Inhibition of Hepatitis C virus ( HCV ) Core protein-induced cell Growth by Non-structural Protein 4 A ( NS 4 A ) is mediated by mitochondrial dysregulation

Hepatitis C virus (HCV) is a signifi cant health problem facing the world. More than  million people are infected with HCV worldwide. HCV encodes a large polyprotein precursor that is processed into at least  distinct products including structural (core, E and E) and non-structural (NS, NS, NSA, NSB, NSA and NSB). Besides its importance in virus replication, NSA functions as a cofactor for NS and contributes to viral pathogenesis by infl uencing cellular functions. Here, we investigated the eff ect of NSA protein on the growth rate induced by core protein in liver cells. Using our established tetracycline inducible system, we demonstrated the ability of NSA protein to inhibit core protein-induced cell growth in Hepatoma cell line, HepG. Induction of both core and NSA proteins in HepGcore/NSA transfectants inhibited core-induced growth advantage in HepG-core transfectants and blocked NSA protein-induced cell growth inhibition in HepG-NSA transfectants. Using both immune fl uorescence staining and Western blot analysis, we confi rmed the localization of NSA protein to the mitochondria in HepG-NSA transfectants expressing NSA protein. Data obtained from fl ow cytometry analysis, using JC- demonstrated the loss of mitochondrial membrane potential (ΔΨm) by the expression of NSA protein in HepG-NSA transfectants, but not by the expression of core protein in HepG-core transfectants. Whereas, the induction of the expression of both core and NSA proteins in HepG-core/NSA transfectants blocked NSA-induced loss of ΔΨm in HepG cells. Taken together, our data suggest an important role for mitochondria in the modulation HCV NSA-induced inhibition of HCV core-mediated cell growth.


Introduction
Hepatitis C virus (HCV) is a signifi cant health problem facing the world.More than  million people are infected with HCV worldwide ().HCV infection causes acute hepatitis, which is naturally cleared in - of patients ().However, in - of cases persists causing chronic hepatitis, which mostly leads to a spectrum of diseases including steatosis, fi brosis, cirrhosis and hepatocellular carcinoma.Up to date there is no eff ective vaccine available for the virus, and the current therapies for many HCV-infected patients show little effi cacy ().HCV genome has a long open reading frame, fl anked with ` and ` untranslated region, which encodes a polyprotein precursor of about , to , amino acids (aa) residues ().Th is polyprotein is cleaved by both host and viral proteases to generate four structural proteins (C, E, E and P) and six non-structural proteins (NS, NS, NSA, NSB, NSA and NSB) ().Core protein is derived from the fi rst  amino acids of the N-terminal of the precursor polyprotein ().Besides its function as a component of viral nucleocapsids, core protein has been shown to act on transactivation of numerous cellular and viral promotors (-), many of which are involved in the regulation of cellular proliferation.In addition, core targets a wide spectrum of cellular factors and diff erent signalling pathways (,).Furthermore, transgenic mice with constitutive expression of core show liver steatosis and eventually develop HCC ().NSA is a -residue amphipathic peptide with a hydrophobic N-terminus and a hydrophilic C-terminus ().Its known functions include acting as cofactor for NS protease, possibly by assisting in the membrane localization of NS and other viral replicase components, and stimulating phosphorylation of NSA ().In addition, the NSA has been reported to inhibit cell proliferation (,) and to enhance mitochondria-mediated apoptosis ().In this study, we demonstrated for the fi rst time the ability of NSA protein to inhibit core protein-induced cell growth by a mechanism including mitochondrial dysregulation.

Construction of HCV core plasmid
Th e construction of the HCV core and HCV NSA plas-mids encoding for the complete HCV core-and HCV NSA proteins respectively was performed as described (,,).Briefly, the viral RNA was extracted from  μl of serum using QIAamp viral RNA extraction kit (Qiagen).The complete HCV cDNA was synthesized with Genscript (Genecraft).Th e HCV ds cDNA region (nt -) was amplifi ed from the complete HCV cDNA by conventional PCR using the following primer pairs: '-GAC CGT GCA CCA TGA GCA CG-` (sense) and `-CTG CCT ACC GAG CAG GCA GCA-` (anti-sense).The PCR products were modifi ed to generate blunt ends and then cloned into the EcoR V site of pcDNA.(+) (Invitrogen) to generate pcDNA.-HCV.Th e HCV core protein-encoding region (nt -) was amplifi ed from the pcDNA.-HCVplasmid comprising the complete HCV cDNA of the genotype A using the following primer pairs having a start codon (bold) and a Hind III restriction site (underlined): '-CCC AAG CTT GGG GAC CGT GCA CCA TGA GCA CG-` (sense) and '-CCC AAG CTT GGG TCG GCG AAG CGG GGA CAG TC-`(antisense).Whereas, the HCV NSA protein-encoding region (nt -) was amplified from the pcDNA.-HCVplasmid comprising the complete HCV cDNA of the genotype A using the following primer pairs having a start codon (bold) and a Cla restriction site (underlined): '-CCA TCG ATA TGG TGA CAA GTA CGT GGG TCT TG -` (sense) and '-CCA TCG ATC TCC TCC ATT TCG TCC AAC TG -`(antisense`).The cDNA sequences of both HCV core and NSA proteins were fi rst sequenced and then inserted into the Hind III and ClaI sites of pRevTRE vector, respectively.

Generation of viruses
Th e generation of viruses was performed as described (,,).Briefl y, the packaging cell line RetroPack TM pT (Clontech) was grown in DMEM with  FCS,  mM L-glutamine (all from Sigma-Aldrich) at °C and in  CO.The cells were transfected with the appropriate retroviral construct e.g.pRev Tet-Off, pRevTRE-core, or pRevTRE-NSA by nucleafec-torTM Kit (Amaxa biosystems).Forty-eight hours post-transfection, the supernatant was collected, filtered through a ,-μm-syringe filter, and spun at .xg for ,h.Pelleted virus was resuspended in a , or , the original volume of medium at °C for h.

Infection of target cells
Th e development of HepG-Tet-Off as well as the double stable Tet-off clones (HepG-Core, HepG-NSA, and HepG-core/NSA) allowing controlled expression of HCV core under the control of tetracycline was performed as described (, , ).G and Hygromycin resistant clones, termed, HepG-core, HepG-core/NSA and HepG-NSA transfectants were screened for expression of HCV core, and NSA proteins by RT-PCR.
Positive clones, with high induction effi ciency, were expanded and rescreened by RT-PCR and immunoblotting using anti-HCV core or anti-NSA antibodies for the expression of HCV core protein as described (, ,).
Immune fl uorescence staining of HCV core transfected cells HepG-core, HepG-NSA, and HepG-core/NSA transfectants were plated in glass bottom culture dishes (MatTek Corporation, USA) at a density of x cells and allowed to grow for h under normal condition in the presence of tetracycline (μg/ml).After the withdrawal of tetracycline for h, the cells were subjected to immunofl uorescence staining as described ().Briefly, cells were fi xed for  min in ice-cold PBS containing  formaldehyde.Blocking and all following procedures were performed in PBS supplemented with  BSA and , saponin.Anti-HCV NSA (Research Diagnostics, Inc) and anti-Tom (SC-; Santa Cruz, USA) antibodies were applied at °C overnight at the appropriate concentrations.Th en, cells were washed three times for  min and incubated with the secondary antibody (:).Finally, samples were washed extensively and mounted in fl uorescent mounting medium (Dako Corporation, Carpintera, CA) with or without  ng/ml ', -diamidino--phenylindole.Pictures were taken on an Axiovert  Microscope (Zeiss, Germany) with an Apochromat x oil immersion lens using OpenLab software (Improvision, Tübingen, Germany).

Immunoblot
Immunoblot analysis was performed according to the standard procedures.Th e following antibodies were used at the indicated dilution: anti-HCV core protein antibody and anti-HCV NSA protein (Research Diagnostic, Inc, USA), :,; anti-Tom (SC-), :; anti-actin (SC-), :, (Santa Cruz Biotechnology, Inc, USA).

MTT assay
The cell number was determined by MTT assay using cell proliferation kit (Roche, Mannheim, Germany) as described (,,,).HepG-core, HepG-NSA and HepG-core/NSA transfectants as (x/well) were plated into a microtiter plate (Nunc) and cultured in medium with (+Tc) or without (-Tc)  μg/ml tetracycline.Th e MTT assays were performed at regular time intervals up to  days.

Preparation of mitochondria and endoplasmatic reticulum (ER) from cultured cells
HepG-core, HepG-NSA and HepG-core/NSA transfectants were allowed to grow in the presence and in the absence of tetracycline (mg/ml), h later, the cells were washed with PBS, and then scraped off with  ml of PBS.Collected cells were precipitated by centrifugation at  g for  min, and washed with HE buff er ( mM Hepes-KOH, pH ,; and  mM EDTA) containing  (wt/vol) sucrose.Th e cells were resuspended in  ml of the same buff er containing  μg/ml -macroglobulin, homogenized by fi ve-times aspiration through a -gauge needle, and then centrifuged at  g for  min to obtain a post-nuclear supernatant.The supernatant was layered over a discontinuous gradient of  and  sucrose in HE buff er ( and  ml, respectively).After centrifugation at , g for  h, ,-ml aliquots were collected from the top of the tube. μl of each fraction was precipitated with  TCA and subjected to SDS-PAGE and immunoblotting using antibodies against Tom (mitochondria) or NSA proteins.Detection of the loss of mitochondrial membrane potential (ΔΨm) using JC- Th e measurement of mitochondrial membrane potential was performed as described ().Briefl y, HepG-core, HepG-NSA and HepG-core/NSA transfectants were allowed to grow in the presence and in the absence of tetracycline (mg/ml), h later, the cells were trypsinized, counted, and washed twice in ice-cold PBS and resuspended in PBS.Th e cells were stained with  μM JC- for  min at room temperature in the dark.Th e intensities of green fl uorescence at - nm (PMT ) and of red fl uorescence at more than  nm (PMT ) of . individual cells were analyzed by using a fl ow cytometer.Th e intensity voltages of the photomultipliers of detector  (PMT ) and detector  (PMT ) were set at  V. Th e value of ΔΨm in response to the test compound was expressed as a ratio of PMT  to PMT .

Tetracycline-regulated expression of HCV core and-NSA proteins in HepG cells
Th e expression of both HCV proteins core and NSA proteins were detected in HepG-core, HepG-NSA and HepG-core/NSA transfectants by Western blot analysis after the withdrawal of tetracycline from the culture medium for h (Figure ).Th e induction expression of HCV core and-NSA proteins was found to be time-dependent, and could be quantitatively regulated by the variation of tetracycline concentration in the culture medium (data not shown).

Inhibition of core protein-induced cell growth by NSA protein
To investigate whether core protein-induced cell sients cultured in the presence and in the absence of tetracycline (μg/) were stained with anti-Tom antibody (mitochondria) and co-stained with anti-NSA.Data obtained from confocal laser scanning microscopy confi rmed the localization of NSA protein to mitochondria (Figure A).In addition, we confi rmed further, the localization of NSA to the mitochondria by Western blot analysis using mitochondrial fractions prepared from HepG-NSA and HepG-NSA/core transfectants cultured in the presence (+Tc) and in the absence (-Tc) of tetracycline for h.Data obtained from Western blot analysis using anti-NSA or anti-Tom antibodies (Figure B) demonstrated the expression of Tom in all mitochondrial fractions obtained from either HepG-NSA or HepG-Core/NSA transfectants cultured in presence and in absence of tetracycline.In contrast, the detection of NSA protein was noted only in the mitochondrial fractions obtained from either HepG-NSA or HepG-core/NSA transfectants cultured in the absence of tetracycline.Th ese data confi rm the localization of HCV NSA protein to the mitochondria and suggest further an important role for the mitochondria in the modulation of NSA-induced cell growth inhibition.Based on the subcellular localization of NSA protein to mitochondria, we hypothesized that NSA may cause the loss of mitochondrial membrane potential and subsequently lead to mitochondrial dysfunction.The HepG-core, HepG-NSA and HepG-core/NSA transfectants were allowed to grow in the presence or in the absence of tetracycline (μg/ml).Forty eight hours later, the cells were subjected to fl ow cytometry analysis for the measurement of mitochondrial membrane potential (ΔΨm) using JC-.Data obtained from fl ow cytometry analysis (Figure C) demonstrated the loss of mitochondrial membrane potential in response to the expression of NSA protein in HepG-NSA transfectants.In contrast, the induction of both NSA and core proteins in HepG-core/NSA transfectants was found to block NSA-induced loss of mitochondrial membrane potential in HepG cells (Figure C).Whereas, in HepG-core transfectants no alteration was noted in response to the expression of core protein.Taken together, these results suggest an essential role for the mitochondrial pathway in the modulation of NSA-induced cell growth inhibition of HepG cells and subsequently the inhibition of core protein-induced cell growth.

Discussion
IIn this study, we demonstrated the ability of NSA protein to inhibit core protein-induced cell growth in HepG cells and specified the role of mitochondria in the modulation of this event.
Many viruses, including HCV, possess viral proteins that either promote or inhibit cell death.Among HCV proteins, core (,), NS (,) and NSA (,) have been reported to enhance cell growth rate or to possess antiapoptotic functions.Also, there are reports showing that core (-), E (,), NS (), NSA (,), NSA and NSB () either cell growth inhibition or possess proapoptotic function.
NSA is known to localize in the endoplasmatic reticulum (ER) (,) and to accumulate on the mitochondria, and subsequently leads to the loss of mitochondrial membrane potential ().In this study, we confi rmed also the localization of NSA protein to the mitochondria and demonstrated further the loss of mitochondrial membrane potential.However, the loss of mitochondrial membrane potential together with the inhibition of NSA protein-induced growth rate in HepG-NSA transfectants suggests an important role for the mitochondria in modulation of NSA-induced cell growth inhibition of HepG cells.Interestingly, the induction of both core and NSA protein expression in HepG-core/NSA transfectants was found to block both NSA-induced loss of mitchondrial membrane potential observed in HepG-NSA transfectants and cell growth advantage induced by the expression of core protein.Our results showed that NSA-induced cell growth inhibition and loss of mitochondrial membrane potential were eliminated by the expression of core protein.However, it should be emphasized, that the mitochondrial morphology and intracellular localization were altered by the expression of both core and NSA proteins in HepG-core/NSA transfectants.These results imply the possibility that both core and NSA proteins, after being transported to mitochondria, exert a signifi cant eff ect on the function of mitochondria without aff ecting mitochondrial membrane potential Previously, the detection of core protein on rough ER surrounding the mitochondria was reported in both core protein-transfected liver cell lines and in core proteintransgenic mice () as well as in direct association with mitochondria (-).Also, this association has been observed in core protein-transgenic mice (,) and was linked to both mitochondrial injury and oxidative stress.
Our data collectively in agreement with a recent study () demonstrate that core protein localizes to the surfaces of mitochondria via its mitochondrial targeting motif and thereby reduces the binding of NSA to the mitochondria and subsequently blocks NSA-induced mitochondrial damage.On the other hand, the accumulation of NSA protein on the mitochondria leads to the induction of mitochondrial damage that finally influences negatively the cell functions and subsequently inhibit core protein induced cell growth advantage in HepG cells.

Conclusion
In conclusion, our present data imply the possibility that NSA protein is responsible at least, in part, for the inhibition of Core protein-induced proliferation of hepatocytes in HCV infected patients.
DENIS SELIMOVIĆ, MOHAMED HASSAN: INHIBITION OF HEPATITIS C VIRUS HCV CORE PROTEIN INDUCED CELL GROWTH BY NONSTRUCTURAL PROTEIN 4A NS4A IS MEDIATED BY MITOCHONDRIAL DYSREGULATION.
DENIS SELIMOVIĆ, MOHAMED HASSAN: INHIBITION OF HEPATITIS C VIRUS HCV CORE PROTEIN INDUCED CELL GROWTH BY NONSTRUCTURAL PROTEIN 4A NS4A IS MEDIATED BY MITOCHONDRIAL DYSREGULATION.growth should be infl uenced by the expression of NSA protein, the transfectants HepG-core, HepG-NSA and HepG-core/NSA were allowed to grow in the presence and in the absence of tetracycline (μg/ml) up to  days and the cell growth rate was determined using cell viability assay (MTT).Data obtained from MTT assay (Figure ) confirmed the ability of core protein to induce cell growth advantage in HepG cells, and demonstrated the inhibition of the same cells by the expression of NSA.Interestingly, the expression of both core and NSA in HepG-core/NSA transfectants suppressed core protein-induced growth rate in HepG-core tranfectants and blocked NSAinduced cell growth inhibition of HepG-NSA transfectants (Figure ).Taken together, these data suggest an important role for NSA protein in the inhibition of core protein-induced growth rate of HepG cells.Subcellular localization of NSA protein to mitochondria triggers the loss of mitochondrial membrane potential in HepG cells To investigate the role of mitochondria in the modulation NSA-induced cell growth inhibition of HepG cells, the HepG transfectants HepG-core, HepG-NSA and HepG-core/NSA were allowed to grow in the presence and in the absence of tetracycline (μg/ml).Forty eight hours later, the cells were subjected either to immune fl uorescence staining, Western blot analysis or to the measurement of mitochondrial membrane potential (ΔΨm) using JC-.To confi rm the subcellular localization of NSA protein, the HepG-NSA tran-DENIS SELIMOVIĆ, MOHAMED HASSAN: INHIBITION OF HEPATITIS C VIRUS HCV CORE PROTEIN INDUCED CELL GROWTH BY NONSTRUCTURAL PROTEIN 4A NS4A IS MEDIATED BY MITOCHONDRIAL DYSREGULATION.DENIS SELIMOVIĆ, MOHAMED HASSAN: INHIBITION OF HEPATITIS C VIRUS HCV CORE PROTEIN INDUCED CELL GROWTH BY NONSTRUCTURAL PROTEIN 4A NS4A IS MEDIATED BY MITOCHONDRIAL DYSREGULATION.
DENIS SELIMOVIĆ, MOHAMED HASSAN: INHIBITION OF HEPATITIS C VIRUS HCV CORE PROTEIN INDUCED CELL GROWTH BY NONSTRUCTURAL PROTEIN 4A NS4A IS MEDIATED BY MITOCHONDRIAL DYSREGULATION.