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 inflammatio [6, 10] . HAb18G/CD147, which was developed and was identifie to be a member of the CD147 family in our laboratory, is involved in tumor metastasis, inflamma tion, and virus infection [11] [12] [13] [14] [15] [16] [17] [18] [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.
for Disease Control and Prevention (China CDC). The cells were cultured in Dulbecco's MEM (Gibco BRL) containing 10% fetal calf serum (Gibco BRL).
Peptides. HAb18G/CD147-antagonistic peptide (AP)-9 (an AP of HAb18G/CD147 obtained in our laboratory, composed of 12 aa residues) [20] [21] [22] [23] [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 195%, 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-YKLP-GHHHHYRP and 1767.69.
Coimmunoprecipitation (Co-IP) analysis. 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 mg 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 mg of mouse anti-human HAb18G/CD147 monoclonal antibody (MAb) HAb18 (prepared in our lab) [25] [26] [27] [28] [29] [30] [31] [32] [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 mg) 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 mg 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.
BIAcore spectroscopy. 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 mg/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 25ЊC, at a flo rate of 5 mL/min for determination of on-rates and equilibrium binding and 50 mL/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 mg/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 affinit 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 firs injection. To correct refractive index changes and nonspecifi 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 fitte to the following reaction mechanism, in which CyPA and N protein are represented by A and B, respectively:
. An equilibrium dissoci-A + B s AB Kd ation rate constant (K d ) was calculated from the kinetic rate constants by nonlinear fittin of the primary sensorgram data by use of BIAevaluation software (version 3.1; BIAcore).
Flow-cytometric analysis. 293 cells, at a density of 10 6 cells/ mL, were incubated with either 10 mL of fluo escein isothiocyanate (FITC)-conjugated anti-CD147 antibody (Pharmingin) or 10 mL of FITC-conjugated mouse IgG1 (control; Pharmingin) in the dark for 30 min at 4ЊC. After being washed once with PBS, cells were fixe with 1% paraformaldehyde and analyzed by use of a FACSCalibur flo 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 (fina concentration, 160 mg/mL) for 60 min, were washed and treated with 5 mL of avidin-FITC (Pharmingin) in the dark for 60 min at 4ЊC. Fixation and data analysis were performed as described above.
Confocal microscopy. 293 cells infected with SARS-CoV (BJ04) were cultured overnight on the sterilized cover slips and then washed in PBS and fixe with cold acetone for 30 min. For analysis of AP-9 binding, the cells were incubated with 25 mg of biotin-AP-9 and avidin-Cy3 (diluted 1:100; Sigma) in the dark overnight at 4ЊC. For analysis of blocking of AP-9, 25 mg of biotin-AP-9 was incubated with 2.5 mg 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 4ЊC. The negative control, which was treated only with avidin-Cy3, was established at the same time. By use of the immuofluo escence double-staining method, the cells were incubated with 25 mg 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 FITClabeled goat anti-mouse IgG (diluted 1:100; Wuhan BOSTER Bioengneer) in the dark overnight at 4ЊC. Careful observations were made by use of a confocal microscope (IX-7; Olympus) with an object lens (UPLAPO; ϫ20 or ϫ40) and image-acquisition software (FLUOVIEW FV 300; Olympus).
Immunoelectronic microscopy. 293 cells infected with SARS-CoV (BJ04) were harvested and pelleted. The cells were treated with 4% glutaral for 30 min at 4ЊC 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% H 2 O 2 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 4ЊC 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% H 2 O 2 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 colloidlabeled 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. with 100 TCID 50 of SARS-CoV (BJ04) for 2 h. After the supernatant was discarded, 2-fold dilution series of TD 0 of AP-9 (range, 973.3-1.9 mmol/L), CB-2 (range, 359.8-0.7 mmol/L), and gancyclovir (range, 23,508.2-45.8 mmol/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 (ϫ10) 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 "++++". TD 50 , TD 0 , TCID 50 , IC 50 , 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.)
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 specifi 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 confi med the interaction between CyPA and HAb18G/ CD147 (figu e 1A). Co-IP analysis further confi med 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 (figu e 1B). The binding kinetics of N protein (in different concentrations) to CyPA was determined by surface plasmon resonance analysis, with a K d of 0.04 mmol/L (figu e 1C).
to them. HAb18G/CD147 was found to be highly expressed on 293 cells, at the expression rate of 98.56% (figu e 2A). AP-9 was shown to be bound to the detected cells, at the binding rate of 98.15% (figu e 2B), which was very close to the expression rate of HAb18G/CD147 on the same kind of cells. AP-9 also had a median fluo escence intensity that was similar to that of HAb18G/CD147.
After being traced by fluo esceins, by use of laser scanning confocal microscopy, AP-9 was found to have high affinit to the detected cells. After incubation with HAb18G/CD147, the binding of AP-9 to the cells obviously decreased (figu e 3A). By use of the immunofluo escence 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 (figu e 3C).
After being traced by gold colloid-labeled antibody, by use of electronic microscopy, CyPA was present on or around the surface of SARS-CoV (figu e 4A), 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 retic-ulum (ER) in cytoplasm (figu e 4B). The gold colloid doublelabeling method was used, and the results showed that AP-9 was present on the same sites as was HAb18G/CD147 (figu e 4C).
The results of analysis of cytopathic effect showed that AP-9, at a concentration of 973.3 mmol/L, was nontoxic to 293 cells-the TD 50 and TD 0 for AP-9 were 1946.7 and 973.3 mmol/L, respectively. The TCID 50 of SARS-CoV to 293 cells was 10 Ϫ3 . When 100 TCID 50 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 IC 50 , MIC, and TI of AP-9 were 60.8 mmol/L, 30.4 mmol/L, and 32, respectively, whereas those of the positive control (with gancyclovir injection) were 91.8 mmol/L, 45.8 mmol/L, and 256, respectively; the TI of the negative control (with CB-2) was only 4.
It has been reported that CyPA can be specificall incorporated into the virions of HIV-1 and can significantl 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 findin seems to be contradictory to the findin that N protein locates in the core of the virus. This difference can be partly explained by the findin 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 confi m 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-cytometri 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 confi med to be on the HAb18G/ CD147 molecule in the infected 293 cells, and AP-9 could efficientl 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 fulllength 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 confi mation.
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.