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Originally published In Press as doi:10.1074/jbc.M402017200 on March 22, 2004

J. Biol. Chem., Vol. 279, Issue 28, 29308-29319, July 9, 2004
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The ORF3 Protein of Hepatitis E Virus Interacts with Liver-specific {alpha}1-Microglobulin and Its Precursor {alpha}1-Microglobulin/Bikunin Precursor (AMBP) and Expedites Their Export from the Hepatocyte*

Shweta Tyagi{ddagger}, Milan Surjit§, Anindita Kar Roy, Shahid Jameel, and Sunil K. Lal

From the Virology Group, International Centre for Genetic Engineering & Biotechnology, P. O. Box 10504, Aruna Asaf Ali Rd., New Delhi 110067, India

Received for publication, February 24, 2004 , and in revised form, March 22, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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, {alpha}1 -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 {alpha}1-microglobulin ({alpha}1m) and bikunin. {alpha}1 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 {alpha}1 m showed a distinct disappearance of the protein from the Golgi compartment, suggesting that ORF3 enhances the secretion of {alpha}1m out of the hepatocyte. Using drugs to block the secretory pathway, we showed that {alpha} m was not degraded in the presence of ORF3. Finally, 1pulse labeling of {alpha}1m 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 {alpha}1m export from the hepatocyte.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepatitis E virus (HEV)1 is a waterborne pathogen and is responsible for sporadic infections as well as large epidemics of acute viral hepatitis in developing countries (1–4). HEV is a plus-stranded RNA virus with a genome of ~7.2 kb, containing three open reading frames called ORF1, ORF2, and ORF3 (5–7). 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 (5, 7–9). 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 (10–14). 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 (15, 16). ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly, associates with the cytoskeleton and shows no major processing (17, 18). The ORF3 protein dimerizes using a 43-amino acid region, interacts with SH3 domains and activates cellular MAP kinase (19, 20). 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 (21). 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 (22–25), and for mapping specific domains involved in protein-protein interactions (20, 21, 26). 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 {alpha}1-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 {alpha}1-microglobulin ({alpha}1m) and bikunin, which are then secreted by the liver cells in free and bound forms. The processed protein, {alpha}1m, 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 {alpha}1m; however, at 46–48 h post-transfection, the perinuclear localization of {alpha}1m disappeared from the cells. Experiments on subcellular localization of {alpha}1m and using drugs that block the secretion pathway of {alpha}1m, we showed in transfected hepatocytes, an ORF3-dependent increase in the export of {alpha}1m from the Golgi compartment. Finally, using pulse-labeled {alpha}1m, we have conclusively proved this observation. The biological significance of this interaction and the possible role for the accelerated export of {alpha}1m in HEV-infected hepatocytes is discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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 (18) 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 (20). DNA manipulations were carried out as described by Sambrook, et al. (27). 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.


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  		TABLE I
Yeast strains, plasmids, and recombinant plasmid constructs used in this study

 
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 (20). 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 x 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 {beta}-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 {beta}-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-{beta}-D-galactopyranoside (X-gal) and 0.27% {beta}-mercaptoethanol. Filters were left for 48 h to develop a blue color, which indicated a positive protein-protein interaction. The liquid {beta}-galactosidase activity, a parameter directly reflecting the strength of protein-protein interactions, was determined using the substrate CPRG assay as described previously (16, 21). Relative {beta}-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 (28). 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 PJ69-4{alpha} haploid yeast strains (29). 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 {alpha}1m; 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 x 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 {alpha}1m, 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 {alpha}1m was detected by direct immunoprecipitation of the growth medium, and the intracellular {alpha}1m 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-{alpha}1m 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, {alpha}1m 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 60x 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-{alpha}1m 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 60x 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)] x 100. For presentation, the original images were processed using Photoshop (Adobe Systems, Mountain View, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (16–19). 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 x 104 Leu+Trp+ transformants, 1 x 102 transformants showed His+ {beta}-gal+ phenotype. These interacting clones (His+ and {beta}-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 {beta}-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 {beta}-galactosidase+ phenotype was specific to the interacting proteins. Liquid {beta}-galactosidase activity was determined for the positive clones along with all appropriate negative and positive controls using the substrate chlorophenol red {beta}-D-galactopyranoside, as shown in Fig. 1B.



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  		FIG. 1.
Yeast two-hybrid screening results. A, the ORF3 protein from HEV interacts with the human liver cDNA library gene product. All appropriate negative and positive controls are shown. Growth on YPD (nonselective) and selective media is shown. Trp– represents SDTrp– media, Leu– represents SDLeu– media, LeuTrp– represents SDLeu–Trp– media, His– represents SDLeu–Trp–His– media, and {beta}-Gal represents results from the {beta}-galactosidase filter assay. B, liquid {beta}-galactosidase ({beta}-gal) assay results. Single transformants and co-transformants were analyzed in a liquid {beta}-galactosidase assay and compared with each other. Values are given in arbitrary units. The numbers above each bar represent the mean from five independent transformants. Y190 corresponds to the untransformed host strain. Transformants with more than one plasmid are separated by a slash. C, genetic verification of the ORF3-LL (liver library) interaction. The haploid host cell is designated per its mating type, a or {alpha}. Diploid cells are designated a/{alpha}. Growth of colonies is shown on YPD (nonselective) and selective media, SDLeu–Trp–, and SDLeu–Trp–His– plates.

 
The AD-LL interacting plasmid was isolated using standard yeast two-hybrid techniques. The interaction was verified using the genetic yeast two-hybrid approach (28). 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 {alpha}1-microglobulin/bikunin precursor (AMBP; gene accession number: XO4494).

AMBP is made up of two unrelated plasma glycoproteins, {alpha}1-microglobulin ({alpha}1m) and bikunin. These are processed following intracellular proteolytic cleavage and are found in a free state as well as complexed with other plasma proteins (Fig. 2A). {alpha}1m is a member of the lipocalin superfamily and is postulated to have a role in immune regulation (30). Bikunin is a Kunitz-type serine protease inhibitor that provides the inter-{alpha}-inhibitor family members with their enzyme inhibitory capacity (31). Despite their lack of any structural or functional relationship, both proteins originate from a shared polypeptide; AMBP, which is encoded by a single copy gene. From rodents to primates, the AMBP gene is exclusively expressed in liver (31). AMBP produces a 1.2-kb mRNA, which is translated into a polyprotein with a 19-amino acid signal peptide. The AMBP cDNA isolated from the human liver library contained an N-terminal deletion of 32 amino acids that would contain the signal sequence comprising of the first 19 amino acids and the remaining 13 amino acids from {alpha}1m (Fig. 2B).



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  		FIG. 2.
The AMBP protein and its processing. A, processing of AMBP in the hepatocyte. AMBP mRNA is translated as a polyprotein with a 19-amino acid N-terminal signal sequence. This signal sequence is responsible for its translocation into the endoplasmic reticulum (ER). The polyprotein undergoes glycosylation and sulfation and gets cleaved into two mature proteins {alpha}1m and bikunin late in the trans-Golgi. Both processed proteins {alpha}1m and bikunin form higher molecular weight complexes with a number of glycoproteins and are eventually secreted from the hepatocyte. ss, signal sequence. B, schematic diagram of the N-terminal truncated AMBP protein isolated from the human liver cDNA library. R352 is an independently cloned full-length AMBP cDNA obtained from Dr. Jean-Philippe Salier. This full-length AMBP cDNA was used for all subsequent studies and for cloning {alpha}1m and bikunin. C, yeast two-hybrid analysis showing ORF3 protein interaction with the full-length, independently cloned AMBP protein (R352) and PCR cloned {alpha}1m. As a positive control, co-transformants showing positive ORF3-ORF3 interaction (57) were used.

 
Interaction of Full-length AMBP with ORF3—To validate our findings we obtained the full-length AMBP cDNA (referred to as R352) as a gift from Dr. Jean-Philippe Salier. R352 was cloned in-frame, into the yeast two-hybrid binding domain vector as an N-terminal fusion (Table I). This BD-R352 fusion protein was cotransformed along with AD-ORF3, into the Y190 yeast strain. Co-transformants were isolated and tested for His+ prototrophy and {beta}-galactosidase activity on filter and liquid assays (Fig. 2C). The full-length AMBP (BD-R352) clearly showed a positive interaction with AD-ORF3. The relative liquid {beta}-galactosidase units of the two clones, namely BD-R352/AD-ORF3 (1.26 units) and AD-AMBP/BD-ORF3 (1.19 units), were also comparable.

{alpha}1-Microglobulin Interacts with ORF3—AMBP is synthesized as a polyprotein and is cleaved post-translationally. {alpha}1m was individually tested for its interaction with ORF3. Yeast two-hybrid analysis showed that {alpha}1m interacted with ORF3 (Fig. 3). Compared with previously observed protein-protein interactions in our laboratory (16, 20, 21), this was a relatively weak interaction.



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  		FIG. 3.
In vitro interaction of ORF3 with AMBP and its processed proteins {alpha}1m and bikunin. A, non-radioactive His6-ORF3 was expressed in vitro and bound to Ni-NTA beads (represented by B). [35S]Methionine-radiolabeled AMBP, full-length AMBP (R352), and {alpha}1m were synthesized using an in vitro coupled transcription-translation system and tested for binding with immobilized ORF3 protein. As control experiment, these proteins were also bound to Ni-NTA beads alone. B, coimmunoprecipitation of {alpha}1m using ORF3 antibodies. COS-1 cells expressing ORF3 alone, {alpha}1m alone, and co-expressing {alpha}1 m and ORF3 were immunoprecipitated using anti-ORF3 antibodies. Lane 1 shows the vector-only control. ORF3 expression is clear in lanes 3 and 4.

 
To further confirm these interactions, we also used an in vitro approach. The full-length ORF3 gene was cloned with an N-terminal His6 tag into the pSGI vector (Table I). The His6-ORF3 protein was expressed using the rabbit reticulocyte lysate-coupled transcription-translation system as described previously (21). In separate reactions, [35S]methionine was used to radiolabel proteins expressed from pSG-AMBP, pSG-R352, and pSG-{alpha}1m (Table I). Unlabeled His6-ORF3 was bound to charged Ni-NTA beads, and these beads were then used to trap 35S-labeled AMBP, R352, or {alpha}1m. In separate control experiments, approximately equal amounts of each 35S-labeled protein was added to tubes with charged Ni-NTA beads in the absence of ORF3. After 4 h of gentle shaking at 4 °C, the beads were washed to remove all unbound protein, and the bound proteins were analyzed by SDS-PAGE and autoradiography. The results of this experiment are shown in Fig. 3A. The control experiments showed clearly that none of the proteins bound to Ni-NTA beads in the absence of ORF3 (Fig. 3A, lanes 1–3). AMBP, R352, and {alpha}1m (Fig. 3A, lanes 5–7) clearly showed good binding to ORF3 in vitro.

We further studied the {alpha}1m-ORF3 interaction in animal cells. Mammalian COS-1 cells were transiently transfected with pMT-ORF3 alone or together with pMT-{alpha}1m (Table I). [35S]Methionine-labeled cell lysates were immunoprecipitated using anti-ORF3 antibodies.

When {alpha}1-microglobulin was co-transfected with ORF3 in COS-1 cells and immunoprecipitated using anti-ORF3 antibodies, this protein was detected in the autoradiogram (Fig. 3B, lane 4). Appropriate single transfection controls did not show immunoprecipitation of {alpha}1m with the anti-ORF3 antibodies (Fig. 3B, lanes 1–3).

Co-localization of {alpha}1-Microglobulin with ORF3—To understand the subcellular localization of {alpha}1m, we used Huh7 hepatoma cells, which endogenously express {alpha}1m. These cells were subjected to immunofluorescent staining followed by fluorescence microscopy. For studying co-localization of ORF3 with {alpha}1m, dual labeling experiments were carried out in which Huh7 cells were transiently transfected with pMT-ORF3. The distribution of ORF3, as observed previously (19, 21), was cytoplasmic and displayed punctate staining (Fig. 4, B and E). The endogenously expressed {alpha}1m protein was cytoplasmic in distribution, with distinct perinuclear staining, possibly in the ER (Fig. 4, A and D). In dual labeling experiments, distinct orange to yellow stained regions were observed in merged images indicating co-localization of ORF3 with {alpha}1m. These yellow regions were primarily perinuclear (Fig. 4, C and F).



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  		FIG. 4.
Co-localization of {alpha}1m with ORF3 in liver cells. Huh7 cells transiently transfected with pMT-ORF3 were doubly labeled with polyclonal anti-{alpha}1m (A–F) and monoclonal anti-ORF3 followed by the Alexa 488- or Alexa 594-conjugated anti-rabbit IgG or anti-mouse IgG antibodies, respectively. Separate images were acquired showing {alpha}1m distribution (A and D) and ORF3 distribution (B and E). Co-localizations are shown in yellow in the MERGE panels (C and F).

 
FRET Measurements of the ORF3-{alpha}1m Interaction—We used fluorescence resonance energy transfer (FRET) to detect the ORF3-{alpha}1m interaction in vivo and to complement the results of in vitro interaction assays. This non-radiative energy transfer follows stringent conditions of distance and dipole orientations between the donor and acceptor fluorophores. Proteins fused to the cyan (ECFP) and yellow (EYFP) colored variants of the enhanced green fluorescent protein (EGFP) were used as the donor-acceptor FRET pair (30). The efficiency of FRET was measured following cotransfection of COS-1 cells as described under "Experimental Procedures." To make measurements independent of the expression levels of the two fusion proteins, we followed an acceptor photobleach protocol and recorded the mean fluorescence intensity from the donor fluorophore (ECFP) before and after photobleaching of the acceptor fluorophore (EYFP) (32–35). Three areas within the same cell were recorded, two regions where both proteins co-localized (A and B) and a third region where no co-localization was observed (C) (Fig. 5). An average percent FRET efficiency of ~12.18 was found in the regions of co-localization as opposed to ~3.76 in the region where the two proteins do not co-localize. The difference in pixel intensity of ECFP before and after photobleaching of EYFP in the areas of co-localization and the region where the two proteins do not co-localize when compared for 16 independent observations showed high levels of significance with a p value of 4.52 x 10–8. Because FRET efficiency between two molecules decreases by the sixth power of the distance between them (33), the presence of FRET indicates an actual protein-protein interaction. Simple co-localization of two proteins is not sufficient to yield energy transfer, and FRET represents a powerful indicator of in vivo protein-protein interactions. The ECFP-EYFP pair is a commonly used donor/acceptor pair, ideally suited for FRET measurements. FRET exploits the ability of the higher energy donor fluorophore (ECFP) to transfer some of its energy to the acceptor fluorophore (EYFP) in its excited state under optimum conditions of spectral overlap and Forsters radius (33). Following FRET, there is a reduction in the donor emission due to the transfer of energy and an increase in the acceptor emission. We show here weak, but significant FRET efficiency between ECFP-ORF3 and EYFP-{alpha}1m, supporting the in vitro and yeast two-hybrid results that the ORF3 protein and {alpha}1m interact with each other.



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  		FIG. 5.
FRET analysis of the ORF3-{alpha}1m interaction. COS-1 cells were co-transfected with ECFP-ORF3 and EYFP-{alpha}1 m fusion proteins and imaged for ECFP (green pseudo color) or EYFP (red pseudo color). The ECFP images before and after EYFP photobleaching are shown. Histograms of the mean fluorescence intensity (MFI) of ECFP in the areas of co-localization (A and B) and in the region where the two proteins do not co-localize (C) are shown, either before or after photobleaching of EYFP. Mean fluorescence intensities (MFI) were determined as described under "Experimental Procedures." Representative images are shown from a total of 16 cells imaged over two separate experiments.

 
Hepatocytes Expressing ORF3 Show Leaching of {alpha}1m— ORF3-expressing liver cells, when stained with anti-{alpha}1m antibodies, showed absence of the distinct perinuclear staining, which was observed in cells not expressing ORF3. The number of cells showing this phenomenon increased with time as observed at 40, 44, and 48 h post-transfection. These observations gave preliminary indications that the intracellular processing and export of processed {alpha}1m increased in the presence of the ORF3 protein within the hepatocyte. Fig. 6 shows a representative field at the 48-h time point. Panel A of Fig. 6 shows expression of the fusion cyan fluorescent protein-ORF3 localizing in the cytoplasm. The same field was observed under the red filter to check the level of {alpha}1m. ORF3-expressing cells (large arrows) and cells not expressing ORF3 (small arrows) can be visualized in the same field in Fig. 6B. In this experiment the perinuclear localization of {alpha}1m can be clearly visualized in non-ORF3-expressing cells (36, 37). This distinct localization of {alpha}1m is completely absent in ORF3-expressing cells. To show that this effect was specific to {alpha}1m, we measured the extracellular levels of a completely unrelated secretory protein, laminin, in ORF3-expressing cells and compared it to non-ORF3-expressing cells. There was no difference in the level of laminin in ORF3-expressing and mock treated cells (data not shown). This data was statistically quantified by visualizing a total of 200 ORF3-expressing cells and scoring them for presence or absence of {alpha}1m (Table II). Only cells showing complete disappearance of {alpha}1m were scored positive. At 40 h post-transfection, only 11/200 cells expressing ORF3 showed this phenomenon, whereas all other cells expressing ORF3 displayed co-localization of ORF3 and {alpha}1m. At 44 h it increased to 60/200, and at 48 h it rose further to 100/200 cells exhibiting complete clearing of {alpha}1m. To establish that the {alpha}1m leaching effect was specific to the presence of ORF3, we used EGFP and ORF2 (capsid protein of HEV) in two independent sets of experiments and checked leaching of {alpha}1m at 40, 44, and 48 h post-transfection. The presence of neither of these proteins in hepatocytes could produce {alpha}1m leaching, which was specific only to ORF3.



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  		FIG. 6.
Shift in distribution of {alpha}1m in liver cells coexpressing the ORF3 protein. A, Huh7 cells were transiently transfected with the CFP-ORF3 construct; B, the same field was analyzed for fluorescence using {alpha}1m antibody bound to Alexa 594. The observations were taken at 48 h post-transfection. {alpha}1m distribution in cells expressing CFP-ORF3 (large arrows) can be easily compared with untransfected cells (small arrows) in the same microscopic field.

 


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  		TABLE II
Statistical analysis of ORF3-mediated leaching of {alpha}1m

Co-transfected cells were stained with Alexa 594 ({alpha}1m-stained), and the levels of {alpha}1m were detected by fluorescence microscopy. A total of 200 ORF3-transfected cells were counted at respective time points and scored for {alpha}1m leaching. Only cells showing clear disappearance of {alpha}1m were counted as positives. Data shown are ± S.E. from three independent sets of experiments.

 
Furthermore, experiments were designed to detect the cellular compartments where ORF3 exerted its effect on {alpha}1m, we designed an immunofluorescence assay with different organelle markers to check the status of {alpha}1m in various subcellular components in the presence or absence of the ORF3 protein.

ORF3 Enhances Clearance of {alpha}1m from the Golgi Apparatus—The CFP-ORF3 plasmid construct was transfected, along with mitochondria, ER, and Golgi-specific fluorescent markers. {alpha}1m localization was visualized in the mitochondria, ER, and Golgi of hepatocytes expressing ORF3 and compared with mock transfected cells that were not expressing ORF3. {alpha}1m was detected by staining with Alexa 488 (green) or Alexa 594 (red) depending on the complementary color used in the experiment. All observations were made at 48 h post-transfection (Fig. 7). Mitochondrial marker (Mito Ds-Red) was used to visualize mitochondria. The {alpha}1m localization in the mitochondria and the effect of CFP-ORF3 were studied (Fig. 7A, panels a –f). Although minute amounts of {alpha}1m were detected in the mitochondria, ORF3 expression showed no effect on its localization. Interestingly, ORF3 expression had no effect on {alpha}1m levels in the ER either (Fig. 7A, panels g–l). However, ORF3 expression resulted in the complete disappearance of {alpha}1m from the Golgi region (Fig. 7A, panels m–r). Moreover, ORF3 itself was found to be concentrated in the Golgi. 200 ORF3-transfected cells from each set of experiments were counted and scored for {alpha}1m status. Cells showing complete disappearance of {alpha}1m were scored positive. Data obtained from this analysis are summarized in Table III. To confirm the observation that ORF3 enhances {alpha}1m transport from the Golgi compartment, we used Brefeldin A and monensin, two commonly used inhibitors of the secretory pathway. Brefeldin A specifically inhibits ER to Golgi transport by binding to ADP ribosylation factor guanine nucleotide exchange factor (38). Monensin blocks protein transport beyond the trans-Golgi vesicle, resulting in intracellular accumulation of protein (39). Fig. 7B shows the effects of Brefeldin A and monensin on {alpha}1m leaching in the presence and absence of ORF3. 47 h post-transfection with ORF3, cells were treated with either of the two drugs for 1 h before fixing the cells for fluorescence microscopy. 500 ORF3-transfected cells were examined for the presence of {alpha}1m within the cells. As expected, ORF3-mediated leaching of {alpha}1m was blocked by Brefeldin A, and {alpha}1m accumulated within the cell (Fig. 7B, panel b). Corresponding untransfected cells stained with {alpha}1m are visible in panel a. Monensin also produced similar effects as Brefeldin A (Fig. 7B, panel d). Corresponding untransfected cells stained with {alpha}1m are visible in panel c. These experiments clearly proved that the disappearance of {alpha}1m from within the hepatocytes was due to ORF3-mediated transport through the Golgi compartment and was not due to {alpha}1m degradation in the presence of ORF3.



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  		FIG. 7.
ORF3 expression in liver cells results in enhanced processing and export of {alpha}1m. A, subcellular localization of {alpha}1-microglobulin. Huh7 cells were transformed with different organelle markers alone or along with CFP-ORF3, fixed, and stained with anti-{alpha}1-m and visualized at 60x magnification in a confocal laser scanning microscope as described under "Experimental Procedures." Panels a and d show localization of Ds-Red in mitochondria. The same field was viewed in the green filter to observe localization of {alpha}1m(panels b and e). Panel c shows the expression of CFP-ORF3 in the same field. Panel f shows the superimposition of panel d over panel e in non-ORF3-expressing cells. Panels h and k show localization of Ds-Red in ER. The same field was visualized in green filter for {alpha}1m localization (panels g and j). Panel i shows expression of CFP-ORF3, and panel l shows a superimposition of j over k. Panels m and p show YFP expression in Golgi. The same field was visualized using a red filter for {alpha}1 m localization (panels n and q). Panel o shows expression of CFP-ORF3. Panel r shows superimposition of panel p over q. B, Huh7 cells transfected with pSGI-ORF3 were treated with 10 µg/ml Brefeldin A or 5 µM monensin 1 h before fixation. Cells were stained with Alexa 488 (green) for {alpha}1m and Alexa594 (red) for ORF3. Panel a shows {alpha}1m staining in BFA treated cells. The small arrow corresponds to non-transfected cells; the large arrow corresponds to ORF3-expressing cells. Panel b shows ORF3-expressing cells in the same field. Panel c shows {alpha}1m expression in monensin-treated cells. Panel d shows ORF3-expressing cells in the same field.

 


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  		TABLE III
No. of cells leaching {alpha}1m from secretory organelles

Liver cells were transfected with Ds-Red ER, YFP Golgi, or Mito Ds-Red alone (mock) or along with CFP-ORF3, stained with anti-{alpha}1m antibody followed by Alexa 488 (for ER and mitochondria) or Alexa 594 (for Golgi). Levels of {alpha}1m were visualized by fluorescence microscopy. Data shown are ± S.E. from three independent sets of experiments.

 
Increased Secretion of {alpha}1m from Hepatocytes Expressing ORF3—Finally, we proposed to study the levels of secreted {alpha}1m from liver cells expressing ORF3. This experiment was conducted by metabolically labeling cells with 35S Promix as described under "Materials and Methods." Growth medium was collected at 40, 44, and 48 h post-transfection and directly subjected to immunoprecipitation using {alpha}1m antibody (Fig. 8A). Significant differences were observed in the {alpha}1m levels, in growth medium between mock-transfected cells (lanes 1, 3, and 5) and transfected cells expressing ORF3 (lanes 2, 4, and 6) at 40, 44, and 48 h, respectively. Densitometric units showing secreted {alpha}1m for all three time-points were significantly different (p < 0.05) between growth media of mock transfected and ORF3-expressing cells, as shown in the bar graph. The lower panel shows corresponding cell lysates immunoprecipitated using anti-ORF3 antibody. To establish the specificity of the enhanced secretion observed in this experiment, we checked secreted levels of {alpha}1m in the presence of EGFP and ORF2, in two separate sets of experiments (Fig. 8B). Densitometric levels were found to be unchanged in both EGFP- and ORF2-expressing cells. As a control in this experiment, we checked the level of secreted laminin in mock transfected or ORF3-transfected cells. As expected, there was no significant difference in the level of laminin in presence of ORF3, supporting our microscopic observations (data not shown).



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  		FIG. 8.
Metabolic labeling and pulse-chase results showing increased secretion of {alpha}1m from hepatocytes expressing ORF3. A, immunoprecipitation of {alpha}1m and ORF3 from growth media at 40 (lanes 1 and 2), 44 (lane 3 and 4), and 48 (lane 5 and 6) h post-transfection. The top panel shows immunoprecipitation of {alpha}1m; the lower panel shows immunoprecipitation of ORF3 protein. Control represents liver cells transfected with empty vector. The graph shows the ± S.E. of densitometry units for {alpha}1m protein levels as examined in three independent sets of experiments. Level of significance was calculated using a standard t test; p values between lanes 1 and 2 was ≤ 0.0012, lanes 3 and 4 was ≤ 0.0033, and lanes 5 and 6 was ≤ 0.0026. B, ORF3 specifically expedites the secretion of {alpha}1 m. Huh7 cells were transfected with pSGI EGFP (lane 1), pSGI ORF3 (lane 2), and pSGI ORF2 (lane 3), and growth media were immunoprecipitated using anti-{alpha}1 m antibody, resolved by 12% SDS-PAGE and detected by fluorography as described under "Experimental Procedures." C, pulse-labeled {alpha}1m being detected at different time intervals of chase (5, 10, 20, and 30 min post-labeling) in control (pSGI transfected) and ORF3 (pSGI ORF3 transfected)-expressing cells. The graph shows densitometric values of corresponding bands on the gels below it. The dotted line represents {alpha}1 m secretion for ORF3-expressing cells, whereas the solid line represents mock transfected cells. D, immunoprecipitation of {alpha}1m from cell lysates (lanes 1–3) and medium (lanes 4 and 5), in the presence and absence of monensin. Faster migration of {alpha}1m in the presence of monensin ({alpha}1m') is probably due to inhibition of Golgi-specific oligosaccharide modification. The image is a representative of three independent sets of experiments. The bottom panel shows the mean densitometry units of {alpha}1 m protein level. p value of lanes 1–3 was ≤0.128.

 
To further confirm that ORF3 indeed expedites the transport of {alpha}1m from hepatocytes, we conducted a pulse-chase assay to study the kinetics of {alpha}1m secretion in the presence and absence of ORF3. Samples of the growth media were analyzed for {alpha}1m at different time points using antibodies against it (Fig. 8C). Data from this experiment showed that pulse-labeled {alpha}1m from ORF3-expressing hepatocytes reached detectable levels in the growth media at 20 min post-chase, whereas {alpha}1m was undetectable at the same time point for mock transfected cells. Interestingly, only the levels of free {alpha}1m were found to increase. The level of {alpha}1m complexes remained unaltered as is visible in the higher molecular weight bands of relatively weaker intensities. Finally, to rule out the possibility of up-regulation of endogenous {alpha}1m expression levels in the presence of ORF3, we conducted the following set of experiments in which we examined the lysate and growth media of mock-transfected and ORF3-expressing liver cells. Both sets of transfected cells were treated with monensin, thus blocking the secretion of all expressed proteins from the cell (Fig. 8C). Results obtained from this experiment showed clearly that mock transfected cells without monensin showed good amounts (195 densitometric units) of {alpha}1m in the media, whereas mock transfected cells with monensin showed very poor (55 densitometric units) levels of secreted {alpha}1m in the media (lane 5), thus proving that the {alpha}1m secretion was completely blocked by monensin. Under the same conditions, we could not detect any difference in the intracellular levels of {alpha}1m between mock transfected (lane 2; 92 densitometric units) and ORF3-transfected cells (lane 3, 91 densitometric units), which proved that ORF3 did not up-regulate the expression of endogenous {alpha}1m. The accelerated migration of {alpha}1m on the SDS-PAGE gel (lanes 2 and 3) in the presence of monensin is most likely due to the inhibition of Golgi-specific oligosaccharide processing by monensin. Thus from all the experiments described in these results, we conclude that the ORF3 protein interacts with {alpha}1m and enhances its export from the hepatocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
HEV cannot be cultured routinely, although it has recently been propagated in primary macaque hepatocytes (6, 40) and a virus resembling HEV has been cultured in A549 cells (41). Studies on HEV protein synthesis, processing, and assembly have hence been limited to heterologous gene expression systems. The ORF3 gene of HEV has been cloned and expressed in bacterial, animal cell, baculoviral, and yeast expression systems (8, 9, 13–15), however no definite function has been assigned to it as yet. The ORF3 protein is ~13.5 kDa (123 amino acid), associates with the cytoskeleton, is a substrate for MAP kinase, self-associates to form dimers, binds various SH3 domain-containing proteins, and interacts with the major structural protein, ORF2 (18, 20, 21). It also activates cellular MAP kinase (19). These observations clearly indicate that the ORF3 protein must have multifarious activities that involve its interactions with its host cell proteins. With this idea in mind, we decided to use the yeast two-hybrid system to isolate and characterize host interacting partners for this protein. Because HEV primarily infects hepatocytes, a human liver cDNA library was screened for cellular partners.

A total of 6 x 104 Leu+Trp+ transformants was screened from which 100 co-transformants showed positive protein-protein interactions. The positive clones were divided into four groups, the strongest and most recurring AD-plasmid was isolated, sequenced, and analyzed using NCBI-BLAST (www.ncbi.nlm.nih.gov/BLAST/). This analysis revealed that the ORF3 protein of HEV interacts with the human {alpha}1-microglobulin/bikunin precursor (AMBP) protein.

AMBP is a liver-specific protein that is processed to give two mature proteins, {alpha}1-microglobulin ({alpha}1m) and bikunin. The AMBP gene has been cloned from various sources, including human (42), mouse (43), cattle (44), and swine (45). The human AMBP gene consists of 10 exons, which span 1.3 kb and 9 introns with an aggregated length of about 16.5 kb and is under the control of a potent and liver-specific enhancer (46). AMBP can dimerize (47) and is cleaved at a tripeptide bridge in the trans-Golgi compartment to yield the two unrelated processed proteins, {alpha}1m and bikunin. Moreover, it has been shown in different expression systems that both {alpha}1m and bikunin can be expressed alone (6, 33, 36, 48).

{alpha}1m is a member of the lipocalin superfamily (30), which is made up of nearly 20 different proteins. Although members of this family display low sequence similarity, all share a conserved folding pattern. {alpha}1m is a glycoprotein found predominantly in free state in plasma or a variety of other plasma proteins (49).

Liver, blood plasma, and kidney are the major sites for {alpha}1m localization. Soon after cleavage, {alpha}1m is released from the hepatocyte. The protein exists in free form as well as in a variety of high molecular weight complexes in serum where its concentration remains unchanged (50). Besides a one-to-one complex with monomeric IgA, {alpha}1m is also linked to albumin and prothrombin (49). In rat serum, it forms complexes with fibronectin (51) and proteinase inhibitor-{alpha}1-inhibitor-3, a homologue of human {alpha}2-macroglobulin (52). Although the biological function of {alpha}1m is not known, it has been shown to possess a number of immunoregulatory properties in vitro. It inhibits the antigen-induced proliferation of peripheral lymphocytes, interleukin-2 production of T-cells, and the migration and chemotaxis of granulocytes (47, 53, 54). These properties of {alpha}1m suggest that it has a general immunosuppressive role.

Our studies show that the ORF3 protein of HEV interacts with AMBP and its processed protein, {alpha}1m. The ORF3-{alpha}1m interaction was found to be weak; nonetheless it was detectable in all the yeast two-hybrid assays, FRET analysis, immunoprecipitations, and in vitro transcription-translation binding assays.

Human hepatoma Huh7 cells expressing AMBP endogenously when stained with anti-{alpha}1m antibodies showed perinuclear staining characteristic of proteins translocating to the ER. AMBP is processed in the trans-Golgi to {alpha}1m, which is in turn transported in a free or bound form, out of the hepatocyte. Using dual staining immunofluorescent microscopy of liver cells, endogenously expressing AMBP, {alpha}1m was shown to co-localize with the ORF3 protein. ORF3 showed punctate staining in the cytoplasm, however on dual staining with anti-{alpha}1m and anti-ORF3 antibodies, co-localization was evident in the perinuclear region. Furthermore, we observed a distinct difference in the cellular localization patterns of {alpha}1m in hepatocytes expressing ORF3, 44 and 48 h post-transfection. An increasing number of transfected hepatocytes showed an absence of distinct perinuclear {alpha}1m staining observed in hepatocytes expressing ORF3. Transfected cells expressing ORF3 showed co-localization with {alpha}1m at 40 h post-transfection; however, at 44 h and further at 48 h post-transfection, increasing numbers of hepatocytes expressing the ORF3 protein showed complete disappearance of {alpha}1m from within the cell. This phenomenon of a "shift" from perinuclear localization to a complete absence of detectable {alpha}1m within the liver cell, steadily increased with increasing post-transfection time. We then further studied in more detail the intracellular secretory pathway through which {alpha}1m gets cleared in ORF3-expressing cells. Experiments using organelle-specific markers clearly proved that {alpha}1m disappearance occurs from the Golgi compartment and that ORF3 expression had no effect on the levels of {alpha}1m inside ER.

Furthermore, experiments using monensin and Brefeldin A, two drugs that disrupt the protein transport machinery within the cell by disrupting ER to Golgi membrane traffic and impairing post-Golgi trafficking, respectively, showed that ORF3-mediated {alpha}1m disappearance was sensitive to both the drugs. There was intracellular accumulation of {alpha}1m due to a block in intracellular traffic. This increase in {alpha}1m within the hepatocyte is evident by a shift from the predominantly perinuclear localization of {alpha}1m to a more diffuse cytoplasmic accumulation of the protein, as observed. Moreover, this experiment also proved that ORF3-mediated disappearance of {alpha}1m with time was not due to the degradation of the latter. Finally by metabolic labeling and pulse-chase experiments, we were able to clearly establish that the ORF3 protein enhances the export of {alpha}1m out of the hepatocyte.

Inhibitory effects of {alpha}1m on the immune system are well studied. {alpha}1m suppresses antigen-induced polyclonal proliferation of cultured lymphocytes (55). It also inhibits spontaneous migration of neutrophil granulocytes in vitro and suppresses the chemotactic attraction of granulocytes by a cytokine concentration gradient released by triggered monocytes, macrophages and B- and T-lymphocytes (56). It is also known that infiltrating lymphocytes in the HEV-infected liver have cytotoxic or suppression immunophenotype (57). Although the plasma levels of {alpha}1m do not change during inflammation, the local effects exerted by them have been well documented (58–62). Our studies showing enhanced export of the {alpha}1m protein in the presence of a viral protein (ORF3) support the hypothesis of an immunosuppressive environment being created around the infected liver cells by virtue of increased localized secretion of {alpha}1m.


   FOOTNOTES
 
* This work was supported in part by internal funds from the International Centre for Genetic Engineering & Biotechnology (ICGEB) and the Wellcome Trust. The Confocal Microscopy Facility at ICGEB is funded through an International Senior Research Fellowship of the Wellcome Trust (to S. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Senior Research Fellow of the University Grants Commission. Back

§ Senior Research Fellow of the Council of Scientific and Industrial Research, India. Back

To whom correspondence should be addressed. Tel.: 91-11-2619-5007; Fax: 91-11-2616-2316; E-mail: sunillal{at}icgeb.res.in.

1 The abbreviations used are: HEV, hepatitis E virus; AMBP, {alpha}1-microglobulin/bikunin precursor; {alpha}1 m, {alpha}1 -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. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge the generous gifts of the yeast two-hybrid vectors and strains from Stephen Elledge and of PJ69-4a and PJ69-4{alpha} strains from Philip James, the R352 gene and antibodies against {alpha}1m from Jean-Philippe Salier. The laboratory assistance of Ravinder Kumar and Purnima Kumar and help with microscopy provided by Preeti Malik and Chetan Chitnis are gratefully acknowledged.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bradley, D. W. (1990) Br. Med. Bull. 46, 442–461[Abstract/Free Full Text]
  2. Emerson, S. U., and Purcell, R. H. (2001) Trends Mol. Med. 7, 462–466[CrossRef][Medline] [Order article via Infotrieve]