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J. Biol. Chem., Vol. 277, Issue 25, 22759-22767, June 21, 2002

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The Phosphorylated Form of the ORF3 Protein of Hepatitis E Virus Interacts with Its Non-glycosylated Form of the Major Capsid Protein, ORF2^*

Shweta Tyagi, Hasan Korkaya, Mohammad Zafrullah, Shahid Jameel, and Sunil K. Lal^§

From the Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 1100067, India

Received for publication, January 8, 2002, and in revised form, April 2, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hepatitis E virus (HEV) is a human RNA virus containing three open reading frames. Of these, ORF1 encodes the viral nonstructural polyprotein; ORF2 encodes the major capsid protein, which exists in a glycosylated and non-glycosylated form; and ORF3 codes for a phosphoprotein of undefined function. Using fluorescence-based colocalization, yeast two-hybrid experiments, transiently transfected COS-1 cell co-immunoprecipitation, and cell-free coupled transcription-translation techniques, we have shown that the ORF3 protein interacts with the ORF2 protein. The domains involved in this ORF2-ORF3 association have been identified and mapped. Our deletion analysis showed that a 25-amino acid region (residues 57-81) of the ORF3 protein is required for this interaction. Using a Mexican HEV isolate, site-directed mutagenesis of ORF3, and a phosphatase digestion assay, we showed that the ORF2-ORF3 interaction is dependent upon the phosphorylation at Ser⁸⁰ of ORF3. Finally, using COS-1 cell immunoprecipitation experiments, we found that the phosphorylated ORF3 protein preferentially interacts with the non-glycosylated ORF2 protein. These findings were confirmed using tunicamycin inhibition, point mutants, and deletion mutants expressing only non-glycosylated ORF2. ORF3 maps in the structural region of the HEV genome and now interacts with the major capsid protein, ORF2, in a post-translational modification-dependent manner. Such an interaction of ORF2 with ORF3 suggests a possible well regulated role for ORF3 in HEV structural assembly.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Hepatitis E is an acute disease endemic in many countries throughout developing parts of the world, in particular on the continents of Africa and Asia, where it causes epidemics and sporadic infections. The causative agent, hepatitis E virus (HEV),¹ is transmitted via the fecal-oral route, predominantly through contaminated water (1). HEV is an RNA virus with a positive-sense genome ~7.2 kb in length with three open reading frames (ORF1, ORF2, and ORF3) encoding three different proteins (2-4). ORF1 (5079 bp) is at the 5'-end of the genome and is predicted to code for putative nonstructural proteins with sequences homologous to those encoding viral methyltransferases, proteases, helicases, and RNA-dependent RNA polymerases (3-6). 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, and yeast and in vitro in a coupled transcription-translation system (7-11). ORF2 encodes the major HEV structural protein, which has been shown to be an 88-kDa glycoprotein that is expressed intracellularly as well as on the cell surface. It is synthesized as a precursor and is processed through signal sequence cleavage into the mature protein, which is capable of self-association (12, 13). When expressed through the baculoviral expression system, ORF2 was shown to assemble into virus-like particles (VLPs), which were cell-associated as well as secreted in the culture medium (14, 15).

ORF3 encodes a small 13.5-kDa phosphoprotein that is expressed intracellularly and shows no major processing. It associates with the cytoskeletal and membrane fractions of cells (16, 17). Recently, the ORF3 protein has been shown to dimerize in a yeast cellular environment, and its dimerization domain has been mapped to a 43-amino acid region overlapping the SH3 binding and phosphorylation domains (13). Furthermore, ORF3 has been recently shown to interact with SH3 domains and to activate MAPK (18).

Heterotypic interactions of the HEV proteins have not been studied as yet. Although ORF3 is located in the 3'-third of the genome and has been termed a structural protein, there is no evidence to date for its involvement in HEV structural assembly. Because ORF2 is the major capsid protein, we undertook studies to examine colocalization of ORF2 and ORF3 in transfected cells and to test for heterotypic interactions between these two viral proteins to evaluate a structural role for ORF3.

In the few years since 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 (19-21), and for mapping specific domains involved in protein-protein interactions (22-24). In this system, two plasmid-borne gene fusions are cotransformed 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.

We have used the yeast two-hybrid system along with fluorescence-based colocalization experiments, transiently transfected COS-1 cell immunoprecipitation, and coupled transcription-translation techniques to show the interaction of ORF3 with ORF2. We have further mapped the interaction domain of ORF3 to a 25-amino acid region. Within this region, we have shown that a single amino acid (Ser⁸⁰) is responsible for this heterotypic protein-protein interaction. Ser⁸⁰ has been shown to be the site for phosphorylation of ORF3. Our yeast two-hybrid analysis using a Mexican HEV isolate, site-directed mutagenesis, and a phosphatase digestion assay revealed that the ORF2-ORF3 interaction is phosphorylation-dependent. Finally, using immunoprecipitation experiments, followed by tunicamycin inhibition, point mutations, and deletion mutations expressing only the non-glycosylated form of ORF2, we have shown that phosphorylated ORF3 preferentially interacts with non-glycosylated ORF2. A possible role of ORF3 in a post-translational modification-dependent interaction with ORF2 is discussed in light of our results.

	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 ORF2 and ORF3 genes of HEV were excised from the pMT-ORF2 and pSG-ORF3 vectors (9, 25), respectively, and cloned into the yeast two-hybrid vectors, resulting in an N-terminal in-frame fusion with either the GAL4 DNA-binding or activation domain. DNA manipulations were carried out as described by Sambrook et al. (26). All deletion constructs were generated by subcloning the full-length ORF2 and ORF3 genes of HEV and are described in Table I. Plasmid constructs not containing fully compatible ends were screened for the correct reading frame by sequencing, whereas all other constructs with fully compatible ends were verified by restriction digestion and sequencing.

Immunofluorescence Analysis-- COS-1 cells were plated at a confluency of ~50% on coverslips 1 day before transfection and grown for 18 h. 40 h post-transfection, phosphate-buffered saline (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 and 0.5% Tween 20 (PBST) containing 1% normal goat serum for 2 h at room temperature. The primary antibodies used were mouse monoclonal anti-ORF3 antibody (1:200 to 1:500 dilution) and rabbit polyclonal anti-ORF2 antibody (1:100 to 1:200 dilution). Cells were washed three times with PBST for 5 min each and then incubated for 1 h at room temperature with a 1:1000 dilution of conjugated secondary antibodies. For colocalization experiments, the secondary antibodies used were goat anti-mouse IgG coupled to Alexa 488 dye and goat anti-rabbit IgG coupled to Alexa 594 dye (Molecular Probes, Inc., Eugene, OR). These were chosen to label the ORF3 protein with Alexa 488 (green) and the ORF2 protein with Alexa 594 (red). Cells were washed as described above and mounted in 90% glycerol in PBS. Fluorescence images were collected using a 60× or 100× 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.

Yeast Two-hybrid Techniques-- The GAL4-based two-hybrid system (kindly provided by Dr. Stephen Elledge) contained pAS2 (DNA-binding domain vector) and pACT2 (activation domain vector), together with yeast reporter Saccharomyces cerevisiae strain Y190 (see Table I). The host strain containing pAS2-SNF1 and pACT2-SNF4 was used as a positive control (27). The Y190 host contains integrated copies of both HIS3 and lacZ reporter genes under the control of GAL4-binding sites. The Y190 yeast strain was transformed with the appropriate plasmids using the lithium acetate procedure and grown on synthetic dextrose (SD) plates in the absence of Trp and Leu (SDTrp and SDLeu, respectively). Protein interaction was tested on SD plates without Leu, Trp, and His (SDLeuTrpHis). After 3 days at 30 °C, individual colonies were streaked out and tested by liquid and filter-lift -galactosidase assays, 3-amino-1,2,3-triazole (3-AT) assay (50 mM), and diploid His assay. The filter -galactosidase assay, a parameter directly reflecting the strength of protein-protein interactions, was performed by streaking doubly transformed yeast colonies onto filter paper and allowing them to grow for 2 days on selection medium. Yeast cells were permeabilized by freezing yeast-impregnated filters in liquid nitrogen and thawing at room temperature. This filter was placed over a second filter that was presoaked in 0.1 M phosphate buffer (pH 7.0) containing 300 mg/ml X-gal and 0.27% -mercaptoethanol. Filters were left for 48 h to develop a blue color, which indicated a positive protein-protein interaction. Liquid -galactosidase activity was determined using the substrate X-gal as described previously (28, 29). Relative enzymatic activity was determined in five independent transformants. Data 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 Y190 host strain containing pAS2-SNF1 and pACT2-SNF4 was used as a positive control (27). The specificity of the in vivo protein-protein interaction was confirmed using a yeast genetic assay for reconfirming positive two-hybrid interactions (30, 31). Plasmid constructs were extracted from the positive Y190 cotransformants (BD-ORF3/AD-ORF2, clones 1 and 2). The plasmids isolated from these clones were separated and verified using E. coli HB101 cells on M9 synthetic medium lacking Leu. Subsequently, these plasmids were singly transformed into the PJ69-4a and PJ69-4 haploid yeast strains (32). After genetic crossing, the His3 prototrophy of the diploid strains was tested by plating for growth on SD plates in the absence of His (SDHis). All possible control transformations were conducted and were verified to be negative for His3 prototrophy.

In Vitro Transcription-Translation Assay-- The full-length ORF2 protein (pRSET-ORF2, encoding 660 amino acids of ORF2 with an N-terminal His₆ tag) and [³⁵S]methionine-radiolabeled full-length ORF3 protein (123 amino acids), along with its deletion mutations (see Table I), were expressed in separate reactions using an in vitro coupled transcription-translation system (TNT coupled reticulocyte lysate system, Promega) following the manufacturer's instructions. The unlabeled ORF2 protein was then bound to Ni²⁺-NTA beads (Amersham Biosciences) and washed three times with PBS (pH 7.4). [³⁵S]Methionine-labeled ORF3 protein (full-length or deletion mutation) was 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 10 ml of SDS-PAGE loading buffer (50 mM Tris-HCl (pH 6.8), 5% 2-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, and 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 [³⁵S]methionine-labeled proteins were detected by autoradiography.

Transfection and Labeling of Cultured Cells-- COS-1 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 20 µg/ml gentamycin. Cells were transfected at a confluency of ~50% with plasmid DNA 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 Dulbecco's modified Eagle's medium without serum or antibiotics, and DNA uptake was allowed to proceed for 6 h at 37 °C in a CO₂ incubator. Forty hours post-transfection, cells were washed with 3 ml of methionine-deficient Dulbecco's modified Eagle's medium (Invitrogen) and metabolically labeled with [³⁵S]methionine (Amersham Biosciences), with each 60-mm diameter plate receiving 100 µCi of label in 1 ml of methionine-deficient Dulbecco's modified Eagle's medium. After 4 h of labeling, cells were washed with ice-cold PBS and harvested for further analysis. In addition to HEV ORF-containing expression plasmids, each experiment also included a control (or mock) transfection, in which the same amount of the parent vector (pSGI) was used. For phosphate labeling, at 40-44 h post-transfection, cells on 60-mm plates were washed once with phosphate-deficient Dulbecco's minimal essential medium (Invitrogen) and incubated in 3 ml of deficient medium for 1 h. Following this step, each plate was labeled for 4 h in a CO₂ incubator at 37 °C with 250 µCi of [³²P]orthophosphate (Amersham Biosciences or PerkinElmer Life Sciences) in 1 ml of deficient medium.

Immunoprecipitation-- PBS-washed transfected COS-1 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, and 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 were added 100 µl of a 10% suspension of GST buffer-washed protein A-Sepharose beads (Amersham Biosciences), 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 for 10 s. Washed beads were resuspended in 50 µl of SDS-PAGE loading buffer, heated at 100 °C for 4 min, and centrifuged, and the supernatant was subjected to SDS-PAGE and autoradiography.

Phosphatase Treatment-- Lysates from cells transfected with the ORF3 expression vectors and labeled with [³²P] were subjected to immunoprecipitation with rabbit polyclonal anti-ORF3 antibody as described above. The immunoprecipitates were washed once with 250 µl of -protein phosphatase reaction buffer (50 mM Tris-HCl (pH 7.8), 5 mM dithiothreitol, 2 mM MnCl₂, and 100 mg/ml bovine serum albumin). The washed immunoprecipitates were resuspended in 50 µl of -protein phosphatase reaction buffer with or without 1 µl of -protein phosphatase (400,000 units/ml; New England Biolabs Inc.) and incubated for 1 h at 30 °C. A control reaction with the same amount of ³⁵S-labeled ORF3 was also conducted to test for protein stability, against -protein phosphatase treatment. After washing the beads with GST binding buffer once, clarified cell lysate containing ³⁵S-labeled ORF2 was applied to the tube with phosphatase-treated ORF3 and incubated at 4 °C for 1 h with gentle shaking. The beads were washed five times with GST binding buffer. A control reaction with the same amount of immunoprecipitated ³²P-labeled ORF3 was subjected to the same treatment to show that ORF2 was capable of binding under the above experimental conditions. The beads were centrifuged, resuspended in SDS-PAGE loading buffer, heated at 100 °C for 4 min, and centrifuged, and the supernatant was subjected to SDS-PAGE, followed by autoradiography.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Colocalization of HEV Structural Proteins ORF2 and ORF3-- Dual labeling immunofluorescence microscopy revealed colocalization of ORF3 and ORF2 in COS-1 cells transiently transfected with the expression vectors pMT-ORF3 and pMT-ORF2 (Fig. 1). The distribution of ORF3 in these cells was cytoplasmic and displayed punctate green staining (Fig. 1, ORF3-488). Distribution of ORF2 was observed in the cytoplasm, too, and was denser around the nucleus, possibly in the endoplasmic reticulum, and stained red (Fig. 1, ORF2-594). Both proteins colocalized in the cytoplasm and did not aggregate in the nucleus or other organelles as shown in yellow (Fig. 1, MERGE). With these initial results showing colocalization of both ORF2 and ORF3, we decided to test the heterotypic interactions of these two HEV structural proteins in vivo and in vitro.

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Colocalization of ORF2 and ORF3 proteins. Two sets of COS-1 cells transiently transfected with pMT-ORF3 and pMT-ORF2 were doubly labeled with mouse monoclonal anti-ORF3 and rabbit polyclonal anti-ORF2 antibodies, followed by Alexa 488-conjugated anti-mouse antibodies (ORF3-488) and Alexa 594-conjugated anti-rabbit antibodies (ORF3-594), respectively. Separate images were acquired showing ORF3 distribution (green) and ORF2 distribution (red) and were merged (yellow) using Adobe Photoshop Version 5.0 software.

The Two Proteins ORF2 and ORF3 Interact with Each Other-- The full-length ORF2 gene was cloned into the yeast two-hybrid vector containing the GAL4 DNA-binding domain, resulting in the expression of a fusion protein with ORF2 fused to the C terminus of the GAL4 DNA-binding domain. Similarly, the full-length ORF3 gene was cloned in-frame into the two-hybrid vector containing the GAL4 activation domain, resulting in the expression of a fusion protein with ORF3 fused to the C terminus of the GAL4 activation domain (Table I). Cotransformation of S. cerevisiae with plasmids encoding BD-ORF3 and AD-ORF3 induced strong GAL4-dependent HIS3 and lacZ expression as determined by growth on SDTrpLeuHis dropout medium and the blue color from filter-lift -galactosidase assays, respectively (Fig. 2). The yeast extract/peptone/dextrose (YPD) plate (Fig. 2B) showed unrestricted growth of all transformants shown in the template (Fig. 2A). Neither plasmid alone induced HIS3 or lacZ expression in yeast. Single transformants, the host strain, and the BD-SNF1/AD-SNF4-positive control (27) were plated on all the restrictive media plates (Fig. 2, C-E). Only transformants that possessed the BD plasmid or constructs containing it grew on SDTrp plates (Fig. 2C), whereas only transformants containing the AD plasmid or constructs derived from it grew on SDLeu plates (Fig. 2D). Only the positive control (BD-SNF1/AD-SNF4) and the transformants containing both BD-ORF2 and AD-ORF3 were able to grow on SDTrpLeuHis plates (Fig. 2E). The second reporter gene (lacZ) was also tested for expression by a filter-lift assay, resulting in a blue color for the positive cotransformants and the positive control (Fig. 2F).

View larger version (72K): [in this window] [in a new window]   Fig. 2.   Two-hybrid results showing heterotypic interactions of the HEV proteins ORF2 and ORF3. A, template for B-F showing transformants streaked in each section of the plate; B-E, growth on YPD, SDTrp, SDLeu, and SDTrpLeuHis plates, respectively; F, results from the filter-lift -galactosidase (-Gal) assay. Growth is represented by white on a black background of the Petri dish. Blue is represented by the black streaks on a white background in the nitrocellulose membrane.

Liquid -galactosidase activity was determined for the positive clones along with all appropriate negative and positive controls using the substrate chlorophenol red -D-galactopyranoside. The mean relative -galactosidase activities are shown in Fig. 3A. The host strain (Y190) along with transformants with single plasmids (AD-ORF2 and BD-ORF3) showed negligible -galactosidase activity. Cotransformants containing none or one of the two ORFs (BD/AD, BD/AD-ORF2, and BD-ORF3/AD) also showed negligible -galactosidase activity; however, cotransformants containing AD-ORF2 and BD-ORF3 together showed a high -galactosidase response.

View larger version (43K): [in this window] [in a new window]   Fig. 3.   A, liquid -galactosidase (-gal) assay results. Single transformants and cotransformants were analyzed in a liquid -galactosidase assay and compared with each other. Values are given in arbitrary units. The numbers above each bar represent the mean of five independent transformants. Y190 corresponds to the untransformed host strain. Transformants with more than one plasmid are separated by slashes. B, measurement of the strengths of the ORF2-ORF3 interaction. Activation of the HIS3 reporter was determined for the host Y190 strain, single transformants (BD, AD, BD-ORF3, and AD-ORF2), and cotransformants (BD-SNF1/AD-SNF4 and BD-ORF3/AD-ORF2). Hundredfold serial dilutions of all of the above-mentioned log-phase cultures were plated on YPD (left), SDHis (middle), and SDHis plus 50 mM 3-AT (right) plates.

We further investigated the level of activation of the HIS3 reporter genes for the full-length ORF2-ORF3 interaction in the presence of 50 mM 3-AT. Hundredfold serial dilutions of log-phase cultures of Y190 strains expressing BD-SNF1 and AD-SNF4 (BD-SNF1/AD-SNF4) and AD-ORF2 and BD-ORF3 (BD-ORF3/AD-ORF2) along with appropriate controls were plated on YPD, SDHis, and SDHis plus 50 mM 3-AT (Fig. 3B). These results indicate the strength of the protein-protein interactions as a function of His prototrophy. The BD-ORF3/AD-ORF2 cotransformants showed growth up to a 10⁴-fold dilution on the SDHis plus 50 mM 3-AT plate. This experiment showed that the ORF2-ORF3 interaction is strong and true.

The specificity of the ORF2-ORF3 interaction was also confirmed using a yeast genetic approach (30). After genetic crossing of the single transformants (haploids), the His3 prototrophy of the diploid strains (a/) was tested. Only the diploids containing both BD-ORF3 and AD-ORF2 (BD-ORF3/AD-ORF2) showed a positive phenotype, similar to the positive diploid control (BD-SNF1/AD-SNF4) (Fig. 4). From all the above experiments, we conclude that the two HEV proteins ORF2 and ORF3 interact with each other in a yeast two-hybrid system.

View larger version (45K): [in this window] [in a new window]   Fig. 4.   Genetic verification of the ORF2-ORF3 interaction. The haploid host cell is designated per its mating type, a or . Diploid cells are designated a/. Growth of colonies is shown on YPD (nonselective) and SDTrpLeuHis plates.

A 25-Amino Acid Region of the ORF3 Protein Binds to the Full-length ORF2 Protein-- To characterize the domains involved in the ORF2-ORF3 interaction, an array of deletion mutations were constructed for both ORF2 and ORF3 and were cloned into the yeast two-hybrid AD and BD vectors as described in Table I. Combinations of full-length fusion constructs and deletion mutants of each fusion construct were tested for in vivo protein-protein interactions as shown in Fig. 5.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Results of interactions of full-length ORF2 and ORF3 along with different combinations of deletion mutants of each versus full-length proteins of the other. AD and BD regions were cloned in-frame with ORF2 (blue) and ORF3 (red). Open boxes represent regions that were deleted from the wild-type sequences of both ORF2 and ORF3. The numbers above the boxes represent the first and last amino acids of the regions included. His represents growth on SDTrpLeuHis plates. The blue dots under the -gal heading represent positive interaction from a filter-lift -galactosidase assay. The numbers in brackets show relative -galactosidase units from the liquid -galactosidase assay. AT signifies growth on SDTrpLeuHis plates with 50 mM 3-AT. Dip His represents growth of diploids tested by the genetic two-hybrid approach.

A phenomenon very clearly observed with the ORF2 protein was that none of its deletion mutants could interact with full-length ORF3 when tested in the yeast two-hybrid system. The ORF2 deletion mutants used in this study represented different parts of the protein and were from a variety of regions and lengths as shown in Fig. 5. On the other hand, ORF3 deletion mutants showed both positive and negative results with various different deletion mutants when tested with full-length ORF2 for two-hybrid interactions. The ORF3-(1-81) deletion fragment showed a positive interaction with full-length ORF2 upon the yeast two-hybrid assay. Consequently, when the ORF3-(83-123) deletion fragment was tested, it showed negative. These initial experiments indicated that amino acids 1-81 of the ORF3 protein contain the interaction domain. Furthermore, when the ORF3-(1-57) deletion fragment was tested with ORF2, the result was again negative, whereas ORF3-(57-123) showed a positive interaction with ORF2. All the above results indicate that the region between amino acids 57 and 81 of ORF3 contains the interaction domain. Our mapping results were confirmed when ORF3-(57-81) was constructed and tested for its in vivo interaction with ORF2. This interaction showed positive upon all yeast two-hybrid assays. From these experiments, we were able to clearly map a 25-amino acid region of the ORF3 protein that is responsible for its interaction with ORF2.

Ser⁸⁰ Plays a Key Role in ORF2-ORF3 Interactions-- The 25-amino acid interaction domain of ORF3 contains Ser⁸⁰, which has earlier been shown by us to be the site for phosphorylation of this protein (17). An S80A point mutant of full-length ORF3 was cloned into the yeast two-hybrid BD vector. When full-length AD-ORF2 was tested with this BD-ORF3(S80A) point mutation, the results showed negative in the yeast two-hybrid analysis (Fig. 6). This result indicates that the ORF2-ORF3 interaction depends on the presence of Ser⁸⁰.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Diagrammatic account of the 25-amino acid interaction domain and its two-hybrid interaction results with full-length ORF2 before and after the S80A point mutation. Shaded boxes represent AD and BD regions. The ORF2 gene is shown as hatched boxes, and the ORF3 deletion mutant and full-length ORF3 are shown as dotted boxes. The open boxes represent regions deleted from the full-length gene. The numbers above the boxes represent the first and last amino acids of the regions included. His represents growth on SDTrpLeuHis plates. -gal represents the filter-lift -galactosidase assay results and the liquid -galactosidase assay results (in brackets) from the yeast two-hybrid analysis. AT represents the 3-AT assay for growth on SDTrpLeuHis plates with 50 mM 3-AT. Dip His represents growth of diploids tested by the genetic two-hybrid analysis.

The above experiments have clearly been able to pinpoint a single amino acid residue responsible for this protein-protein interaction. We designed a co-immunoprecipitation procedure to study the ORF2-ORF3 interaction and to validate our two-hybrid findings. Heterotypic interactions of the two HEV proteins ORF2 and ORF3 were studied by transiently transfecting COS-1 cells with either pMT-ORF2 alone (as a control) or in combination with one of the following: pMT-ORF3 (full-length, containing Ser⁸⁰), pSG-BD-ORF3-(57-81) (containing the binding domain fused to the 25-amino acid interaction domain from ORF3), or pSG-ORF3(S80A) (full-length, containing an S80A point mutation) (Fig. 7). [³⁵S]Methionine-labeled cell lysates were then immunoprecipitated with polyclonal antibodies. In a control experiment, the expression of both ORF2 and ORF3 was detected by their respective antibodies (Fig. 7, lanes 1-3). All binding studies were conducted with anti-ORF2 antibodies (Fig. 7, lanes 4-10).

View larger version (39K): [in this window] [in a new window]   Fig. 7.   Detection of ORF3 and ORF3-(57-81) by immunoprecipitation of the ORF2 protein with anti-ORF2 antibodies. COS-1 cells coexpressing ORF2 and ORF3 proteins are shown in lanes 10 and 7, respectively. The ORF3(S80A) mutant was not detected by immunoprecipitation of the ORF2 protein (lane 9). Lanes 1-6 and 8 are appropriate expression and binding controls for experiments analyzed on 8% (A) and 15% (B) SDS-polyacrylamide gels. Both anti-ORF2 and anti-ORF3 antibodies (Ab) were polyclonal antibodies. Lane M shows molecular mass markers (in kilodaltons) as indicated.

Upon cotransfection of COS-1 cells with pMT-ORF3 and pMT-ORF2 constructs and immunoprecipitation with anti-ORF2 antibodies, both full-length proteins ORF2 and ORF3 were detected (Fig. 7, lane 10). In another set of cotransfection experiments, pSG-BD-ORF3-(57-81) and pMT-ORF2 were immunoprecipitated with anti-ORF2 antibodies, and bands corresponding to both ORF2 and BD-ORF3-(57-81) were detected (Fig. 7, lane 7), thus providing in vitro evidence that the 25-amino acid region (residues 57-81) contains the interaction domain. As a control, pSG-BD and pMT-ORF2 were immunoprecipitated with anti-ORF2 antibodies, which showed negative (Fig. 7, lane 8). Upon testing the pSG-ORF3(S80A) point mutant with pMT-ORF2 and immunoprecipitation with anti-ORF2 antibodies, only ORF2 was detected. All appropriate binding (Fig. 7, lanes 4-6 and 8) and expression (lanes 1-3) controls used in these experiments are shown. These results very clearly show that Ser⁸⁰ is indispensable for the ORF2-ORF3 interaction.

Subsequently, we tested for ORF2 interaction with full-length ORF3, the 25-amino acid interaction domain of ORF3, and the S80A point mutation of ORF3 by an in vitro coupled transcription-translation immobilization assay. The full-length ORF2 protein was cloned into the pRSET cloning vector, thus expressing a fusion protein with a His₆ tag fused to its N-terminal end. The ORF2 protein was immobilized to Ni²⁺-NTA-charged beads for these experiments. The full-length ORF3 protein and the BD-ORF3-(57-81) (containing the 25-amino acid interaction domain) and ORF3(S80A) point mutation constructs were individually transcribed and translated using [³⁵S]methionine. Fig. 8 (A and B) shows the results from these experiments. Lanes 1 and 7 show the full-length ORF2-ORF3 interaction in vitro after radiolabeled ORF3 was allowed to bind to immobilized ORF2, washed, and analyzed by SDS-PAGE. Lane 4 shows the positive interaction of the full-length ORF2 protein with BD-ORF3-(57-81) (containing the 25-amino acid interaction domain), and lane 8 shows the loss of positive signal when the immobilized ORF2 protein was tested for binding with [³⁵S]methionine-labeled ORF3(S80A) mutant protein. Lanes 2, 3, 5, and 6 show appropriate negative and expression controls.

View larger version (33K): [in this window] [in a new window]   Fig. 8.   A and B, in vitro coupled transcription-translation assays for detecting heterotypic interactions of the full-length HEV structural proteins ORF2 and ORF3 and their deletions. B represents Ni2+-NTA beads in the figure. The His6-ORF2 protein was produced by coupled transcription-translation in the absence of [35S]Met and immobilized on Ni2+-NTA beads. Full-length ORF3, BD-ORF3-(57-81), and ORF3(S80A) were transcribed-translated in the presence of [35S]Met and tested for interaction as shown in lanes 1/7, 4, and 8, respectively. Appropriate expression and binding controls are shown in lanes 2, 3, 5, and 6. C, a yeast two-hybrid comparison of interactions between the full-length ORF2 protein and ORF3-(57-81), ORF3(S80A), and the Mexican ORF3 isolate is shown. SDHis and -Gal assay represent the two-hybrid assays. The numbers represent liquid -galactosidase assay results.

Ser⁸⁰ of ORF3 is conserved in all known isolates of HEV, except the Mexican isolate (33). We thus cloned the ORF3 coding region of the Mexican isolate of HEV into the yeast two-hybrid BD vector and tested it for an interaction against the AD-ORF2 (full-length) protein. The results for this experiment are shown in Fig. 8C. As positive and negative controls in this experiment, we used the ORF3-(57-81) protein (containing the 25-amino acid interaction domain) and the ORF3(S80A) point mutant, respectively. The Mexican ORF3 protein clearly showed negative upon yeast two-hybrid assays. Hence, we have proved through various in vivo and in vitro methods that Ser⁸⁰ is essential for the ORF2-ORF3 interaction.

Phosphorylation at Ser⁸⁰ of the ORF3 Protein Is Essential for the ORF2-ORF3 Interaction-- Ser⁸⁰ of ORF3 has been shown to be the major site for phosphorylation (17). Our experiments described above showed that Ser⁸⁰ is essential for the ORF2-ORF3 interaction. We thus designed assays using -protein phosphatase to investigate the requirement of phosphorylation of Ser⁸⁰ for the ORF2-ORF3 interaction. In these experiments, two aliquots of COS-1 cells were starved for phosphate and sulfate separately. These cultures were radiolabeled with [³²P] and [³⁵S], respectively. The ORF3 protein was immunoprecipitated using anti-ORF3 antibodies from the ³²P-labeled cell lysate.

Upon -protein phosphatase treatment of ³²P-labeled ORF3, the protein was allowed to interact with the ³⁵S-labeled ORF2-expressing cell lysate (Fig. 9, lane 6). ³⁵S-labeled ORF3 was subjected to -protein phosphatase and shown to be unaffected (data not shown). On the other hand, phosphatase treatment of ³²P-labeled ORF3 resulted in no visible ORF3 band on the autoradiogram (Fig. 9, lane 5), showing complete removal of the phosphate group from ORF3. Lanes 1-4 show all required expression and immunoprecipitation controls for this experiment. Thus, phosphorylation at Ser⁸⁰ of ORF3 is required for the ORF2-ORF3 interaction.

View larger version (56K): [in this window] [in a new window]   Fig. 9.   Phosphatase assays used to study the effects of phosphorylation on the ORF2-ORF3 interaction. Reactions with a plus sign represent -protein phosphatase treatment. 32P-Labeled ORF3 protein was immunoprecipitated using anti-ORF3 antibodies and allowed to interact with 35S-labeled ORF2 in vitro (lane 6). Lane 5 shows complete phosphatase activity of the enzyme. Lanes 1-4 show all required expression and immunoprecipitation controls for these experiments.

The Phosphorylated ORF3 Protein Preferentially Interacts with the Non-glycosylated Form of ORF2-- ORF2 is a glycoprotein with three glycosylation sites (Asn¹³⁷, Asn³¹⁰, and Asn⁵⁶²). The glycosylation site at Asn³¹⁰ is the major site for ORF2 glycosylation. We designed experiments to investigate whether phosphorylated ORF3 binds primarily to the glycosylated or non-glycosylated fraction of ORF2. Fig. 10A shows the results of our initial experiments. Lane 1 shows the glycosylated (gORF2) and non-glycosylated (ORF2) forms of the ORF2 protein expressed in COS-1 cells and immunoprecipitated by anti-ORF2 antibodies. Upon cotransfection with both ORF2- and ORF3-expressing vectors and immunoprecipitation with anti-ORF3 antibodies, we found primarily the non-glycosylated form of ORF2 binding to ORF3 (Fig. 10A, lane 2). When -protein phosphatase was added to the lysate coexpressing ORF2 and ORF3 and immunoprecipitated with antibodies against ORF3, none of the forms of the ORF2 protein were detected (Fig. 10A, lane 3). These results gave us preliminary evidence that ORF3 preferentially binds to the non-glycosylated fraction of ORF2.

View larger version (49K): [in this window] [in a new window]   Fig. 10.   The non-glycosylated form of the ORF2 protein interacts with the phosphorylated form of the ORF3 protein of HEV. A, immunoprecipitation of ORF3 using antibodies against the ORF3 protein was examined for the ORF2 protein. Lane 1 shows the glycosylated (gORF2) and non-glycosylated (ORF2) forms of the ORF2 protein as detected by antibodies against ORF2. Lane 2 shows the non-glycosylated form of the ORF2 protein preferentially interacting with the ORF3 protein, which was immunoprecipitated by its antibody. Upon phosphatase treatment of this lysate, non-glycosylated ORF2 did not bind to non-phosphorylated ORF3 (lane 3). B and C, shown is the preferential interaction of the ORF3 protein with the non-glycosylated form of ORF2. B is an expression control for C. Plus and minus signs indicate the presence and absence of tunicamycin in the reactions, respectively. 137,310, 310-562, and 2-34 represent the two double point mutants (ORF2(N137A,N310A) and ORF2(N310A,N562A)) and the deletion mutant (ORF2(2-34)) of ORF2.

We subsequently used the ORF2 mutants ORF2(N137A,N310A), ORF2(N310A,N562A), and ORF2(2-34) described in Table I. ORF2(N137A,N310A) and ORF2(N310A,N562A) are double point mutations (Asn-to-Ala) at positions 137, 310, and 562 in different combinations. ORF2(2-34) is an N-terminal deletion of the putative signal sequence of the ORF2 protein, preventing its transport into the endoplasmic reticulum and thus rendering it non-glycosylated.

Fig. 10B (lane 1) shows transient coexpression in COS-1 cells of the full-length ORF3 protein along with the two forms of ORF2: non-glycosylated (ORF2) and glycosylated (gORF2). Lane 2 shows tunicamycin inhibition of glycosylation, with only the non-glycosylated form of ORF2. Lanes 4-6 correspond to expression of the non-glycosylated forms of ORF2 by mutants ORF2(N137A,N310A), ORF2(N310A,N562A), and ORF2(2-34), respectively. All samples were co-immunoprecipitated using antibodies against the ORF2 protein. When these coexpression lysates were analyzed for the ORF3 protein, each one of the corresponding lysates containing the non-glycosylated form of ORF2 pulled out the ORF3 protein from the lysates (Fig. 10C). This clearly proves that the non-glycosylated form of ORF2 is capable of binding to ORF3. The combined results in this report prove that the phosphorylated ORF3 protein interacts preferentially with the non-glycosylated form of the ORF2 protein of HEV.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HEV cannot be cultured routinely, although it has recently been propagated in primary macaque hepatocytes (34, 35), and a virus resembling HEV has been cultured in A549 cells (33). As a result, studies of HEV protein synthesis, processing, and assembly have been limited to heterologous expression systems. We chose the yeast two-hybrid system to study the heterotypic interactions of the two proteins encoded by ORF2 and ORF3 located in the structural part of the HEV genome. Our interests in this interaction increased significantly when we found these proteins to colocalize upon immunofluorescence microscopy of cotransfected cells. Using the yeast two-hybrid approach, we showed interactions between these two proteins, mapped the interaction domains, and showed that Ser⁸⁰ of ORF3 protein is responsible for this interaction. Results thus obtained were verified using other in vitro binding and immunoprecipitation techniques. Furthermore, we showed that the ORF2-ORF3 interaction is dependent upon phosphorylation at Ser⁸⁰ of the ORF3 protein. Because the ORF2 protein exists in both glycosylated and non-glycosylated forms (25), we designed experiments to observe any preference shown by ORF3 for the glycosylated and non-glycosylated forms of ORF2. Analysis using ORF2 mutants and tunicamycin inhibition revealed that ORF3 preferentially interacts with the non-glycosylated form of ORF2.

There are three sites for ORF2 glycosylation (Asn¹³⁷, Asn³¹⁰, and Asn⁵⁶²), with Asn³¹⁰ being the primary one (25). Torresi et al. (36) have shown that the glycosylated ORF2 species is much less stable than non-glycosylated ORF2, which is present in the cytosol and represents the major product accumulated in the cell. It is postulated from this work that the non-glycosylated form may be involved in capsid assembly. Our results show that ORF3 preferentially binds with the non-glycosylated form of ORF2.

Our data also show that ORF3 interacts only with full-length ORF2 and not with any of its deletion mutants. Using baculovirus constructs expressed in insect Tn5 cells, Xing et al. (37) have shown that post-translational proteolytic cleavage is required for particle formation. Similarly, Li et al. (15) have shown that an N-terminally 111-amino acid truncated ORF2 protein shows empty particle formation. Both these studies suggest that there is proteolytic processing of ORF2 prior to capsid assembly. Our data show that full-length ORF3 interacts only with full-length ORF2 and not with any of its deletions mutants, including ORF2-(112-660). This result indicates that, during the course of ORF2 processing in the viral replication cycle, the ORF2-ORF3 interaction occurs prior to the processing of the ORF2 protein into its ~50-kDa processed form, which later forms VLPs.

To date, there is no evidence for an RNA-binding activity for either ORF2 or ORF3. Sequence analysis reveals, however, that the N-terminal 111 residues of native ORF2 contains basic residues and hence may be involved in RNA binding. Alternatively, ORF3 may be the RNA-binding protein and thus may interact with ORF2 during capsid assembly. The ORF3 dimerization (13) is independent of the ORF2-ORF3 interaction domain and the phosphorylation domain. So, it is possible that the ORF3 protein forms a dimer prior to interacting with full-length ORF2. After dimerization, ORF3 probably gets phosphorylated, which makes it capable of binding to ORF2. If the ORF3 protein were to bind RNA and get phosphorylated, the ORF2-ORF3 interaction may have a major role to play in RNA packaging.

The stability of VLPs after proteolytic modification of the ORF2 protein has been shown to decrease (37). It has been thus postulated from previous work that recombinant HEV particles lack a stabilizing scaffold and thus become fragile and easily damaged during purification. This suggests the possibility of another protein interacting with ORF2 that may provide it more stability.

The ORF2 protein has been shown to self-associate in the absence of ORF3 to form dimers (12, 13) and VLPs (15, 37). Although these VLPs mimic the HEV virion, there are detectable differences in size, and the internal cavity thus formed is too small to accommodate the ~7.2-kb HEV genomic RNA (37). Virions of this size have not been found in the bile or stool of patients suffering from hepatitis E or of experimentally infected monkeys (1). Also, the calculated capacity of these VLPs for packaging RNA is only ~1 kb in size, whereas the size of the HEV genome is 7.5 kb. Together with the size assessment of the capsid and the calculated indispensable volume of the viral RNA, the possibility of the involvement of another viral protein, ORF3 in this case, may be a possibility for correct HEV capsid assembly. With ORF3 selectively binding to the non-glycosylated form of ORF2, which is the one involved in capsid formation, our study shows ORF3 to be an important candidate for participation in capsid assembly.

The ORF3 phosphoprotein has been shown to self-associate (13), to bind proteins containing SH3 domains, and to activate cellular MAPK (18). Although ORF3 maps in the structural region of the HEV genome, it has, in these recent reports, shown indications of regulatory functions. Also, with this report showing ORF2 interacting with ORF3, the possibility of a nonstructural function for the ORF2 protein also exists.

In this report, we have observed that the ORF3 protein from the Mexican isolate, which contains leucine instead of serine at position 80, fails to associate with ORF2. Upon close examination of the sequence of the Mexican ORF3 amino acid sequence in that region, we observed that it was considerably different, with an upstream serine at position 76 also replaced with leucine. Either the ORF2-ORF3 interaction is not critical for virion assembly or infection, as suggested by the Mexican HEV isolate, and has a nonstructural role, or the Mexican isolate has a weak serine-free ORF2-ORF3 interaction that we were unable to detect as fusion proteins in a yeast environment. In the absence of comparative infectivity data on the prototypic and Mexican isolates of HEV, it is difficult to assess the effects of lack of Ser⁸⁰ phosphorylation on viral pathogenesis. Finally, because the ORF2-ORF3 interaction is phosphorylation-dependent, this seems like a good post-translational control mechanism to check the level of ORF2-ORF3 interaction within the hepatocyte.

	ACKNOWLEDGEMENTS

We gratefully acknowledge the generous gifts of the yeast two-hybrid vectors and strains from Stephan Elledge and of the PJ69-4a and PJ69-4 strains from Philip James. The laboratory assistance of Ravinder Kumar is greatly appreciated.

	FOOTNOTES

^* This work was supported by the International Centre for Genetic Engineering and Biotechnology.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research Fellow from the University Grants Commission.

^§ To whom correspondence should be addressed. Tel.: 91-11-6177357; Fax: 91-11-6162316; E-mail: sunillal@icgeb.res.in.

Published, JBC Papers in Press, April 4, 2002, DOI 10.1074/jbc.M200185200

	ABBREVIATIONS

The abbreviations used are: HEV, hepatitis E virus; ORF, open reading frame; VLPs, virus-like particles; SH3, Src homology 3; MAPK, mitogen-activated protein kinase; PBS, phosphate-buffered saline; SD, synthetic dextrose; 3-AT, 3-amino-1,2,3-triazole; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; BD, binding domain; AD, activation domain; Ni²⁺-NTA, nickel-nitrilotriacetic acid; GST, glutathione S-transferase; YPD, yeast extract/peptone/dextrose.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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