The ORF3 Protein of Hepatitis E Virus Interacts with Liver-specific α1-Microglobulin and Its Precursor α1-Microglobulin/Bikunin Precursor (AMBP) and Expedites Their Export from the Hepatocyte
2004; Elsevier BV; Volume: 279; Issue: 28 Linguagem: Inglês
10.1074/jbc.m402017200
ISSN1083-351X
AutoresShweta Tyagi, Milan Surjit, Anindita Roy, Shahid Jameel, Sunil K. Lal,
Tópico(s)Liver Disease and Transplantation
ResumoHepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, α1 -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins α1-microglobulin (α1m) and bikunin. α1 m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His6 pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing α1 m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of α1m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that α m was not degraded in the presence of ORF3. Finally, 1pulse labeling of α1m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing α1m export from the hepatocyte. Hepatitis E virus (HEV), a plus-stranded RNA virus contains three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein and ORF3 codes for a phosphoprotein of undefined function. Using the yeast two-hybrid system to screen a human cDNA liver library we have isolated, an N-terminal deleted protein, α1 -microglobulin/bikunin precursor (AMBP) that specifically interacts with the ORF3 protein of HEV. Independently cloned, full-length AMBP was obtained and tested positive for interaction with ORF3 using a variety of in vivo and in vitro techniques. AMBP, a liver-specific precursor protein codes for two different unrelated proteins α1-microglobulin (α1m) and bikunin. α1 m individually interacted with ORF3. The above findings were validated by COS-1 cell immunoprecipitation, His6 pull-down experiments, and co-localization experiments followed by fluorescence resonance energy transfer analysis. Human liver cells showing co-localization of ORF3 with endogenously expressing α1 m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of α1m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that α m was not degraded in the presence of ORF3. Finally, 1pulse labeling of α1m showed that its secretion was expedited out of the liver cell at faster rates in the presence of the ORF3 protein. Hence, ORF3 has a direct biological role in enhancing α1m export from the hepatocyte. Hepatitis E virus (HEV) 1The abbreviations used are: HEV, hepatitis E virus; AMBP, α1-microglobulin/bikunin precursor; α1 m, α1 -microglobulin; AD, activation domain; BD, binding domain; ORF, open reading frame; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; MAP, mitogen-activate protein; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium. is a waterborne pathogen and is responsible for sporadic infections as well as large epidemics of acute viral hepatitis in developing countries (1Bradley D.W. Br. Med. Bull. 1990; 46: 442-461Crossref PubMed Scopus (176) Google Scholar, 2Emerson S.U. Purcell R.H. Trends Mol. Med. 2001; 7: 462-466Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 3Purcell R.H. Emerson S.U. Lancet. 2000; 355: 578Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 4Purcell R.H. Ticehurst J.R. Zukerman A.J. Viral Hepatitis and Liver Disease. Alan R. Liss, Inc., New York, NY1988: 131-137Google Scholar). HEV is a plus-stranded RNA virus with a genome of ∼7.2 kb, containing three open reading frames called ORF1, ORF2, and ORF3 (5Koonin E.V. Gorbalenya A.E. Purdy M.A. Rozanov M.N. Reyes G.R. Bradley D.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8259-8263Crossref PubMed Scopus (406) Google Scholar, 6Tam 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 (886) Google Scholar, 7Tsarev S.A. Emerson S.U. Rees G.R. Tsareva T.S. Letgers L.J. Malik I.A. Iqbal M. Purcell R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 559-563Crossref PubMed Scopus (249) Google Scholar). ORF1 (5079 bp) is at the 5′-end of the genome and is predicted to code for putative non-structural proteins with sequences homologous to those encoding a viral methyltransferase, a cysteine protease, a RNA helicase, and a RNA-dependent RNA polymerase (5Koonin E.V. Gorbalenya A.E. Purdy M.A. Rozanov M.N. Reyes G.R. Bradley D.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8259-8263Crossref PubMed Scopus (406) Google Scholar, 7Tsarev S.A. Emerson S.U. Rees G.R. Tsareva T.S. Letgers L.J. Malik I.A. Iqbal M. Purcell R.H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 559-563Crossref PubMed Scopus (249) Google Scholar, 8Ansari I.H. Nanda S.K. Durgapal H. Agrawal S. Mohanty S.K. Gupta Jameel D.S. Panda S.K. J. Med. Virol. 2000; 60: 275-283Crossref PubMed Scopus (82) Google Scholar, 9Reyes G.R. Huang C.C. Tam A.W. Purdy M.A. Arch. Virol. 1993; 7: 15-25Crossref Scopus (61) Google Scholar). In the absence of a reliable in vitro culture system for HEV, fundamental studies on its replication and expression strategy have not been undertaken. ORF2 and ORF3 have been expressed in Escherichia coli, animal cells, baculovirus, yeast, and in vitro in a coupled transcription-translation system (10He J. Tam A.W. Yarbough P.O. Reyes G.R. Carl M. J. Clin. Microbiol. 1993; 31: 2167-2173Crossref PubMed Google Scholar, 11Jameel S. Zafrullah M. Ozdener M.H. Panda S.K. J. Virol. 1996; 70: 207-216Crossref PubMed Google Scholar, 12Khudyakov Y.E. Favorov M.O. Jue D.L. Hine T.K. Fields H.A. Virology. 1994; 198: 390-393Crossref PubMed Scopus (70) Google Scholar, 13Lal S.K. Tulasiram P. Jameel S. Gene (Amst.). 1997; 190: 63-67Crossref PubMed Scopus (22) Google Scholar, 14Panda S.K. Ansari H.I. Durgapal H. Agrawal S. Jameel S. J. Virol. 2000; 74: 2430-2437Crossref PubMed Scopus (115) Google Scholar). ORF2, a 88-kDa glycoprotein, is expressed intracellularly as well as on the cell surface and is the major capsid protein for HEV. It is synthesized as a precursor that is processed through signal sequence cleavage into the mature protein, which is capable of self-association (15Khudyakov Y.E. Khudyakova N.S. Fields H.A. Jue D. Starling C. Favorov M.O. Krawczynski K. Polish L. Mast E. Margolis H. Virology. 1993; 194: 89-96Crossref PubMed Scopus (72) Google Scholar, 16Tyagi S. Jameel S. Lal S.K. J. Virol. 2001; 75: 2493-2498Crossref PubMed Scopus (36) Google Scholar). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton and shows no major processing (17Aye T.T. Uchida T. Ma X.-Z. Iida F. Shikata T. Zhuang H. Win K.M. Nucleic Acids Res. 1992; 20: 3512Crossref PubMed Scopus (146) Google Scholar, 18Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar). The ORF3 protein dimerizes using a 43-amino acid region, interacts with SH3 domains and activates cellular MAP kinase (19Korkaya 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, 20Tyagi S. Jameel S. Lal S.K. Biochem. Biophys. Res. Commun. 2001; 284: 614-621Crossref PubMed Scopus (13) Google Scholar). Recently the phosphorylated form of the ORF3 protein has also been shown to interact with the non-glycosylated form of the ORF2 (capsid) protein of HEV (21Tyagi S. Korkaya H. Zafrullah M. Jameel S. Lal S.K. J. Biol. Chem. 2002; 277: 22759-22767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). These properties of ORF3 clearly indicate that this protein may have multiple roles in HEV pathogenesis. To delineate the functions of this viral protein, studies were conducted to screen and characterize ORF3-interacting host proteins from a human liver cDNA library. Since a few years of its introduction, the yeast two-hybrid system has proven invaluable for studying physical interactions between genetically defined partners, for identifying contacts among the subunits of multiprotein complexes (22Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1226) Google Scholar, 23Fields S. Sternglanz R. Trends Genet. 1994; 10: 286-291Abstract Full Text PDF PubMed Scopus (524) Google Scholar, 24Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4863) Google Scholar, 25Jackson A.L. Pahl P.M. Harrison K. Rosamond J. Sclafani R.A. Mol. Cell Biol. 1993; 13: 2899-2908Crossref PubMed Scopus (214) Google Scholar), and for mapping specific domains involved in protein-protein interactions (20Tyagi S. Jameel S. Lal S.K. Biochem. Biophys. Res. Commun. 2001; 284: 614-621Crossref PubMed Scopus (13) Google Scholar, 21Tyagi S. Korkaya H. Zafrullah M. Jameel S. Lal S.K. J. Biol. Chem. 2002; 277: 22759-22767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 26Kalpana G.V. Goff S.P. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10593-10597Crossref PubMed Scopus (55) Google Scholar). In this system, two plasmid-borne gene fusions are co-transformed into yeast cells, and the interaction between these two fusion proteins is measured by the reconstitution of a functional transcriptional activator that triggers the expression of reporter genes lacZ and HIS3. Using this system we have isolated an interacting partner from the human liver cDNA library, for the ORF3 protein of HEV. This interaction partner is α1-microglobulin/bikunin precursor (AMBP), a liver-specific precursor protein. This interaction was verified using an independently cloned full-length AMBP gene, obtained from another laboratory and tested for positive interaction with ORF3, using yeast two-hybrid techniques and in vitro binding experiments. AMBP gets processed in the trans-Golgi region to give α1-microglobulin (α1m) and bikunin, which are then secreted by the liver cells in free and bound forms. The processed protein, α1m, was individually tested for interaction with ORF3 using the yeast two-hybrid approach, in vitro binding, immunoprecipitation, and FRET. Dual-labeling immunofluorescent staining followed by fluorescence microscopy in human liver cells showed co-localization of the ORF3 protein with α1m; however, at 46–48 h post-transfection, the perinuclear localization of α1m disappeared from the cells. Experiments on subcellular localization of α1m and using drugs that block the secretion pathway of α1m, we showed in transfected hepatocytes, an ORF3-dependent increase in the export of α1m from the Golgi compartment. Finally, using pulse-labeled α1m, we have conclusively proved this observation. The biological significance of this interaction and the possible role for the accelerated export of α1m in HEV-infected hepatocytes is discussed. Strains, Media, and Plasmid Constructs—All strains, plasmids, and plasmid constructs used in this study are described in Table I. The full-length ORF3 gene of HEV was excised from the pSG-ORF3 vector (18Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar) and cloned into the yeast two-hybrid BD vector resulting in an N-terminal in-frame fusion with the GAL4 DNA binding domain, as described before (20Tyagi S. Jameel S. Lal S.K. Biochem. Biophys. Res. Commun. 2001; 284: 614-621Crossref PubMed Scopus (13) Google Scholar). DNA manipulations were carried out as described by Sambrook, et al. (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). All deletion constructs were generated by subcloning the full-length ORF3 genes of HEV (Table I). All constructs were verified by restriction digestion and sequencing.Table IYeast strains, plasmids, and recombinant plasmid constructs used in this studyStrain/plasmid/constructsDescription/referenceStrainsYeastY190MATa trp1-901 his3 leu2-3, 112 ura3-52 ade2 gal4 gal80URA3::GAL-lacZ LYS2::GAL-HIS3PJ69.4aMATa trp 1-901 leu2-3, 112 ura 3-52 his 3-200 gal4Δ gal80Δ Lys2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZPJ69.4αMATa trp 1-901 leu2-3, 112 ura 3-52 his 3-200 gal4Δ gal80Δ Lys2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZPlasmidspGAD424/pACT2GAL4 AD vector [GAL4(768-881)]; LEU2, 2 μm, AmprpGBT9/pAS2/pAS2-1GAL4 DNA-BD vector [GAL4(1-147)]; TRP1, 2pGBT9-ORF3pSG-ORF3 digested with SmaI and BamHI, fragment ligated in pGBT9pGAD424-ORF3pSG-ORF3 digested with SmaI and BamHI, fragment ligated in pGAD424pAS2-1-ORF3pSG-ORF3 digested with EcoRI and BamHI, fragment ligated in pAS2-1pAS2-ORF3pGBT9-ORF3 digested with SmaI and BamHI, fragment ligated in pAS2pAS2-R352pR352 [Rouet et al. (44Lindqvist A. Akerstrom B. Biochim. Biophys. Acta. 1996; 1306: 98-106Crossref PubMed Scopus (15) Google Scholar)] digested with EcoRI and SmaI fragment ligated in pAS2pACT2-α1mPCR amplified and cloned from PACT2-AMBP using SmaI and BamHI sites (underlined) for forward (5′-CCCATGGCCCCGGGGATGGAAAACTTCAATAT-3′) and reverse (5′-CGGGATCCTGCAGCAGCGCTCCGGACTCTCG-3′), respectivelypSG-R352pR352 digested with EcoRI and SmaI, fragment ligated in pSGIpSG-AMBPpACT2-AMBP digested with BamHI and BglII, fragment ligated in pSGIpMT-α1mpACT2-α1m digested using SmaI and EcoRI sites, fragment ligated in pMTpSG-α1mpACT2-α1m digested using SmaI and BamHI sites, fragment ligated in pSGpYFP-α1mpAS2-R352 was digested using SmaI and BamHI sites, fragment ligated in pEGYFP-N1pCFP-ORF3pSG-ORF3 was digested using EcoRI and AccI sites, fragment ligated in pECFP-N1pSG-ORF3Jameel et al. (12Khudyakov Y.E. Favorov M.O. Jue D.L. Hine T.K. Fields H.A. Virology. 1994; 198: 390-393Crossref PubMed Scopus (70) Google Scholar)pMT-ORF3Jameel et al. (12Khudyakov Y.E. Favorov M.O. Jue D.L. Hine T.K. Fields H.A. Virology. 1994; 198: 390-393Crossref PubMed Scopus (70) Google Scholar)pSG-His6ORF3Zafarullah et al. (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) Open table in a new tab Yeast Two-hybrid Techniques—The GAL-4-based two-hybrid system, kindly provided by Dr. Stephen Elledge, containing pAS2 (DNA binding domain vector) and pACT2 (activation domain vector), together with the yeast reporter strain Saccharomyces cerevisiae Y190 (Table I) were used. The host strain containing pGBT9-ORF3 and pGAD424-ORF3 was used as a positive control (20Tyagi S. Jameel S. Lal S.K. Biochem. Biophys. Res. Commun. 2001; 284: 614-621Crossref PubMed Scopus (13) Google Scholar). The GAL4 activation domain fusion, human liver cDNA library was purchased from Clontech. The number of independent clones, as mentioned by the manufacturer, was 1 × 106. The average size of the cDNA insert ranged from 0.5 to 2.5 kb. The cDNA library was repeatedly transformed with the BD-ORF3 construct and transformants thus obtained were screened for Leu+Trp+ phenotype. The Y190 host contains integrated copies of both HIS3 and lacZ reporter genes under the control of GAL4 binding sites. This yeast strain, was co-transformed with the appropriate plasmids, using the lithium acetate procedure and grown on SD plates lacking Trp and Leu (SDTrp–Leu–). Protein interaction was tested on SD plates lacking Leu, Trp, and His (SDLeu–Trp–His–). After 3 days at 30 °C, individual colonies were streaked out and tested for liquid and filter-lift β-galactosidase activity, 50 mm 3-amino-1,2,3-trizole assay and for diploids showing His+ phenotype, using standard yeast two-hybrid procedures. The filter β-galactosidase assay was performed by streaking doubly transformed yeast colonies onto filter paper and allowing them to grow for 2 days on selection medium. Yeast was permeabilized by freezing yeast-impregnated filters in liquid nitrogen and thawing at room temperature. This filter was placed over a second filter that was pre-soaked in a 0.1 m phosphate buffer (pH 7.0) containing 300 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal) and 0.27% β-mercaptoethanol. Filters were left for 48 h to develop a blue color, which indicated a positive protein-protein interaction. The liquid β-galactosidase activity, a parameter directly reflecting the strength of protein-protein interactions, was determined using the substrate CPRG assay as described previously (16Tyagi S. Jameel S. Lal S.K. J. Virol. 2001; 75: 2493-2498Crossref PubMed Scopus (36) Google Scholar, 21Tyagi S. Korkaya H. Zafrullah M. Jameel S. Lal S.K. J. Biol. Chem. 2002; 277: 22759-22767Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Relative β-galactosidase activity for quantitative assays were corrected for yeast cell number and are the mean ± S.E. of triplicate assays. Appropriate positive/negative controls and buffer blanks were used. The specificity of the in vivo protein-protein interaction was confirmed using a yeast genetic assay for reconfirming positive two-hybrid interactions (28Tyagi S. Lal S.K. Biochem. Biophys. Res. Commun. 2000; 277: 589-593Crossref PubMed Scopus (16) Google Scholar). Plasmid constructs were extracted from the positive Y190 co-transformants, separated, and verified using E. coli HB101 cells on M9 synthetic media lacking Leu. Subsequently, these plasmids were singly transformed into the PJ69-4a and PJ69-4α haploid yeast strains (29James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). After genetic crossing, the His3 prototrophy of the diploid strains was tested by plating for growth on SDHis– media. All possible control transformations were conducted and were verified to be negative for His3 prototrophy. In Vitro Transcription/Translation Assay—The full-length ORF3 protein (pSG-HisORF3, encoding 123 amino acids and ORF3 with an N-terminal His6 tag) and radiolabeled 35[S]methionine proteins (AMBP, R352, and α1m; described in Table I) were expressed in separate reactions using a coupled in vitro transcription-translation system (TnT coupled reticulocyte lysate system; Promega) as per the manufacturer's instructions. The unlabeled ORF3 protein was bound to Ni-NTA beads (Amersham Biosciences) and washed thrice with PBS (pH 7.4). The [35S]methionine-labeled proteins were then added to the same tube and incubated for 4 h at 4 °C with gentle shaking. The beads were washed three times with PBS, resuspended in 25 μl of SDS-PAGE loading buffer (50 mm Tris-HCl, pH 6.8, 5% 2-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and boiled for 4 min to dissociate the bound proteins. Aliquots (10 μl) of the supernatants were subjected to SDS-PAGE, and the 35[S]methionine-labeled proteins were detected by autoradiography. Transfection and Labeling of Cultured Cells—Human hepatoma (Huh7) cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum and 20 μg of gentamicin per ml. Cells were transfected at about 50% confluency with plasmid DNA by using Lipofectin (Invitrogen) according to the manufacturer's guidelines. For each 60-mm diameter culture dish, 2.5 μg of DNA and 10 μl of Lipofectin were used in 1.2 ml of DMEM without serum or antibiotics, and DNA uptake allowed to proceed for 6 h at 37 °CinaCO2 incubator. Forty hours post-transfection, cells were washed with 3 ml of methionine-deficient DMEM (Invitrogen) and metabolically labeled with 35[S]methionine (Amersham Biosciences), with each 60-mm diameter plate receiving 100 μCi of label in 1 ml of methionine-deficient DMEM. After 4 h of labeling, cells were washed with ice-cold phosphate-buffered saline (PBS) and harvested for further analysis. Immunoprecipitation—Transfected, PBS-washed Huh7 cells were harvested directly in 0.5 ml of GST binding buffer (20 mm Tris (pH 7.9), 180 mm KCl, 0.2 mm EDTA, 5 mm MgCl2, 1 mm phenylmethylsulfonyl fluoride, 0.01% Nonidet P-40, 1 mm dithiothreitol containing 1 μg/ml bovine serum albumin) after incubation on ice for 15 min. Lysates were clarified at 10,000 × g for 10 min, and the supernatant was incubated on ice for 1 h with 5 μl of rabbit antiserum. To this 100 μl of a 10% suspension of binding buffer-washed protein A-Sepharose beads (Amersham Biosciences) were added, and the mixture was incubated with constant shaking at 4 °C for 1 h. The beads were washed five times, each time with 0.5 ml of GST buffer, after being centrifuged at 10,000 rpm at 4 °C for 10 s. Washed beads were resuspended in 50 μl of SDS-PAGE loading buffer, heated at 100 °C for 4 min, centrifuged, and the supernatant was subjected to SDS-PAGE and autoradiography or fluorography. For detection of α1m, human hepatoma (Huh7) cells were mock transfected with vector only or with a ORF3-expressing plasmid and allowed to grow for 39 h following which they were starved in cysteine methionine-deficient medium for 1 h. These cells were labeled with 100 μCi of 35S Promix (PerkinElmer Life Sciences) in 1 ml of cysteine– methionine– medium for 1 h. The secreted α1m was detected by direct immunoprecipitation of the growth medium, and the intracellular α1m level was detected by lysing the cells as described above. Pulse-chase Analysis—Forty-four hours post-transfection, Huh7 cells were starved in cysteine– methionine– medium for 1 h. These cells were subsequently supplemented with 200 μCi of 35S Promix in 1 ml of cysteine– methionine– medium for 5 min. The cells were washed in PBS and chased in complete medium for respective time periods. Immunoprecipitation was conducted as described above. Immunofluorescence Analysis—Huh7 cells were plated at a confluency of about 50% on coverslips a day before the transfection and grown for 18 h. 40 h post-transfection (or as specified), the PBS-washed cells were fixed with 2% paraformaldehyde in PBS at room temperature for 10 min, permeabilized with 100% methanol at –20 °C for 3 min and then rehydrated with PBS for 20 min at room temperature. The cells were blocked with 5% normal goat serum for 2 h at room temperature and then incubated with appropriately diluted primary antibodies in PBS/0.5% Tween 20 (PBST) containing 1% normal goat serum for 2 h at room temperature. The primary antibodies used were: monoclonal anti-ORF3 at 1:200 dilution, polyclonal anti-α1m at 1:1000 dilution, or anti-bikunin at 1:500 dilution. Cells were washed thrice with PBS for 5 min each and then incubated for 1 h at room temperature with a 1:1000 dilution of conjugated secondary antibodies. For co-localization experiments, the secondary antibodies used were goat anti-rabbit IgG or goat anti-mouse IgG coupled to either Alexa594 (red) or Alexa488 (green) dyes (Molecular Probes, Eugene, OR). The secondary antibodies used for labeling ORF3, α1m or bikunin, are specified in individual experiments. For localization studies using different organelle markers, fluorescent protein-expressing constructs were transfected alone or with pSGORF3, Ds-Red that was targeted to ER (ER Ds-Red), Ds-Red that was targeted to mitochondria (Mito Ds-Red), yellow fluorescent fusion protein targeted to the Golgi apparatus (YFP-Golgi) were obtained from Clontech. For experiments using Brefeldin A and monensin, these drugs were added 1 h before fixing the cells. The final concentration used was 10 μg/ml for Brefeldin A and 5 μm for monensin. Cells were processed as earlier and mounted in 90% glycerol in PBS. Fluorescence images were collected using a 60× planapo objective in a Bio-Rad 1024 LSM attached to a Nikon inverted microscope. To prevent cross-talk in dual labeling experiments, only one dye was excited at a time, keeping the other channel completely closed. The images were processed using Confocal Assistant followed by Adobe Photoshop version 5.0. FRET Analysis—COS-1 cells were plated on coverslips and transfected with Lipofectin as described above with expression vectors for the ECFP-ORF3 and EYFP-α1m fusion proteins. Forty-eight hours posttransfection, the coverslips were washed with PBS, fixed in 4% paraformaldehyde for 15 min at room temperature, and washed once again in PBS. These were then mounted using Antifade (Bio-Rad) and sealed with silicon sealant. A planapo 60× numerical aperture/1.4 oil immersion objective (Nikon, Japan) with a 2100 Radiance Unit confocal microscope (Bio-Rad) was used for all experiments. Confocal images were acquired sequentially using the 457-nm (ECFP) and the 514-nm (EYFP) laser lines of the argon laser. Images of the ECFP emission were collected using a 500 DCLPXR dichroic mirror with an HQ 485/30 emission filter. The EYFP emission images were collected using a 560 DCLPXR dichroic mirror with an HQ 545/40 emission filter. FRET was detected using the acceptor photobleaching approach as follows. Cells expressing the ECFP and EYFP fusion proteins were first imaged sequentially, followed by specific photobleaching of the acceptor fluorophore (EYFP) by 10–15 min of continuous illumination with the 514-nm laser line at 500 lines per second speed with a 80% laser intensity to ensure complete photobleaching of EYFP. At the end of 15 min, cells were imaged once again. Laser Pix 2000 software (Bio-Rad) was used for quantitating the mean fluorescence intensity of ECFP emission in areas of co-localization before and after photobleaching of EYFP. Change in mean fluorescence intensity before and after photobleaching of areas where the two proteins did not co-localize served as an internal control. The increase in ECFP emission, which is a direct measure of FRET efficiency, was calculated as E% = [1–(ECFP emission before EYFP photobleach/ECFP emission after EYFP photobleach)] × 100. For presentation, the original images were processed using Photoshop (Adobe Systems, Mountain View, CA). Screening of the Human Liver cDNA Library for Cellular Proteins That Interact with ORF3—Although the function of the ORF3 protein of HEV is yet unknown, preliminary indications clearly point to its involvement in various cellular pathways and functions (16Tyagi S. Jameel S. Lal S.K. J. Virol. 2001; 75: 2493-2498Crossref PubMed Scopus (36) Google Scholar, 17Aye T.T. Uchida T. Ma X.-Z. Iida F. Shikata T. Zhuang H. Win K.M. Nucleic Acids Res. 1992; 20: 3512Crossref PubMed Scopus (146) Google Scholar, 18Zafrullah M. Ozdener M.H. Panda S.K. Jameel S. J. Virol. 1997; 71: 9045-9053Crossref PubMed Google Scholar, 19Korkaya 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). To identify the cellular proteins that might interact with the ORF3 protein, we used the yeast two-hybrid system to screen a human liver cDNA library. Full-length ORF3, fused in-frame to the GAL4 binding domain in pAS2 vector, was used as "bait" to screen the cDNA library, which contained in-frame fusions with the GAL4 activation domain cloned into the pACT2 vector. The plasmids, containing the BD-ORF3 fusion (pAS2-ORF3) and the liver library activation domain fusion (pACT2-LL) DNA, were co-transformed into the yeast host strain (Y190) and selected for growth on SDLeu–Trp– plates for co-transformants. Colonies, thus obtained, were then tested for His+ prototrophy. Out of 6 × 104 Leu+Trp+ transformants, 1 × 102 transformants showed His+ β-gal+ phenotype. These interacting clones (His+ and β-gal+) were divided into four groups depending on their restriction analysis (data not shown). For this study, two clones from the group showing the strongest His+ and β-gal+ phenotype were selected. Activation domain plasmids from these two clones showed identical restriction digestion patterns (data not shown). Results of yeast two-hybrid studies on these two identical interacting clones are shown in Fig. 1A. All appropriate positive and negative controls were used as shown. Cells transformed with the vector alone did not activate the HIS3 gene. Similarly AD alone transformed with BD-ORF3 did not show growth on SDLeu–Trp–His– plates. This showed that the His+ phenotype and β-galactosidase+ phenotype was specific to the interacting proteins. Liquid β-galactosidase activity was determined for the positive clones along with all appropriate negative and positive controls using the substrate chlorophenol red β-d-galactopyranoside, as shown in Fig. 1B. The AD-LL interacting plasmid was isolated using standard yeast two-hybrid techniques. The interaction was verified using the genetic yeast two-hybrid approach (28Tyagi S. Lal S.K. Biochem. Biophys. Res. Commun. 2000; 277: 589-593Crossref PubMed Scopus (16) Google Scholar). Our genetic crosses of the haploid strains, containing singly transformed putative interacting fusion partners BD-ORF3 and AD-LL, resulted in diploids containing both interacting plasmids and showed positive His3 reporter gene activity (Fig. 1C). The interacting plasmid AD-LL was digested using restriction enzymes, and the insert size was estimated at 1.1 kb. This 1.1-kb insert was sequenced from one end, and the sequence obtained was subjected to the NCBI-BLAST search. This revealed a 100% homology with a gene encoding α1-microglobulin/bikunin precursor (AMB
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