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• W2144429812 abstract "The biology and pathogenesis of hepatitis E virus are poorly understood due to the lack of an in vitro culture or infection models. The viral Orf3 protein activates the cellular mitogen-activated protein kinase pathway and is likely to modulate the host cell environment for efficient viral replication. We screened for cellular genes whose transcription was differentially up-regulated in an Orf3-expressing stable cell line (ORF3/4). The gene for mitochondrial voltage-dependent anion channel (VDAC) was one such candidate. The up-regulation of VDAC in ORF3/4 cells was confirmed by Northern and Western blotting in various cell lines. Transfection of ORF3/4 cells with an ORF3-specific small interfering RNA led to a reduction in VDAC protein levels. VDAC is a critical mitochondrial outer membrane protein, and its overexpression results in apoptosis. Surprisingly, Orf3-expressing cells were protected against staurosporine-induced cell death by preservation of mitochondrial potential and membrane integrity. A small interfering RNA-mediated reduction in Orf3 and VDAC levels also made cells sensitive to staurosporine. Chemical cross-linking showed Orf3-expressing cells to contain higher levels of oligomeric VDAC. These cells also contained higher levels of hexokinase I that directly interacted with VDAC. This interaction is known to preserve mitochondrial potential and prevent cytochrome c release. We report here the first instance of a viral protein promoting cell survival through such a mechanism. The biology and pathogenesis of hepatitis E virus are poorly understood due to the lack of an in vitro culture or infection models. The viral Orf3 protein activates the cellular mitogen-activated protein kinase pathway and is likely to modulate the host cell environment for efficient viral replication. We screened for cellular genes whose transcription was differentially up-regulated in an Orf3-expressing stable cell line (ORF3/4). The gene for mitochondrial voltage-dependent anion channel (VDAC) was one such candidate. The up-regulation of VDAC in ORF3/4 cells was confirmed by Northern and Western blotting in various cell lines. Transfection of ORF3/4 cells with an ORF3-specific small interfering RNA led to a reduction in VDAC protein levels. VDAC is a critical mitochondrial outer membrane protein, and its overexpression results in apoptosis. Surprisingly, Orf3-expressing cells were protected against staurosporine-induced cell death by preservation of mitochondrial potential and membrane integrity. A small interfering RNA-mediated reduction in Orf3 and VDAC levels also made cells sensitive to staurosporine. Chemical cross-linking showed Orf3-expressing cells to contain higher levels of oligomeric VDAC. These cells also contained higher levels of hexokinase I that directly interacted with VDAC. This interaction is known to preserve mitochondrial potential and prevent cytochrome c release. We report here the first instance of a viral protein promoting cell survival through such a mechanism. Hepatitis E virus (HEV) 4The abbreviations and trivial name used are: HEV, hepatitis E virus; ORF, open reading frame; ERK, extracellular signal-regulated kinase; VDAC, voltage-dependent anion channel; OMM, outer mitochondrial membrane; EGFP, enhanced green fluorescence protein; TBS, Tris-buffered saline; STS, staurosporine; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; HK, hexokinase; siRNA, small interfering RNA; JC-1, 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl benzimidazolcarbocyanine iodide; cytc, cytochrome c. 4The abbreviations and trivial name used are: HEV, hepatitis E virus; ORF, open reading frame; ERK, extracellular signal-regulated kinase; VDAC, voltage-dependent anion channel; OMM, outer mitochondrial membrane; EGFP, enhanced green fluorescence protein; TBS, Tris-buffered saline; STS, staurosporine; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; HK, hexokinase; siRNA, small interfering RNA; JC-1, 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl benzimidazolcarbocyanine iodide; cytc, cytochrome c. is the causative agent of hepatitis E, a major form of viral hepatitis in developing countries (1Purcell R.H. Ticehurst J.R. Viral Hepatitis and Liver Disease. 1988; (Zuckerman, A. J., ed) pp. , Alan R. Liss, New York: 131-137Google Scholar, 2Ramalingaswami V. Purcell R.H. Lancet. 1988; 1: 571-573Abstract PubMed Scopus (69) Google Scholar, 3Krawczynski K. Hepatology. 1993; 17: 932-941Crossref PubMed Scopus (220) Google Scholar). It is a waterborne pathogen that is transmitted primarily through the feco-oral route, causing rampant sporadic infections and large outbreaks in endemic areas (1Purcell R.H. Ticehurst J.R. Viral Hepatitis and Liver Disease. 1988; (Zuckerman, A. J., ed) pp. , Alan R. Liss, New York: 131-137Google Scholar, 2Ramalingaswami V. Purcell R.H. Lancet. 1988; 1: 571-573Abstract PubMed Scopus (69) Google Scholar, 3Krawczynski K. Hepatology. 1993; 17: 932-941Crossref PubMed Scopus (220) Google Scholar). Although the infection is self-limited and never proceeds to chronicity, fulminant hepatitis with high rates of mortality is known to occur in a small fraction of patients, especially in pregnant women (4Khuroo M.S. Teli M.R. Skidmore S. Sofi M.A. Khuroo M. Am. J. Med. 1981; 70: 252-255Abstract Full Text PDF PubMed Scopus (437) Google Scholar, 5Nayak N.C. Panda S.K. Datta R. Zuckerman A.J. Guha D.K. Madanagopalan N. Buckshee K. J. Gastroenterol. Hepatol. 1989; 4: 345-352Crossref PubMed Scopus (39) Google Scholar). The HEV was recently classified as the only member of Hepevirus in the family Hepeviradae (6Emerson S.U. Anderson D. Arankalle A. Meng X.J. Purdy M. Schlauder G.G. Tsarev S.A. Fauquet C.M. Mayo M.A. Maniloff J. Desselberger U. Ball L.A. Virus Taxonomy, VIIIth Report of the ICTV. 2004: 851-855Google Scholar). It is a nonenveloped virus with a single-stranded positive sense RNA genome of ∼7.2 kb and contains three open reading frames (ORFs) (7Tam A.W. Smith M.M. Guerra M.E. Huang C.C. Bradley D.W. Fry K.E. Reyes G.R. Virology. 1991; 185: 120-131Crossref PubMed Scopus (872) Google Scholar). The orf1 encodes the major nonstructural polyprotein, orf2 codes for the major viral capsid protein, and orf3 codes for a small protein whose functions are not fully understood. Due to the lack of reliable cell culture systems or small animal models of infection and disease, subgenomic expression strategies have been employed to functionally characterize HEV-encoded proteins (8Jameel S. Zafrullah M. Ozdener M.H. Panda S.K. J. Virol. 1996; 70: 207-216Crossref PubMed Google Scholar, 9Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar, 10Zafrullah M. Ozdener M.H. Kumar R. Panda S.K. Jameel S. J. Virol. 1999; 73: 4074-4082Crossref PubMed Google Scholar).The orf3 overlaps the other two ORFs and encodes a protein of 123 amino acids (Orf3). A recent report proposes that Orf2 and Orf3 are translated from the same subgenomic RNA and that the latter protein was 9 amino acids shorter at its N-terminal end than previously reported (11Graff J. Torian U. Nguyen H. Emerson S.U. J. Virol. 2006; 80: 5919-5926Crossref PubMed Scopus (195) Google Scholar). The Orf3 protein is phosphorylated at a single serine residue by the cellular mitogen-activated protein kinase (9Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar). In its N-terminal half, Orf3 contains two hydrophobic domains, the first shown to be responsible for its cytoskeletal association (9Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar). In its C-terminal half, the protein contains two proline-rich regions, of which one carries the phosphorylated serine residue (9Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar). The other region was shown to contain a PXXPXXP motif and to bind several proteins that contain Src homology 3 domains (12Korkaya H. Jameel S. Gupta D. Tyagi S. Kumar R. Zafrullah M. Mazumdar M. Lal S.K. Xiaofang L. Sehgal D. Das S.R. Sahal D. J. Biol. Chem. 2001; 276: 42389-42400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Such PXXP motifs are part of polyproline helices found in a number of viral and cellular proteins involved in signal transduction and bind the Src homology 3 domains found in a diverse group of signal-transducing molecules (13Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2217) Google Scholar, 14Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (924) Google Scholar). Previously, we have also shown Orf3 to activate the extracellularly regulated kinase (ERK), a member of the mitogen-activated protein kinase superfamily, supporting a prosurvival role for it. The Orf3 protein binds Pyst1, an ERK-specific member of dual specificity mitogen–activated protein kinase phosphatases; this interaction inhibits ERK dephosphorylation, thereby prolonging its activated state (15Kar-Roy A. Korkaya H. Oberoi R. Lal S.K. Jameel S. J. Biol. Chem. 2004; 279: 28345-28357Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar).In the present study, we found increased expression of the voltage-dependent anion channel (VDAC) protein in Orf3-expressing cells. The VDAC protein, also known as porin for its pore forming ability, is a resident outer mitochondrial membrane (OMM) protein of 30–35 kDa that regulates the transport of solutes such as Ca2+ and ATP across the OMM (16Colombini M. J. Membr. Biol. 1989; 111: 103-111Crossref PubMed Scopus (253) Google Scholar, 17Bernardi P. Physiol. Rev. 1999; 79: 1127-1155Crossref PubMed Scopus (1327) Google Scholar). The mitochondrion plays an important and fateful role in the apoptotic death of mammalian cells by releasing apoptogenic proteins into the cytoplasm (18Halestrap A.P. Doran E. Gillespie J.P. O'Toole A. Biochem. Soc. Trans. 2000; 28: 170-177Crossref PubMed Scopus (283) Google Scholar). The release of cytochrome c from mitochondria into the cytoplasm activates caspases, setting the cell on an apoptotic path (19Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4433) Google Scholar). The VDAC protein regulates OMM permeability and is in turn regulated by direct interaction with Bcl-2 family members as well as other proteins, such as hexokinase and mitochondrial creatine kinase (20Tsujimoto Y. Shimizu S. Biochimie (Paris). 2002; 84: 187-193Crossref PubMed Scopus (223) Google Scholar). Further, VDAC is known to be up-regulated in cancer cells (21Shinohara Y. Ishida T. Hino M. Yamazaki N. Baba Y. Terada H. Eur. J. Biochem. 2000; 267: 6067-6073Crossref PubMed Scopus (69) Google Scholar) and exhibits elevated binding to hexokinase I and/or hexokinase II. Due to their higher energy requirements, cancer cells are characterized by a high rate of glycolysis (22Arora K.K. Pedersen P.L. J. Biol. Chem. 1988; 263: 17422-17428Abstract Full Text PDF PubMed Google Scholar, 23Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (297) Google Scholar). This in turn requires various genetic and biochemical adaptations, including increased expression of mitochondria-bound isoforms of hexokinase (HK I and HK II) (23Pedersen P.L. Mathupala S. Rempel A. Geschwind J.F. Ko Y.H. Biochim. Biophys. Acta. 2002; 1555: 14-20Crossref PubMed Scopus (297) Google Scholar, 24Rempel A. Bannasch P. Mayer D. Biochim. Biophys. Acta. 1994; 1219: 660-668Crossref PubMed Scopus (74) Google Scholar, 25Sebastian S. Kenkare U.W. Biochem. Biophys. Res. Commun. 1997; 235: 389-393Crossref PubMed Scopus (14) Google Scholar). Purified HK I has been shown to interact directly with purified VDAC reconstituted into a planar lipid bilayer, leading to channel closure. This prevents opening of the permeability transition pore and release of cytochrome c, thus inhibiting the mitochondrial phase of apoptosis (26Azoulay-Zohar H. Israelson A. Abu-Hamad S. Shoshan-Barmatz V. Biochem. J. 2004; 377: 347-355Crossref PubMed Scopus (327) Google Scholar). A dynamic equilibrium exists between monomeric and oligomeric forms of VDAC that determines its interactions with other proteins and the regulated formation of pores large enough to allow the release of apoptogenic factors from mitochondria (27Zalk R. Israelson A. Garty E.S. Azoulay-Zohar H. Shoshan-Barmatz V. Biochem. J. 2005; 386: 73-83Crossref PubMed Scopus (174) Google Scholar).We show here a prosurvival effect of the HEV Orf3 protein and relate this to its ability to modulate VDAC expression and oligomerization. We also show that VDAC oligomerization in Orf3-expressing cells protects against an apoptotic insult and is accompanied by enhanced hexokinase I expression and its interaction with VDAC. These findings are discussed in the context of HEV pathogenesis.EXPERIMENTAL PROCEDURESPlasmids, Cell Lines, and Antibodies—The expression vectors for orf3, pSG-ORF3 (8Jameel S. Zafrullah M. Ozdener M.H. Panda S.K. J. Virol. 1996; 70: 207-216Crossref PubMed Google Scholar), and pORF3-ECFP (15Kar-Roy A. Korkaya H. Oberoi R. Lal S.K. Jameel S. J. Biol. Chem. 2004; 279: 28345-28357Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) have been described earlier. The orf3 and vector control stable cell lines (12Korkaya H. Jameel S. Gupta D. Tyagi S. Kumar R. Zafrullah M. Mazumdar M. Lal S.K. Xiaofang L. Sehgal D. Das S.R. Sahal D. J. Biol. Chem. 2001; 276: 42389-42400Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) as well as polyclonal antibodies to Orf3 (8Jameel S. Zafrullah M. Ozdener M.H. Panda S.K. J. Virol. 1996; 70: 207-216Crossref PubMed Google Scholar) have also been described earlier. The VDAC1, VDAC2, and VDAC3 genes were generously provided by Dr. Michael Forte (Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland, OR). The VDAC antibody was purchased from Cell Signaling Technology (Beverly, MA), whereas the Alexa dye-conjugated secondary antibodies were procured from Molecular Probes, Inc. (Eugene, OR). The EGFP-cytochrome c expression vector was kindly provided by Dr. Israrul Haq Ansari (University of Nebraska, Lincoln, NE).Suppressive Subtractive Hybridization—Suppressive subtractive hybridization was performed using orf3 and vector control stable cell lines according to Mishra et al. (28Mishra R.N. Ramesha A. Kaul T. Nair S. Sopory S.K. Reddy M.K. Anal. Biochem. 2005; 345: 149-157Crossref PubMed Scopus (23) Google Scholar). Briefly, total RNA was isolated from the cells and mRNA prepared by magnetic separation after annealing with biotinylated oligo(dT) primer and immobilizing it onto streptavidin-linked paramagnetic beads. Five μg of mRNA from each cell line was used to synthesize first strand cDNA by separately priming with P1-dT and P2-dT oligonucleotide primers (28Mishra R.N. Ramesha A. Kaul T. Nair S. Sopory S.K. Reddy M.K. Anal. Biochem. 2005; 345: 149-157Crossref PubMed Scopus (23) Google Scholar). After reverse transcription, mRNA and excess primers were removed before ligating the P1–3′ and P2–3′ adaptor oligonucleotides to the 3′-ends of first strand cDNAs with T4 RNA ligase (28Mishra R.N. Ramesha A. Kaul T. Nair S. Sopory S.K. Reddy M.K. Anal. Biochem. 2005; 345: 149-157Crossref PubMed Scopus (23) Google Scholar). The first strand cDNA population from control samples was PCR-amplified using 5′ biotinylated P1 forward and nonbiotinylated P1 reverse primers (28Mishra R.N. Ramesha A. Kaul T. Nair S. Sopory S.K. Reddy M.K. Anal. Biochem. 2005; 345: 149-157Crossref PubMed Scopus (23) Google Scholar). The PCR conditions included 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min. The biotinylated double-stranded cDNAs thus obtained were immobilized onto streptavidin-linked paramagnetic beads, whereas the nonbiotinylated complementary antisense strands were denatured with 0.15 m NaOH for 10 min at room temperature and separated out from the beads with a magnet. The beads were washed twice with 0.15 m NaOH and used for the first round of cDNA subtraction. The unamplified first strand cDNAs that were made from ORF3/4 cells were hybridized for 5 h at 65 °C with PCR-amplified sense strand cDNAs of control cells that were immobilized onto the magnetic beads. The hybrids between the sense strand and the common complementary antisense cDNA strands, as well as the beads, were magnetically separated. Differentially up-regulated genes in ORF3/4 cells remained in the hybridization solution and were PCR-amplified in a sequence-independent manner with P2 forward and P2 reverse primers. The PCR conditions included 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 3 min. The PCR-amplified differentially expressed cDNA population was then cloned into the pGEMT vector (Promega, Madison, WI). The recombinant plasmids thus obtained were digested with NotI; depending on insert size, six different clones were selected and sequenced, and the genes were identified by BLAST analysis.Northern Blotting—For probe preparation, the VDAC1 clone was digested with AccI-BglI to give a 230-bp fragment, the VDAC2 clone was digested with AccI-HindIII for a 530-bp fragment, and the VDAC3 clone was digested with HindIII for a 470-bp fragment. The probe fragments corresponded to the 3′-ends of the VDAC genes and showed very low nucleotide homology, enabling specific detection of VDAC RNAs by Northern blotting. Total RNA was isolated with Trizol (Invitrogen) as per the manufacturer’s guidelines. Samples of total RNA (20 μg/lane) were denatured in 17.4% (v/v) formaldehyde, 50% (v/v) formamide, 20 mm 3-(N-morpholino) propanesulfonic acid, 5 mm sodium acetate, and 1 mm EDTA (pH 7.0) for 5 min at 65 °C and separated on a 1% (w/v) agarose, 0.66 m formaldehyde gel. Following electrophoresis, the RNA was transferred to a nylon membrane (HyBond-N+; Amersham Biosciences) by capillary blotting for 24 h in 10× SSC (1× SSC: 150 mm NaCl and 15 mm sodium citrate, pH 7.0) and UV-cross-linked to the nylon membrane. The blot was stained with methylene blue for RNA integrity and equivalence by determining transfer of 28 and 18 S rRNA from the gel. Membranes were prehybridized at 42 °C for 5 h in hybridization solution containing 50% (v/v) deionized formamide, 50 mm sodium phosphate, 0.8 m NaCl, 2% (w/v) SDS, 100 μg salmon sperm DNA/ml, 20 μg of transfer RNA/ml, and 1× Denhardt’s solution (50× Denhardt’s solution: 1% each of bovine serum albumin, Ficoll, and polyvinylpyrrolidone). The VDAC probes were labeled with [α-32P]dCTP using a random priming labeling kit (Hexalabel DNA labeling kit; Fermentas). Hybridization was carried out at 42 °C for 20 h in hybridization solution containing the radiolabeled VDAC isoform-specific probe. Membranes were washed sequentially once in 2× SSC at room temperature for 10 min and twice with 0.5× SSC, 0.1% SDS at 65 °C for 30 min. The bands were visualized by autoradiography.Immunoprecipitation and Western Blotting—Cells were lysed with a buffer containing 20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, and a protease inhibitor mixture (Roche Applied Science). The cell lysates were normalized for protein content, and 1 mg of total proteins in 500 μl of lysis buffer were incubated with 10 μl of Protein G-agarose (Amersham Biosciences) beads for 1 h at 4 °C. The precleared lysate was then incubated with 2 μg of anti-hexokinase I polyclonal antibody (sc-6518; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4 °C. This was followed by incubation with 10 μl of Protein G-agarose beads for 2 h at 4 °C. After five washings in lysis buffer, the beads were boiled in Laemmli buffer, and the proteins were separated by SDS-PAGE. For Western blotting, the separated proteins were transferred to a nitrocellulose membrane (MDI, Advanced Microdevices Pvt. Ltd., Ambala, India). After blocking with Tris-buffered saline (TBS) containing 5% nonfat milk (Nestle) for 1–2 h at room temperature, the membrane was washed with TBST (TBS containing 0.1% Tween 20) and incubated overnight at 4 °C with the primary antibody appropriately diluted in TBST containing 5% bovine serum albumin. The blot was then washed three times for 10 min each with TBST and then incubated with horseradish peroxidase-linked anti-rabbit or anti-goat IgG diluted in TBST containing 5% nonfat milk. Chemiluminescent detection of proteins was carried out using the Phototope horseradish peroxidase Western blot detection system (Cell Signaling Technology, Beverly, MA) according to the supplier’s protocol. In all Western blotting experiments, total ERK levels were evaluated as loading controls.Cell Viability Assay—For the cell survival assay, cells were treated with 1 μm staurosporine (STS) for 10 h, followed by two washes with PBS. The treated cells were then stained with 0.25% Coomassie Brilliant Blue R-250 in 10% acetic acid, 50% methanol (v/v) and washed, and the stained colonies of live cells were counted on an Elispot Reader (Bioreader 4000; Biosys). The cell viability was also evaluated in attached cells using a colorimetric assay based on 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) staining (29Mosmann T. J. Immunol. Methods. 1983; 65: 55-63Crossref PubMed Scopus (45530) Google Scholar) that measures mitochondrial function (30Carmichael J. DeGraff W.G. Gazdar A.F. Minna J.D. Mitchell J.B. Cancer Res. 1987; 47: 936-942PubMed Google Scholar, 31Vistica D.T. Skehan P. Scudiero D. Monks A. Pittman A. Boyd M.R. Cancer Res. 1991; 51: 2515-2520PubMed Google Scholar). The cells were either mock-treated or treated with various concentrations of STS for the indicated time(s) followed by the MTT assay. All data are expressed as percentages with respect to mock-treated cells and represent the mean ± S.E. of at least three independent experiments.Mitochondrial Membrane Potential Measurement—The change in mitochondrial membrane potential (ΔΨm) was analyzed by flow cytometry using the ΔΨm-sensitive dye 5,5′,6,6′-tetrachloro-1,1′3,3′-tetraethyl benzimidazolcarbocyanine iodide (JC-1; Sigma) (32Cossarizza A. Baccarani-Contri M. Kalashnikova G. Franceschi C. Biochem. Biophys. Res. Commun. 1993; 197: 40-45Crossref PubMed Scopus (946) Google Scholar). Briefly, cells were harvested, washed once in PBS, and then resuspended in complete culture medium containing 1 μm JC-1 at 37 °C for the indicated time(s) in the presence or absence of 2 μm STS. Stained cells were then washed with PBS and analyzed by flow cytometry. The emission maxima of JC-1 monomers and aggregates are 527 nm (FL-1 channel) and 590 nm (FL-2 channel), respectively.Microscopy—For immunofluorescence staining and colocalization experiments, Huh-7 cells were seeded at about 30% confluence on coverslips in 12-well plates, grown for 18 h, and then cotransfected with Ds-Red Mito and EGFP-cytc, either with or without ECFP-ORF3. Also, pCN and ORF3/4 cells were cotransfected to express Ds-Red Mito and EGFP-cytc. At 48 h post-transfection, the cells were treated with 2 μm STS for 2 h, washed with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and washed once again in phosphate-buffered saline. The cells were then mounted using Antifade (Bio-Rad) and sealed with a synthetic rubber-based adhesive, Fevibond (Fevicol; Pidilite Industries). Confocal images were collected using a 60× planapo objective on a Bio-Rad Radiance 2100 system attached to a Nikon inverted microscope. Multiple randomly selected cells from each group were analyzed, and the amount (percentage) of EGFP-cytochrome c that colocalized with Ds-Red Mito was quantitated with the LaserPix software (Bio-Rad). Such colocalization calculations are independent of the relative intensities in the green and red channels.Cross-linking Experiments—The pCN and ORF3/4 cells were treated with or without 5 μm STS for 30 min followed by a PBS wash. Cells were lysed by five or six repeated freeze-thaw cycles in liquid nitrogen and at room temperature. The unclarified lysate was then incubated with 1 mm DSP (Pierce) dissolved in Me2SO for 30 min at 30 °C according to manufacturer’s protocol. The Me2SO concentration in control and reagent-containing samples was up to 2.5% (v/v). The reaction was stopped by adding 50 mm Tris, pH 7.5, and the samples were analyzed on nonreducing as well as reducing SDS-PAGE.siRNA-mediated Knockdown of Orf3 and VDAC—The orf3 siRNA 5′-UCACGUCGUAGACCUACCAUU-3′ was designed using the “Dharmacon siDESIGN Center” program available on the World Wide Web and was obtained from Dharmacon (Lafayette, CO) as a duplex. The VDAC1-specific and GFP siRNAs were obtained as siGENOME SMARTpool reagent from Dharmacon. Cells were transfected using RNAiFect (Qiagen, Hilden, Germany) with 200 nm VDAC siRNA (si-V), 300 nm orf3 siRNA (si-3), and 200 nm GFP siRNA (si-G). All siRNA-related experiments were performed in 12-well culture plates, and GFP-specific siRNA was used as nonspecific control. For siRNA-based survival experiments, cells were transfected, with either the orf3 siRNA for 48 h or the VDAC siRNA pool for 36 h followed by treatment with 2 μm STS for 2 h. The cell viability assays were then performed as described above.Reverse Transcription-PCR Analysis—Total RNA was isolated from pCN and ORF3/4 cells using the Trizol reagent (Invitrogen). Four μg of RNA in a 25-μl reaction mixture was used for cDNA synthesis with Reverse Transcriptase (Promega, Madison, WI) according to the supplier’s protocol. Of this, 0.5 μl of cDNA was used as a template for PCR amplification of target genes. The PCRs were performed in a 50-μl volume containing 1× reaction buffer, 200 μm dNTPs, 10 pmol of each primer, and 1.25 unit of TaqDNA polymerase (Real Biotech Corp., Taipei, Taiwan), for 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 2 min. The primer sequences for hexokinase I and II were retrieved from the World Wide Web and custom-synthesized. Primer pairs HK1-F (5′-TCCTGGCCTATTACTTCACGG-3′) and HK1-R (5′-GGACCTTACGAATGTTGGCAA-3′) and HK2-F (5′-GAGCCACCACTCACCCTACT-3′) and HK2-R (5′-ACCCAAAGCACACGGAAGTT-3′) were used to amplify hexokinase I and II, respectively. The histone H4 gene was amplified as a reference control using the primers HISTH4-F (5′-TGAGAGACAACATTCAGGGCATCAC-3′) and HISTH4-R (5′-CGCTTGAGCGCGTACACCACATCCAT-3′). The amplified products were resolved on a 2% agarose gel and visualized following staining with ethidium bromide. The sizes of the amplified fragments were as follows: hexokinase I, 194 bp; hexokinase II, 130 bp; histone H4, 211 bp.RESULTSSuppression subtractive hybridization was performed using control (pCN) and Orf3-expressing (ORF3/4) stable cell lines as described under “Experimental Procedures.” The differentially expressed cDNAs were PCR-amplified and cloned. A number of clones were randomly picked and analyzed by restriction digestion. Clones with varying sizes of inserts were identified and sequenced to determine the identity of the up-regulated genes they represented. One such up-regulated gene was VDAC1.To validate Orf3-mediated increased expression of VDAC, we carried out Northern and Western blot analyses. The ORF3/4 cell line showed higher levels of VDAC1 RNA (Fig. 1A). Using probes from the 3′-end of the VDAC gene, where minimal homology was observed between VDAC1 and its two other isoforms, VDAC2 and VDAC3, increased expression of all three VDAC isoforms was observed in Orf3-expressing cells (Fig. 1A). To confirm that the higher RNA levels were also reflected in higher levels of the VDAC protein, Western blotting was performed on lysates prepared from pCN and two independent Orf3-expressing cell lines, ORF3/1 and ORF3/4. Western blotting using anti-VDAC antibodies showed higher expression of the VDAC protein in Orf3-expressing cells (Fig. 1B, top, lanes 1–3). Since the anti-VDAC antibodies reacted with all three isoforms of the protein, it was not possible to study the effects on individual isoforms. In another experiment, human hepatoma Huh-7 cells that were transiently transfected with an orf3 expression vector (pSG-ORF3) also showed higher levels of the VDAC protein compared with Huh-7 cells transfected with the empty vector (Fig. 1B, top, lanes 4–6). We checked a HeLa/Tet-OFF-inducible stable cell line 5M. Rajala and S. Jameel, unpublished observations. to show that compared with control cells, the Orf3-expressing cells showed higher levels of the VDAC protein (Fig. 1B, bottom, lanes 1–3). These cells showed leaky expression of Orf3 even in the presence of tetracycline (lane 2); this was also reflected in VDAC expression in HeLa/Tet-OFF/ORF3 cells in the presence (lane 2) and absence of tetracycline (lane 3). Thus, using multiple cell line systems, either transiently transfected or stably expressing Orf3, we show that increased VDAC expression correlated with Orf3 expression.The VDAC protein is the principal mitochondrial channel protein, and its regulated expression is important for cell survival. A higher VDAC expression level in cells results in cell death by apoptosis (33Abu-Hamad S. Sivan S. Shoshan-Barmatz V. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 5787-5792Crossref PubMed Scopus (187) Google Scholar). For insight into VDAC overexpression in Orf3-stable cell lines, we treated the cells with STS, a potent apoptogenic stimulus. Apoptosis mediated by STS occurs through a mitochondrial pathway and leads to loss of mitochondrial transmembrane potential. Cells that survived STS-initiated death were initially scored by staining with Coomassie Blue and counting. Surprisingly, significantly reduced cell death was observed in ORF3/4 cells compared with the control pCN cells (Fig. 2A), which was in contras" @default.
• W2144429812 created "2016-06-24" @default.
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• W2144429812 date "2007-07-01" @default.
• W2144429812 modified "2023-09-28" @default.
• W2144429812 title "The Hepatitis E Virus Orf3 Protein Protects Cells from Mitochondrial Depolarization and Death" @default.
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