SARS coronavirus protein 7a interacts with human Ap₄A-hydrolase

In order to identify cellular proteins that interact with the 7a protein, we performed a yeast two-hybrid screening of the Hybrid Hunter™ Premade cDNA library (human adult lung) constructed in pYESTrp2 (Invitrogen). Approximately 1 × 10⁶ transformants were screened. From these, 35 clones were able to grow in the selective media (-Trp, -Leu, -His) and gave a strong signal when analyzed for β-galactosidase activity. Plasmid DNA from the positive yeast clones was purified and used for transformation of the E. coli strain DH5α. Next, the DNA of the plasmids was sequenced and analyzed using BLAST. One of the clones contained a complete ORF that showed sequence identity to the human Ap₄A-hydrolase gene [GenBank: U30313].

To verify interactions established by the yeast two-hybrid screening, we used a co-immunoprecipitation assay. HEK 293 cells were either transfected with pXJ3'-ORF7a-HA, or co-transfected with both pXJ3'-ORF7a-HA and pcDNA3.1/V5-His -Ap₄A. The transfected cells were lysed 24-48 h after transfection. Protein extracts were immunoprecipitated using monoclonal Anti-V5 antibody (Fig. 1, lanes 1 and 2). As a negative control, the same protein extracts were immunoprecipitated using an unrelated antibody - mouse monoclonal anti-β-actin (Fig. 1, lane 4). Antibody-antigen complexes were immobilized on protein A and G-sepharose bead mixture. The immobilized proteins were eluted by boiling the beads in SDS-PAGE loading buffer. The samples were subsequently analyzed by Western blot, using an anti-HA monoclonal antibody, to detect HA tagged co-immunoprecipitated proteins. As shown in Fig. 1, the HA-tagged 7a protein co-immunoprecipitated with V5-tagged Ap₄A-hydrolase. As described earlier [12], the 7a protein was detected in the transfected cells in two forms: unprocessed and processed. The 7a protein was not detected when unrelated antibody was used for co-immunoprecipitation. Our results indicate that 7a specifically interacts with Ap₄A-hydrolase in human cells.

There is no consensus opinion in subcellular localization of 7a protein [for review see [11]]. 7a protein was found co-locolaze with trans-Golgi marker, Golgin97 [9], in the Golgi compartment [14]. Other reports demonstrated co-localization with endoplasmic reticulum marker GRP94 or the intermediate compartment marker Sec 31 [7, 17]. Since the co-immunoprecipitation experiment showed interaction between HA- tagged 7a and V5-tagged Ap₄A-hydrolase, we hypothesized that these proteins may co-localize in human cells. To visualize these proteins, we tagged them with fluorescent proteins: 7a with EGFP and Ap₄A-hydrolase with DsRed2. HEK 293 cells were co-transfected with pEGFP-C2-ORF7a and pDsRed2-Ap₄A, and sub-cellular distribution of proteins was examined by confocal microscopy.

Our data demonstrated that both proteins had similar cytoplasmic localization (Fig. 2, left and middle panels). Co-localization analysis showed an overlap in the subcellular distribution of both proteins, as can be seen by the appearance of the yellow color in the overlaid image (Fig. 2, right panel).

The plasmid pXJ3'-ORF7a-HA containing full-length SARS-CoV ORF 7a with C-terminal HA-tag was a generous gift from Dr. Yee-Joo Tan (Institute of Molecular and Cell Biology, Singapore) and has been described elsewhere in detail [12].

In our study, the ORF 7a gene was amplified from RNA of SARS-CoV-infected Vero E6 cells (strain Tor 2) using a one step RT PCR kit (Qiagen, Mississauga, Canada) (primers 5'-GGGGTACC ATGAAAATTATTCTCTTCCT-3' and 5'-GGAATTC TCATTCTGTCTTTCTCTTAA-3') and cloned into the Kpn I and Eco R I sites (underlined) of the pH5L vector [40] to create pH5L-ORF7a. Next, ORF 7a was amplified from the plasmid pH5L-ORF7a using primers 5'-CGGAATTC ATGTTTCATCTTGTTGACTT-3' and 5'-CGCTCGAG TTATGGATAATCTAACTCCA-3'. The PCR-product was digested with Eco R I and Xho I (recognition sites are underlined) and subcloned into pHybLex/Zeo (Invitrogen, Burlington, Canada) in frame with LexA to create the "bait" pHybLex/Zeo-ORF7a plasmid for yeast two-hybrid library screening. Then, pH5L-ORF7a was digested with Eco R I and Xho I and the DNA fragment containing ORF 7a was cloned into Eco R I and Xho I sites of pEGFP-C2 (Clontech, Mountain View, USA) resulting in pEGFP-C2-ORF7a where ORF 7a was fused to the C-terminus of EGFP.

The plasmid containing the human full-length Ap₄A-hydrolase gene was recovered from the yeast clone as described below. The gene was amplified by PCR using the specific primers 5'-GCGGATCC AGATGGCCTTGAGAGCATG-3' and 5'-CGCTCGAG GGGCCTCTATGGAGCAAAGA-3' and subcloned into Bam H I and Xho I site (underlined) of pcDNA3.1/V5-HisA vector (Invitrogen) to construct pcDNA3.1/Ap₄A V5-His where V5-His tag was attached to the C-terminus of Ap₄A-hydrolase. Also, the gene encoding Ap₄A-hydrolase was subcloned into the Hind III and Eco R I sites of pDsRed2 (Clontech) (primers 5'-TTAAGCTT ATGGCCTTGAGAGCATGTG-3' and 5'-GTGAATTC GTGCCTCTATGGAGCAAAG-3') resulting in pDsRed2-Ap₄A where DsRed2 was fused to the C-terminus of Ap₄A-hydrolase. The sequences of all constructs were confirmed by sequencing.

The Hybrid Hunter™ yeast two-hybrid kit was purchased from Invitrogen, and all experiments were carried out as recommended by the manufacturer. Briefly, the full-length cDNA of ORF 7a was cloned into pHybLex/Zeo to create a "bait" gene construct pHybLex/Zeo-ORF7a as described above. Hybrid Hunter™ Premade cDNA library (human adult lung) constructed in pYESTrp2 (Invitrogen) was used as a source of "prey" genes.

The plasmid pHybLex/Zeo-ORF7a was used to transform the yeast strain EGY48/pSH18-34, using the lithium acetate method to create a "bait" yeast strain. Next, the "bait" yeast strain was transformed with the cDNA library. Positive clones were initially selected for growth in the absence of uracil, leucine, histidine and tryptophane and further tested for β-galactosidase activity using a filter assay, as suggested by Invitrogen.

HEK 293 cells (ATCC CRL-1573) were maintained in minimal Eagle medium (Invitrogen) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% HEPES and 0.05 mg/ml gentamicin (Invitrogen). Cells were cultured at 37° in an incubator supplied with 5% CO₂.

Cells were transiently transfected with expression vectors using the Profection Mammalian Transfection System-Calcium Phosphate (Promega, Madison, USA), in accordance with the manufacturer's instruction. From 24 to 48 h after transfection, the expression of proteins was screened by Western-blot and confocal microscopic analysis.

The following antibodies were used in the present study: monoclonal mouse Anti-V5 antibody (Invitrogen), dilution 1:1000; monoclonal mouse anti-HA Tag antibody (Upstate, Temecula, USA), dilution 1:5000; monoclonal mouse anti-β-actin (Sigma-Aldrich, Oakville, Canada), dilution 1:2000; and goat anti-mouse horseradish peroxidase conjugated IgG (BioRad, Mississauga, Canada), dilution 1:2000.

At 48 h after transfection, HEK293 cells were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 1% deoxicholate Na, 0.1% SDS and 1 mM PMSF). After incubation on ice for 30 min, the samples were briefly sonicated on ice and centrifuged. Supernatants were subjected to 12% SDS-PAGE and were transferred to a nitrocellulose membrane (Ready Gel™ Blotting Sandwiches from BioRad). After incubation with blocking buffer (5% non-fat dry milk in TBST (20 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.05% Tween-20)), membranes were incubated with specific primary antibodies, washed three times in TBS-T, and then incubated with horseradish-peroxidase-conjugated secondary antibodies. The resulting signals were visualized by the ECL- Plus Western Blotting Detection System (GE Healthcare, Baie d'Urfe, Canada).

Cells in 6-well culture plates (5 × 10⁵ cells per well) were transiently co-transfected with 3 μg pXJ3'-ORF7a-HA and 3 μg pcDNA3.1/V5-His-Ap₄A using the Profection Mammalian Transfection System-Calcium Phosphate (Promega, Madison, USA). At 48 h after transfection, cells were harvested and washed twice with ice-cold PBS. Cell lysates were prepared in IP-lysis buffer (10 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM PMSF, 1% Nonident-40). The supernatants were incubated with monoclonal Anti-V5 antibody, or anti-HA Tag antibody or anti-β-actin antibody overnight at 4°C. After the incubation, protein A/G Sepharose Fast Flow beads (GE HealthCare) were added and the samples were incubated for additional 1 h at 4°C. The beads were washed five times with IP-lysis buffer, boiled in SDS sample buffer, and subjected to Western blot analysis.

HEK 293 cells, co-transfected with pDsRed2-Ap₄A and pEGFP-C2-ORF7a, were grown on 35 mm glass bottom culture dishes (Mattek, Ashland, USA). At 24 h post transfection, cells were analyzed using a confocal microscope Zeiss LSM410 equipped with external Argon Ion Laser. EGFP was excited by Argon Ion Laser Beam (488 nm) while DsRed2 was excited by Helium/Neon Laser beam (594 nm). Both signals were detected simultaneously, and separate images were taken and superimposed.