, MTP depleted cells supported HCV replication as well as control cells. As discussed above, HCV infection itself suppresses MTP protein expression and lipid -oxidation. So that the effect of shRNA mediated knockdown can be over-shadowed by this endogenous knockdown.
For “gain of function” study, we took advantage of lentiviral vector to transduce Huh7.5 cells to express MTP proteins. Huh7.5 cells were infected by lentiviral vector and selected by Hygromycin B containing medium to obtain stable transformant expressing MTPα, β and both α and β (α/β). These transformants were infected with reporter virus of Jc1-p7NLuc2A at the M.O.I=0.05 and followed for 3 days. Result showed that MTP expressing cells were more resistant to HCV infection. A transformant expressing both MTPα and β (α/β) was mostly resistant to HCV, suppressing HCV replication by approximately 50%. This implies that host cells can resist against viral invasion, when lipid -oxidation is compensated by ectopic expression of MTP proteins. To confirm this, Huh7.5 cells were transduced with lentiviral vector for MTP protein expression. Although it is yet transient gene transduction without any drug selection in this case, cells were evenly transformed. These cells were infected with tissue culture generated JFH1 virus at M.O.I=0.03 and followed for 7 days. Again, MTP overexpression, either α or β showed slight resistance to HCV infection. Finally, cells infected with lentiviral vectors were electroporated with in vitro transcribed JFH1 RNA (5µg). Transfected cells were maintained for 3 weeks. Results were more evident as MTP expressing cells were more resistant to viral RNA replication. At 21 days post electroporation, abundance of HCV RNA was 2 log less for MTPα expressing cells and 1 log less for MTPβ expressing cells. MTP protein expression was sustaining over the entire period, while endogenous MTP expression can be severely depleted by HCV infection when they were not compensated by lentiviral vector infection.