Courtney Ewart's Research at Davidson
Molecular Analysis of the Mouse Hepatitus Virus Receptor, MHVR

The murine coronavirus mouse hepatitis virus strain A59 (MHV), which affects mice, has a primary cellular receptor called MHVR. MHVR isa transmembrane murine biliary glycoprotein, which exists naturally as a 2- or 4-domain protein. The primary binding site for MHV-A59 is a small sequence of amino acids in domain 1. However, when domain 4 is absent, MHVR is no longer a functional receptor for MHV. This research project involves investigating what regions other than domain 1 of MHVR are important in the virus-binding event of MHV-A59, and serves as a model for interactions between viruses and their host receptors.

Two different constructs of MHVR are being used in this investigation. One construct, containing domains 1 and 2 of MHVR (1,2), is a non-functional receptor. The other construct (1,4), which contains domains 1 and 4, functions normally as a receptor for the mouse hepatitis virus. The general procedure for this project is to make the (1,2) receptor look more like the (1,4) MHVR by mutating small sequences in domain 2, and then monitoring the receptor activity of the mutants.

The two MHVR constructs were initially PCR amplified, extracted and purified from their original insect expression vectors because they eventually need to find their way into a mammalian expression plasmid. The MHVRs were then ligated into a cloning vector (pGEM-T), as an intermediate step before cloning them into their mammalian vector. Southern blot analysis showed that only MHVR(1,2) was successfully cloned into pGEM-T. Thus, MHVR(1,4) was extracted from a different plasmid, and its presence has been indicated so far on an agarose gel. Southern blot analysis remains to be done.

A mutagenesis protocol was then chosen and primers designed to create one deletion and one mismatch mutation in MHVR(1,2) so that it resembles MHVR(1,4) more closely. The mutations are in the process of being made. Once the mutagenesis protocol is complete, the constructs will be sequenced to ensure the mutations occurred. Finally, these mutated receptors will be cloned into a mammalian expression plasmid (pcDNA3.1) so that their activity can be monitored in gerbil cells. The gerbil cells will be induced to express the plasmids containing the mutated receptors. These cells will then be exposed to MHV to monitor the activity of the mutant receptors.


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