Research Description

Identification of Binding Proteins on Portions of the Latency Associated Transcript in Herpes Simplex One using the Yeast One-Hybrid Model

Introduction

Herpes Simplex One is a double stranded DNA virus that initially infects the epithelial cells before traveling up the sensory neurons to establish latency in the ganglia. During latency, only the Latency Associated Transcript (LAT) is expressed. All other genes, referred to as the lytic genes, are essentially turned off. Although LAT’s mechanism is not expressly known, it plays an important part in reactivation since mutants lacking LAT transcription are unable to reactivate in animal models. The aim of this project is to identify host proteins that bind to LAT's Promoter and Enhancer and, therefore, might influence the reactivation cycle by controlling transcription.

Yeast One-Hybrid System

This project's goal is to uncover host proteins that bind to either the DNA or surrounding chromatin of LAT’s Promoter and Enhancer using the Yeast One-Hybrid Model. This model uses two different plasmids within a mutant yeast cell that cannot produce leucine or histidine to screen a 106 clone cDNA library (isolated from non-infected rabbit trigeminal ganglia) for binding proteins. One plasmid, pGAD-10, has a segment of cDNA library cloned into it, along with a necessary protein for leucine synthesis. This plasmid is freely transcribed in the yeast cell. The plasmid pHisi-1 contains the bait DNA (fragment of LAT’s Promoter or Enhancer) and a transcript for an essential enzyme for histidine production. This plasmid is minimally transcribed. If the cDNA protein (from pGAD-10) does bind to the bait DNA, it induces transcription of pHisi-1 so the cell can now create a colony on agar deficient in leucine and histidine.

Importance of Project

This project is important since it will identify host proteins that interact with the virus to control its reactivation cycle. Clinical symptoms of HSV-1 reactivation include painful lesions at the original point of infection such as the eye and lip. Repeated sores on the eye can lead to Herpes Keratitis, a leading cause of infectious blindness. Current therapies are only effective when the virus has already reactivated. Finding the proteins that regulate LAT can lead to a better therapy where HSV-1 is kept in a state of latency.