Use of short interfering RNAs (siRNAs) to investigate which equine herpesvirus type 1 (EHV-1) glycoproteins are essential for infection.
Presentation
Overview
Overview
Description
The goal of our project is to identify which equine herpesvirus type 1 (EHV-1) glycoproteins are necessary and sufficient for productive infection. There are twelve EHV-1 glycoproteins embedded within the lipid envelope of the virus. To investigate the functions of these glycoproteins, and to assess which are essential for infection, we will employ short interfering RNA (siRNA) technology. Short-interfering RNA molecules are 18-25 base pairs long and they disrupt expression of a particular gene by inhibiting the proper translation of messenger RNA (mRNA). From a viral DNA template, the glycoprotein genes gK, gN, gB, gH, gM, gL, gG, and gE were amplified via polymerase chain reaction. Two of these genes, gK and gL, were inserted into the expression vector, LITMUS 28i and the resulting plasmid was transformed into competent E.coli. The plasmids were isolated from bacteria, purified, and then digested with specific restriction enzymes to verify that they contained gK or gL. siRNA specific for gK and gL will be generated from these plasmids and tested for their ability to knockdown expression of the targeted glycoprotein and for their ability to inhibit EHV-1 infection. Similar experiments will be performed with the other 10 glycoproteins.
