Investigating HSV-1 Host-Viral Protein Complexes and Expression Patterns using Quantitative Proteomics

Abstract

Herpes Simplex Virus 1 (HSV-1) is a prevalent, contagious pathogen that is historically known to cause facial ulcers, but has also been shown to rarely cause encephalitis and keratitis. HSV-1 has a complicated life cycle with three known stages of gene transcription: Immediate-Early (IE), Early, and Late. HSV-1 structural proteins have been shown to transactivate IE gene activation, leading to the creation of a protein concentration gradient through the activation and degradation of different host and viral factors for efficient replication. In our first project, we used fluorescently-labeled viruses in conjunction with immunoaffinity purification and mass spectrometry to study virusvirus and virus-host protein interactions during HSV-1 infection in primary human fibroblasts and found an interesting nuclear association between viral capsid proteins and the de novo DNA methyltransferase DNMT3A, which we confirmed by reciprocal isolations and microscopy. We therefore identified DNMT3A as a possible target for future drug design. In addition to the host perspective, I was interested in how the coordinated expression of viral proteins orchestrate viral replication and assembly. To begin to answer this broad question, we must have comprehensive, quantitative profiles of viral gene expression. Currently, this cannot be achieved with antibody-based methods. Thus, I undertook a second project to continue development of a mass spectrometry-based method for absolute quantification of the HSV-1 proteome using heavy-labeled viral protein concatemers (QconCATs). This is the first attempt to design a method for analysis of viral, not host, protein abundance during infection to understand the epigenetic changes necessary for replication.