Invasion of virus into the cell is by the receptor and ligand.In what way to verify the receptor?
Depends on the nature of the receptor. For influenza virus the receptors on host cells are oligosaccharides attached to proteins or proteoglycans. The interaction can be detected by a glycan array which has multiple short sequences of sugars joined in various ways attached to different grid squares on a chip. One then adds the virus and then a labeled anti-virus antibody and measures fluoresence. One could also do immunoprecipitation with host cell membrane extract + virus particles + labeled antibody to virus followed by western blot. The band can be cut from a gel and analyzed by mass spectrometry. However, my understanding is that the identification of specific oligosaccharide sequences by this method is not as advanced as for proteins and requires considerable expertise. If the receptor is a protein, the latter option (immunoprecipitation followed by mass spec) should be relatively straightforward, as many universities have proteomics core facilities that could identify the protein(s) bound by the virus.
Depends on the nature of the receptor. For influenza virus the receptors on host cells are oligosaccharides attached to proteins or proteoglycans. The interaction can be detected by a glycan array which has multiple short sequences of sugars joined in various ways attached to different grid squares on a chip. One then adds the virus and then a labeled anti-virus antibody and measures fluoresence. One could also do immunoprecipitation with host cell membrane extract + virus particles + labeled antibody to virus followed by western blot. The band can be cut from a gel and analyzed by mass spectrometry. However, my understanding is that the identification of specific oligosaccharide sequences by this method is not as advanced as for proteins and requires considerable expertise. If the receptor is a protein, the latter option (immunoprecipitation followed by mass spec) should be relatively straightforward, as many universities have proteomics core facilities that could identify the protein(s) bound by the virus.
Flow cytometry is a straithforward technique to check for the presence of a viral receptor in cell membrane, receptor facing the outside of the cell. This technique requires specific antibodies or ligands specific for binding to the receptor on living or fixed cells, this is the crucial point. These antibodies or ligands can also be visualized by secondary labelled antibodies, with the advantage of amplification of the signal. The sensitivity of the technique is quite good, with the advantage, compared to biochemical techniques, that you can distinguish and characterize subpopulations of cells which express the receptor in your sample. you can also sort these cells alive for further experiments...
1) Cells lacking the receptor should be resistant to infection
2) Antibodies against the receptor moiety should/could block infection
3) Purified ectodomain of the receptor should directly interact with virus glycoprotein or viral capsid. Can confirm by in vitro binding studies (pull-down assays, ELISA, SPR etc)
4) Soluble purified receptor ectodomain added to susceptible cells should/could block infection.
5) Introduction of a cDNA clone of the receptor molecule to a non-susceptible cell line should/could confer susceptibility.
Not always easy to show a receptor molecule is real.
- If the receptor is not a protein, you can use enzymes such as sialidases for removing receptors from the cell surface.
- If the recepto contains oligosaccharides you can use specific lectins that recognizes specific oligosaccharide sequences (including fluorescence labelled lectins in FACS assays) for charecterizing
Thanks for the answers from Stephen Pruett,Vincent François , John S L Parker, Isabel Muñoz-Barroso.It's interesting about the receptor. I thnki i should read more basis materials about it.
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