The Production and Characterization of a Mutant Form of Herpes Virus Entry Mediator (HVEM)

Abstract

Herpes Simplex Virus (HSV) entry is a multistep process involving viral glycoproteins and cellular receptors. Viral glycoproteins bind cell-surface glycosaminoglycans, but this binding is not sufficient for virus entry. A cellular coreceptor is also essential for virus entry. Herpes Virus Entry Mediator (HVEM), an integral membrane protein, is a co-receptor that can mediate HSV entry. Hybrid proteins composed of HVEM and CD40 have shown that only one specific region, Cysteine Rich Repeat Domain 1 (CRRD1), of HVEM is essential to mediate HSV entry. The purpose ofthis project was to identify amino acids within CRRD1 essential for HVEM-mediated HSV entry. Site-directed mutagenesis using long PCR was utilized to introduce mutation(s) into a mammalian expression vector encoding HVEM. Mutation(s) were constructed to change specific amino acids in CRRD1. Mutant vectors were transfected into mammalian cells that are normally resistant to HSV entry. Transfected cells were then challenged with reporter virus to assay for entry-mediating ability. Results show one mutant was created that has virus entry-mediating ability at a lower level than wild type HVEM. However, the mutation(s) carried by this mutant protein are not consistent with the mutations that were specifically designed to be inserted into the new HVEM protein. This thesis describes the creation and characterization of the new HVEM mutant.