Identification of NCAM that interacts with the PHE-CoV spike protein

Abstract

Background: The spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies.

Results: We identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection.

Conclusions: A novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

Figure 1

The results of display of the cDNA library from N2a cells on T7 phage. (A) Lane1:The result of extracted total RNA of N2a cells. The electrophoresis results show 28 S and 18 S bands were clear, indicating the total RNA extraction without degradation. M: DL2000 Marker. (B) Lane1:The result of purified mRNA of N2a cells. OD260/OD280 = 1.950. The data show that the purified mRNA could be used for cDNA synthesis. M: DL2000 Marker. (C) Lane1 to 10: The PCR identified result of randomly picked phage clones of the library. Amplification of inserts in randomly selected clones revealed that the library contained >90% recombinants with average insert size of >300 bp. M: DL2000 Marker.

Figure 2

Detection of inserted fragments of phage clones in the fifth round of selection library by PCR. M: DL2000 Marker; Lane 1 to 5: The PCR result of randomly picked phage clones of the fifth round of selection library. (A) Approximately 38% of the phage clones had an insert size of 830 bp. (B) 25% of the phage clones had an insert size of 750 bp. (C) 22% of the phage clones had an insert size of 400 bp. (D) 15% of the phage clones had an insert size of 250 bp.

Figure 3

Western blot analysis of proteins expression in total extracts of 293T cells transfected with the pcDNA3.1 (+) expression plasmid. Lane 1: Western blot analysis of NCAM protein expression. The full lengths cDNA of NCAM gene was used to construct the transfect plasmid. Cell lysates from 293T cells were run on a 10% SDS-PAGE gel and blotted onto polyvinylidene difluoride membranes. The blots were probed with a 1:10 dilution of the rabbit anti-NCAM polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies and chemiluminescence. Lane 2: Immunoblots for Hdac2 protein. The blot was probed with a 1:10 dilution of the rabbit anti-Hdac2 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-rabbit IgG antibodies. Lane 3: Immunoblots for RPS13 protein. The blot was probed with a 1:10 dilution of the goat anti-RPS13 polyclonal IgG (200 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated mouse anti-goat IgG antibodies. Lane 4: Immunoblots for Sf3b2 protein. The blot was probed with a 1:5 dilution of the mouse monoclonal anti-Sf3b2 IgG2a (100 μg/ml). The antibodies were detected by horseradish peroxidaseconjugated goat anti-mouse IgG antibodies. Lane 5: The 293T cells transfected with vector alone.

Figure 4

The NCAM binding to PHE-CoV S protein. Lane 1-3, NCAM involves in recognition by PHE-CoV S protein. Supernatants of 293T cells transfected with plasmid encoding soluble NCAM. The 293T cells were added fusion S protein (6 mg) and incubate for 2 h at 4°C. The cells were lysed in 500 μl of radioimmune precipitation buffer. The 10-ml aliquot of lysate was incubated with 300 μl of glutathione-Sepharose beads conjugated with fusion anti-S protein antibody and gently rocking on a orbital shaker overnight at 4°C. The sepharose beads are boiled for 5 min to dissociate the immunocomplexes from the beads. The supernatant was electrophoresed through 12% sodium dodecyl sulfate-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The blots were blocked at room temperature for 3 h with 3% BSA in PBS containing Tween 20 (0.05%) and then incubated overnight with the anti-NCAM protein antibody. The proteins was analyzed by western blotting. Lane 4-6, Sf3b2, Hdac2 and RPS13 were not immunoprecipitated with S protein. Lane 7, 293T cells transfected with vector alone were negative controls.

Figure 5

Anti-NCAM antibody inhibition of PHE-CoV binding to N2a cells. PHE-CoV binding assay using various concentrations of anti-NCAM antibody. The 10 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 75%. With the increased anti-NCAM antibody concentration in the blocking, the inhibition rate increased accordingly. The 25 μg/ml anti-NCAM antibody inhibited PHE-CoV binding to N2a cells by 95.7%. However, the proliferation of virus could not be suppressed completely.

Figure 6

Double-stranded siRNA could effectively inhibit NCAM expression in N2a cells. N2a cells were transfected with siRNA targeted against NCAM. The cells were harvested after siRNA transfection and analyzed by FACS with rabbit anti-NCAM antibody and FITC-conjugated goat anti-rabbit IgG (H+L). Mock-transfected siRNA N2a cells served as a control. There appeared to be a slight decrease of the positive rate of N2a KD cells compared to that of controls within 72 hours.

Figure 7

The NCAM siRNAs inhibit PHE-CoV infection by indirect immunofluorescence. After a 48 h viral infection, the N2a cells were fixed with 80% acetone for 10 min at -20°C, rehydrated in PBS, labeled with rabbit PHE-CoV antiserum, and washed three times with PBS. FITC-conjugated goat anti-rabbit IgG (H+L) (1:50 dilution) was added to the N2a cell mixtures for 30 min at room temperature, and the cells were washed and observed with an Olympus FV1000 laser scanning confocal microscope. Microscopic magnification, 400×. (A) Mock transfection (stained with PHE-CoV-positive serum); (B) Mock transfection (stained with PHE-CoV-negative serum); (C) siCtrl transfection; (D) siNCAM79 transfection; (E) siNCAM81 transfection; (F) siNCAM90 transfection.

Figure 8

The NCAM siRNAs could inhibit PHE-CoV infection in a period of time. Culture supernantants were collected 120 hours after the PHE-CoV challenge. The supernatants harvested at indicated timings were subjected to plaque assay. There was significant difference (p < 0.05) in virus titres. Knock-down of NCAM caused a marked reduction of PHE-CoV infection within 84 hours.