Scott S. Terhune, PhD
Microbiology and Molecular Genetics & Biotechnology and Bioengineering Center
Medical College of Wisconsin
Research Focus: Human Cytomegalovirus-Host Cell Protein Interactions during Infection
PhD: Northwestern University Medical School (2000)
Postdoctoral Training: Princeton University, Princeton, NJ
The objective of my research is to determine the functions of human cytomegalovirus (HCMV) proteins through identifying protein-protein interactions during infection using mass spectrometry. HCMV is a member of the beta-herpesvirus family. Infections in healthy children and adults are generally asymptomatic. However, primary infection or reactivation of latent virus in immunologically immature or compromised individuals results in life threatening disease. The HCMV genome contains greater than 200 open reading frames (ORF) but it is unclear which ORFs are expressed during infection. Expression of viral genes occurs through a regulated cascade with immediate early genes first followed by early and late genes.
My work focuses upon the role of viral proteins expressed at immediate early and early times during HCMV infection that are necessary to maintain the cell in a permissive state for virus replication. Using powerful genetics available for manipulating infectious BAC (bacterial artificial chromosome) clones of HCMV strains, we have constructed a library of viruses with each virus containing a different epitope tagged open reading frame (Fig. 1). These tags include the TAP (tandem affinity purification), YFP (yellow fluorescence protein), and FLAG epitope. Viral and cellular interacting proteins are then isolated from infected cells using a rapid one-step immunoaffinity purification and then identified by mass spectrometry (Fig. 2).
This approach of combining viral genetics and proteomics has been successfully used to demonstrate the function of the viral early protein, pUL38. During infection, HCMV pUL38 interacts with multiple protein complexes including tuberous sclerosis protein complex (TSC1/TSC2) and the HDAC1-containing nucleosome remodeling and deacetylation complex (Mi-2/NuRD). TSC1/2 functions by integrating cellular stress signals resulting, in part, in a block in protein translation. pUL38 is necessary and sufficient to interfere with TSC1/2 function during infection. The pUL38/NuRD interaction is necessary for efficient virus replication and we hypothesis that this occurs through modulating both viral and cellular gene expression.
Projects in my lab include: Further characterizing the role of pUL38/NuRD complex during infection; determining the relationship between the pUL38/TSC and pUL38/NuRD complexes; and using genetics and proteomics to determine the role of additional HCMV proteins of unknown function during infection. I am also interested in investigating the role of class I and II histone deacetylases as well as members of the interferon-inducible p200 family using epitope tagged cellular proteins and mass spectrometry during HCMV infection.
Tagged HCMV viral proteins expressed at various time during infection.
Methodology for identifying viral and cellular interacting proteins during HCMV infection using MS analysis.
Scott Terhune, Ph.D.
Department of Microbiology and Molecular Genetics
8701 Watertown Plank Road, Milwaukee, WI 53226
Room: TBRC C2890