Vaccinia virus (VACV) strain Western Reserve gene A40R encodes a type II membrane glycoprotein with a C-type lectin-like domain at the C terminus. The A40 protein is not incorporated into virions, is nonessential for virus replication in cell culture and does not affect virus virulence in a murine intranasal model of infection. However, A40 does affect the outcome of infection in an intradermal infection model in which the virus lacking gene A40R produced smaller lesions and alterations in the host immune response. A40 has amino acid similarity to C-type lectins, such as NKG2A and DC-SIGN. This observation together with its location on the infected cell surface and its ability to bind to the surface of cells of the immune system is consistent with A40 functioning as an immunomodulator. It is possible that A40 might function by mimicking native host lectins or by modulating recognition of VACV-infected cells by cells of the immune system. To investigate the mechanism by which A40 affects the outcome of infection in vivo, a cloning and experimental strategy was devised to search for its ligand(s) and try to determine its structure in collaboration with protein crystallographers. To achieve these goals, the recombinant A40 protein has been produced in E. coli, mammalian cells, and from insect cells infected with recombinant baculoviruses. Bacterially expressed recombinant A40 was used to generate an antibody specific to A40 and this was then purified, characterised and used to further the characterisation of A40. The potential for A40 to interact with a ligand on the surface of another cell was investigated by a cell binding assay using recombinant A40 protein. This protein was produced in a mammalian system and was found to bind to the surface of immune cells but not to epithelial cells. In cytotoxicity assays, the absence or over-expression of A40 was found to modulate the ability of NK cells to kill VACV infected cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:508477 |
| Date | January 2009 |
| Creators | Jarmin, Susan Anne |
| Contributors | Smith, Geoffrey |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/4717 |
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