Function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome coronavirus.

To identify the function of HAb18G/CD147 in invasion of host cells by severe acute respiratory syndrome (SARS) coronavirus (CoV), we analyzed the protein-protein interaction among HAb18G/CD147, cyclophilin A (CyPA), and SARS-CoV structural proteins by coimmunoprecipitation and surface plasmon resonance analysis. Although none of the SARS-CoV proteins was found to be directly bound to HAb18G/CD147, the nucleocapsid (N) protein of SARS-CoV was bound to CyPA, which interacted with HAb18G/CD147. Further research showed that HAb18G/CD147, a transmembrane molecule, was highly expressed on 293 cells and that CyPA was integrated with SARS-CoV. HAb18G/CD147-antagonistic peptide (AP)-9, an AP of HAb18G/CD147, had a high rate of binding to 293 cells and an inhibitory effect on SARS-CoV. These results show that HAb18G/CD147, mediated by CyPA bound to SARS-CoV N protein, plays a functional role in facilitating invasion of host cells by SARS-CoV. Our findings provide some evidence for the cytologic mechanism of invasion by SARS-CoV and provide a molecular basis for screening anti-SARS drugs.

Severe acute respiratory syndrome (SARS) coronavirus (CoV), the pathogen ascertained to be responsible for SARS, caused disastrous results around the world at the end of 2002 [1, 2]. How SARS-CoV infects host cells still remains unclear, and the results of studies of the other CoVs suggest that CoVs infect host cells by direct or indirect interaction between viral proteins and cellular proteins expressed on the unit membrane [2–5 ]. Cyclophilin A (CyPA) is a ubiquitously distributed cellular protein and is thought to assist protein folding and to function as a chaperone. It can be integrated with virions of some kinds of viruses, such as HIV-1 [6] and vaccinia virus [7]. By interacting with cellular receptors or through other pathways, integrated CyPA plays a role in viral invasion or replication. CD147 (called “EMMPRIN” in [8] and “basigin” in [9]) is a transmembrane glycoprotein and belongs to the immunoglobulin superfamily. It is a receptor for CyPA and contributes to viral infection or inflammation [6, 10]. HAb18G/CD147, which was developed and was identified to be a member of the CD147 family in our laboratory, is involved in tumor metastasis, inflammation, and virus infection [11–19 ]. Inspired by the known relationship between CD147 and HIV-1, we conducted the present study to investigate the possible function of HAb18G/CD147 in invasion of host cells by SARS-CoV

Materials and Methods

SARS-CoV (BJ04) and 293 cells (ATCC CRL-1573) were supplied by the Chinese Center for Disease Control and Prevention (China CDC). The cells were cultured in Dulbecco’s MEM (Gibco BRL) containing 10% fetal calf serum (Gibco BRL)

HAb18G/CD147–antagonistic peptide (AP)–9 (an AP of HAb18G/CD147 obtained in our laboratory, composed of 12 aa residues) [20–24 ], CB-2 (a randomly synthesized peptide also containing 12 aa residues, used as negative control), and biotin–AP-9 were synthesized by CL Bio-Scientific. Their purity, assayed by high-pressure liquid chromatography, was >95%, and their amino-acid sequences and molecular weights, respectively, were as follows: AP-9, YKLPGHHHHYRP and 1541.09; CB-2, LHRHSHGHSYTS and 1389.50; and biotin–AP-9, biotin-YKLPGHHHHYRP and 1767.69

The ProFound mammalian Co-IP kit (Pierce) and the ECL Plus Western blotting detection system (Amersham Pharmacia) were used in accordance with the manufacturers’ instructions. To analyze the interaction between HAb18G/CD147 and CyPA, 100 μg of HAb18G/CD147 (a recombinant extracellular fragment of HAb18G/CD147, prepared in our lab) was incubated with an equivalent amount of CyPA (Alexis Biochemical) overnight at room temperature (RT). The mixture was then added to the gel, which was coupled with 200 μg of mouse anti–human HAb18G/CD147 monoclonal antibody (MAb) HAb18 (prepared in our lab) [25–33 ], for incubation with gentle end-over-end mixing for 4 h. After the bound proteins were eluted, aliquots of the eluent were analyzed by a Western blot developed with rabbit anti–human CyPA antibody (diluted 1:2000; Calbiochem-Novabiochem) and horseradish peroxidase (HRP)–conjugated goat anti–rabbit IgG (diluted 1:5000; Amersham Pharmacia). Purchased CyPA (2.5 μg) was used as the positive control

To analyze the interaction between CyPA and SARS-CoV structural proteins (spike [S], membrane [M], envelope [E], and nucleocapsid [N]; provided by China CDC), the gel was coupled with 200 μg of CyPA, which was used as the bait protein. Mouse antibodies to the 4 proteins (diluted 1:1000) and HRP-conjugated goat anti–mouse IgG (diluted 1:5000) were used in the subsequent Western blot analyses. Five micrograms of the respective protein was used as the positive control. Blank controls were established in accordance with the manufacturer’s instructions

Surface plasmon resonance analysis was performed, by use of a BIAcore X biosensor system (BIAcore), as described elsewhere [34]. HAb18G/CD147 or CyPA was immobilized on research-grade CM5 sensor chips (BIAcore), with a concentration of 100 μg/mL in 10 mmol/L sodium acetate (pH 6.0), by use of the amine coupling kit supplied by the manufacturer. Approximately 2000 resonance units of HAb18G/CD147 or CyPA was immobilized under these conditions. Unreacted moieties on the surface were blocked with ethanolamine, and the chips were measured in HEPES-buffered saline containing 10 mmol/L HEPES (pH 7.4)

Analyses were performed at 25°C, at a flow rate of 5 μL/min for determination of on-rates and equilibrium binding and 50 μL/min for determination of off-rates. To determine which protein of SARS-CoV could be captured by CyPA or HAb18G/CD147, the S, M, E, and N proteins of SARS-CoV were diluted to 1 μg/mL in HEPES-buffered saline for capture by the CyPA or HAb18G/CD147 surface. Surfaces were typically regenerated with 100 mmol/L hydrochloric acid and 0.2 mol/L Tris buffer

To assess the affinity of N protein to CyPA, N protein was diluted to 40, 32, 24, 16, and 8 nmol/L for capture by the CyPA surface. Biosensor data were prepared for kinetic analysis, by zeroing the time and response before the first injection. To correct refractive index changes and nonspecific binding, the binding responses generated in the control experiments were subtracted from the responses generated in the CyPA–N protein interaction. The binding data were then globally fitted to the following reaction mechanism, in which CyPA and N protein are represented by A and B, respectively: . An equilibrium dissociation rate constant (Kd) was calculated from the kinetic rate constants by nonlinear fitting of the primary sensorgram data by use of BIAevaluation software (version 3.1; BIAcore)

293 cells, at a density of 106 cells/mL, were incubated with either 10 μL of fluorescein isothiocyanate (FITC)–conjugated anti-CD147 antibody (Pharmingin) or 10 μL of FITC-conjugated mouse IgG1 (control; Pharmingin) in the dark for 30 min at 4°C. After being washed once with PBS, cells were fixed with 1% paraformaldehyde and analyzed by use of a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson)

For analysis of AP-9 binding, the cells, blocked by goat serum for 30 min and incubated with biotin–AP-9 (final concentration, 160 μg/mL) for 60 min, were washed and treated with 5 μL of avidin-FITC (Pharmingin) in the dark for 60 min at 4°C. Fixation and data analysis were performed as described above

293 cells infected with SARS-CoV (BJ04) were cultured overnight on the sterilized cover slips and then washed in PBS and fixed with cold acetone for 30 min. For analysis of AP-9 binding, the cells were incubated with 25 μg of biotin–AP-9 and avidin-Cy3 (diluted 1:100; Sigma) in the dark overnight at 4°C. For analysis of blocking of AP-9, 25 μg of biotin–AP-9 was incubated with 2.5 μg of HAb18G/CD147 for 2 h at RT. Then, the mixture (biotin–Ap-9 and HAb18G/CD147) and avidin-Cy3 were added to the cells and incubated in the dark overnight at 4°C. The negative control, which was treated only with avidin-Cy3, was established at the same time. By use of the immuofluorescence double-staining method, the cells were incubated with 25 μg of HAb18 and an equivalent amount of biotin–AP-9 for 2 h at RT. After being washed with PBS, the cells were incubated with avidin-Cy3 (diluted 1:100) and FITC-labeled goat anti–mouse IgG (diluted 1:100; Wuhan BOSTER Bioengneer) in the dark overnight at 4°C. Careful observations were made by use of a confocal microscope (IX-7; Olympus) with an object lens (UPLAPO; ×20 or ×40) and image-acquisition software (FLUOVIEW FV 300; Olympus)

293 cells infected with SARS-CoV (BJ04) were harvested and pelleted. The cells were treated with 4% glutaral for 30 min at 4°C and then were washed with phosphate buffer twice and treated with 1% osmium tetroxide (Polysciences) for 1.5 h at RT. After they had been dehydrated and embedded, the ultrathin sections of the cells were prepared

The indirect gold colloid–labeling method was used to detect the localization of CyPA and HAb18G/CD147. After being washed in distilled water for 3 min, the ultrathin sections were treated with 1% H2O2 for 5 min and incubated with normal goat serum (diluted 1:75) for 10 min at RT and with rabbit anti–human CyPA antibody (diluted 1:25; Calbiochem-Novabiochem) or mouse anti–human CD147 antibody (diluted 1:50; Abcam) for 16 h at 4°C and for 1 h at RT. After being washed in PBS 3 times and in distilled water once, the sections were treated with PBS (containing 1% bovine serum albumin [BSA] [pH 8.2]) for 5 min and then were incubated with gold colloid–labeled protein A (diluted 1:50; diameter of the gold particle, 10 nm; prepared by the Academy of Military Medical Sciences [China]) for 1 h at RT and consecutively stained with 5% uranium acetate and lead acetate

Then, the gold colloid double-labeling method was used to determine the colocalization of HAb18G/CD147 and AP-9. After being washed, the sections were treated with 1% H2O2 and normal goat serum (diluted 1:75), as described above. After being incubated with PBS (containing 1% BSA [pH 8.2]) for 5 min, the cells were incubated with the mixture of gold colloid–labeled AP-9 (diluted 1:400; diameter of the gold particle, 20 nm; prepared by the Academy of Military Medical Sciences [China]) and mouse anti–human CD147 antibody (diluted 1:50) and then were incubated with the gold colloid–labeled protein A (diluted 1:50) for 1 h at RT and were consecutively stained with 5% uranium acetate and lead acetate. Careful electronic microscopic observations were made by use of a JEM-100SX microscope (JEDL)

293 cells (4×105 cells/mL) were planted into 96-well plates (Costar) and kept in 5% CO2 for 24 h at 37°C. For analysis of the toxicity of AP-9 to 293 cells, 2-fold dilution series of AP-9 (range, 3893.4–7.6 μmol/L), CB-2 (negative control; range, 4318.1–8.4 μmol/L), and gancyclovir (an antivirus chemical drug purchased from Hubei Keyi Pharmacy) injection (positive control; range, 23,508.2–45.8 μmol/L) were added to the cells. The TD0 and TD50 were determined. For analysis of the virulence of SARS-CoV (BJ04) to 293 cells, viruses in 8 dilutions (10−1–10−8) were added to the cells, to determine the TCID50 of SARS-CoV (BJ04). For the inhibitory-effect analysis of AP-9, the cells were incubated with 100 TCID50 of SARS-CoV (BJ04) for 2 h. After the supernatant was discarded, 2-fold dilution series of TD0 of AP-9 (range, 973.3–1.9 μmol/L), CB-2 (range, 359.8–0.7 μmol/L), and gancyclovir (range, 23,508.2–45.8 μmol/L) injection were added to the appropriate wells. Normal cell controls and virus controls were established. Cytopathic effects on the above cells were observed daily by use of an inverted microscope (XSZ-D; Nanjing Optical Instrument Factory of China) with an object lens (×10) and a camera (Ricon-5). Morphological changes observed in <25% of total cells were marked as “+”, in 25%–50% were marked as “++”, in 51%–75% were marked as “+++”, and in 76%–100% were marked as “++++”. TD50, TD0, TCID50, IC50, MIC, and treatment index (TI) were calculated by use of the Reed-Muench method. (For the inhibitory-effect analysis, when “+++” or “++++” was marked in virus control, the test was stopped.)

Results

Surface plasmon resonance analysis showed that none of the SARS-CoV S, M, E, or N proteins directly bound to HAb18G/CD147. However, N protein was found to interact with CyPA. In Co-IP analysis, when the mixture of HAb18G/CD147 and CyPA was added, CyPA was found to be indirectly bound to the gel, mediated by the specific interaction of HAb18G/CD147 to its MAb, HAb18, which was used as the capture antibody. Thus, the HAb18-HAb18G/CD147-CyPA complex was formed. After the coimmunoprecipitated CyPA was eluted, it was visible by Western blot analysis performed with anti-CyPA antibody, which confirmed the interaction between CyPA and HAb18G/CD147 (figure 1). Co-IP analysis further confirmed the interaction between N protein and CyPA. Since CyPA was used as the bait protein, the coimmunoprecipitated N protein was visible by Western blot analysis performed with anti–N protein antibody (figure 1). The binding kinetics of N protein (in different concentrations) to CyPA was determined by surface plasmon resonance analysis, with a Kd of 0.04 μmol/L (figure 1)

Figure 1

Analysis of protein-protein interaction. A Coimmunoprecipitation (Co-IP) analysis–revealed interaction between HAb18G/CD147 and cyclophilin A (CyPA). Blank, blank control; eluate, coimmunoprecipitated CyPA in the eluate; standard, 2.5 μg of purchased CyPA. B Co-IP analysis–revealed interaction between CyPA and severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid (N) protein. Blank, blank control; eluate 1, 2, and 3, coimmunoprecipitated N protein in orderly eluting; standard, 5 μg of N protein expressed in Escherichia coli. C Binding kinetics of SARS-CoV N protein to CyPA, determined by surface plasmon resonance analysis. N protein, at different concentrations, could bind to CyPA. The lines with different colors are the binding kinetics curves of N protein to CyPA, at different concentrations: 40, 32, 24, 16, and 8 nmol/L. RU, resonance unit

HAb18G/CD147 was found to be highly expressed on 293 cells, at the expression rate of 98.56% (figure 2). AP-9 was shown to be bound to the detected cells, at the binding rate of 98.15% (figure 2), which was very close to the expression rate of HAb18G/CD147 on the same kind of cells. AP-9 also had a median fluorescence intensity that was similar to that of HAb18G/CD147

Figure 2

Flow-cytometric analysis of HAb18G/CD147 and antagonistic peptide (AP)–9. A Expression of HAb18G/CD147 on 293 cells, analyzed by use of fluorescein isothiocyanate (FITC)–conjugated anti-CD147 antibody. B Binding of AP-9 to 293 cells, analyzed by use of biotin–AP-9 and avidin-FITC

After being traced by fluoresceins, by use of laser scanning confocal microscopy, AP-9 was found to have high affinity to the detected cells. After incubation with HAb18G/CD147, the binding of AP-9 to the cells obviously decreased (figure 3). By use of the immunofluorescence double-staining method, both HAb18G/CD147 and AP-9 were seen on the same binding sites (the cell membrane and the cytoplasm) of SARS-CoV–infected 293 cells (figure 3)

Figure 3

Confocal microscopic analysis of HAb18G/CD147 and antagonistic peptide (AP)–9. A Severe acute respiratory syndrome coronavirus (SARS-CoV)–infected 293 cells stained with biotin–AP-9 and avidin-Cy3 (red). The result showed that AP-9 was bound to the detected cells. The binding of AP-9 to the detected cells was partially blocked by HAb18G/CD147. B Immunofluorescence double-labeling method in SARS-CoV–infected 293 cells. Two kinds of fluorescence that indicated HAb18G/CD147 (green) and AP-9 (red) simultaneously presented in the cells, as observed by confocal imaging

After being traced by gold colloid–labeled antibody, by use of electronic microscopy, CyPA was present on or around the surface of SARS-CoV (figure 4), indicating that CyPA was integrated with SARS-CoV. HAb18G/CD147 was located on the detected cell surface and the unit membrane, especially on the membrane of endoplasmic reticulum (ER) in cytoplasm (figure 4). The gold colloid double-labeling method was used, and the results showed that AP-9 was present on the same sites as was HAb18G/CD147 (figure 4)

Figure 4

Subcellular localization of cyclophilin A (CyPA), HAb18G/CD147, and antagonistic peptide (AP)–9 on severe acute respiratory syndrome coronavirus (SARS-CoV)–infected 293 cells. A Gold particles representing CyPA (arrow) located on the virus surface in a cluster or around the virus. B Gold particles representing CD147 (arrow) located on the infected cell surface, across the membrane, or on the unit membrane in cytoplasm (especially on the membrane of endoplasmic reticulum) in cluster. C Gold colloid double labeling. Gold colloid particles of 2 different sizes that indicated AP-9 (20 nm) and HAb18G/CD147 (10 nm), respectively, were located on the same site (arrow)

The results of analysis of cytopathic effect showed that AP-9, at a concentration of 973.3 μmol/L, was nontoxic to 293 cells—the TD50 and TD0 for AP-9 were 1946.7 and 973.3 μmol/L, respectively. The TCID50 of SARS-CoV to 293 cells was 10−3. When 100 TCID50 of SARS-CoV was added to the normally cultured 293 cells, the cells became infected and necrotic. After an MIC of AP-9 was added, the infected cells gradually recovered, whereas the AP-9–untreated virus control cells remained necrotic. The IC50, MIC, and TI of AP-9 were 60.8 μmol/L, 30.4 μmol/L, and 32, respectively, whereas those of the positive control (with gancyclovir injection) were 91.8 μmol/L, 45.8 μmol/L, and 256, respectively; the TI of the negative control (with CB-2) was only 4

Discussion

It has been reported that CyPA can be specifically incorporated into the virions of HIV-1 and can significantly enhance an early step of cellular HIV-1 infection. CD147 can facilitate HIV-1 infection by interacting with virus-associated CyPA [6]. The present study has shown that HAb18G/CD147, a member of the CD147 family, can interact with CyPA, which may be associated with the SARS viruses and play a functional role in invasion of host cells by SARS-CoV. AP-9 has an inhibitory effect on SARS-CoVs

SARS-CoV is known to have 4 structural proteins: S, M, E, and N. S, M, and E proteins are envelope proteins of SARS-CoV, and N protein is bound to viral RNA in the core. S protein of the other known CoVs is regarded to be responsible for both binding to receptors on host cells and membrane fusion [2]. In the present study, we have found that N protein may play a role in invasion of host cells by SARS-CoV. N protein was bound to CyPA, but S, M, and E proteins were not. However, CyPA was present only on the surface of mature SARS-CoVs, as observed by electronic microscopy. This finding seems to be contradictory to the finding that N protein locates in the core of the virus. This difference can be partly explained by the finding of a previous report that CyPA bound to viral proteins in the core can be relocated from the core to the viral surface during maturation of the virus [35]. The results of the present study also confirm the interaction between CyPA and HAb18G/CD147, as determined by Co-IP analysis, indicating that CyPA may act as a mediator between SARS-CoV N protein and HAb18G/CD147 in the process of invasion of host cells by SARS-CoV. By flow-cytometric analysis, confocal microscopy, and immunoelectronic microscopy, we found that HAb18G/CD147 was highly expressed in the SARS-CoV–permissive 293 cells and that it was located on the cytomembrane and the unit membrane in the cytoplasm (especially on the membrane of ER) as a transmembrane molecule. In the present study, the binding site of AP-9 was confirmed to be on the HAb18G/CD147 molecule in the infected 293 cells, and AP-9 could efficiently block the binding sites of HAb18G/CD147 on the cytomembrane and the unit membrane in the cytoplasm of the 293 cells and had an inhibitory effect on SARS-CoV in vitro. From this finding, we can conclude that HAb18G/CD147 is a functional molecule in SARS-CoV infection of host cells

The mechanism of HAb18G/CD147 as a functional molecule can be inferred as follows: (1) CyPA is bound to N protein after invasion of host cells by SARS-CoV, and CyPA relocates to the virus surface during the maturation of the virus [35]; (2) the exposed CyPA molecules interact with HAb18G/CD147 on the cell membrane, which leads to the infection of other host cells; and (3) AP-9 blocks HAb18G/CD147, to prevent virus infection after those viruses complete their life cycle

It also has been reported that the life cycle of CoVs that invade host cells includes N protein assembling with the full-length replicated RNA to form the RNA protein complex, which is associated with the M protein embedded in the membrane of ER and virus particles, which are formed as the nucleocapsid complex buds into ER [2, 36]. SARS-CoV is believed to have a similar process in the life cycle [36]. Therefore, we also inferred that SARS-CoV N protein associated with CyPA could interact with HAb18G/CD147 located on the membrane of ER and that the interaction could facilitate the virus particles in forming or budding into ER. AP-9, the small peptide, could enter the cells to prevent the virus particles from forming or budding into ER, by blocking HAb18G/CD147 located on ER. Thus, HAb18G/CD147 plays an important role in the process of invasion of host cells by SARS-CoV

Both the function of HAb18G/CD147 in invasion of host cells by SARS-CoV and a clearer understanding of the mechanism responsible for the process need further confirmation. However, the present study may provide some evidence for the cytologic mechanism of invasion by SARS-CoV and may provide an important molecular basis for screening some anti-SARS drugs, such as antibody, polypeptide, immunocomplex, and small-molecule compounds