Cloning and Characterization of the Mouse Hepatitis Virus Receptor
Abstract
The attachment of mouse hepatitis virus (MHV), a coronavirus, to the host cell membrane is the key first step leading to viral infection. The cellular receptor for MHV has been previously characterized as a 100 -120 k Damembrane glycoprotein, found in colon, small intestine and liver. This receptor has been shown to be the only portal of entry for MHV-A59. Identification of the mouse gene for the MHV receptor is essential in understanding the mechanism of host cell-virus interaction. To this end, a new cloning strategy based on the polymerase chain reaction technology was developed using RNA as starting material (RNAPCR). I employedglyceraldehyde-3-phosphate dehydrogenase as a control gene for the establishment of this cloning strategy. Amino acid homology and antibody reactivity had pointed to the murinecarcinoembryonic antigen (CEA) family as a candidate for the cellular receptor for MHV. Using the RNAPCR system with information obtained from the partial N-terminal amino acid sequence for the MHV receptor and a partial murine CEA cDNA sequence, a 710 bp product was obtained. Nucleic acid sequencing confirmed that this clone was a portion of the receptor. This fragment was then used as a probe to screen a BALB/c liver lambda gtl 1 cDNA library, from which a clone was obtained that begins at amino acid 10 and ends with a poly A tail. Using an alternative PCR technique, the sequence of the first 10 amino acids of the mature receptor protein and part of the leader peptide were then identified. The partial MHV receptor cDNA was transcribed and translated in vitro. The in vitro synthesized protein had the predicted size based on the amino acid sequence, and was immuno precipitated with polyclonal antibody directed against affinity-purified MHV receptor. This polyclonal antibody has been shown to block MHV infection of murine tissue culture cells to a dilution greater than1/1,200.
Document Details
- Document Type
- Technical Report
- Publication Date
- Feb 11, 1991
- Accession Number
- AD1010992
Entities
People
- Gabriela S. Dveksler
Organizations
- Uniformed Services University of the Health Sciences