Scott S. Terhune, PhD
Microbiology and Immunology & Biotechnology and Bioengineering Center
Medical College of Wisconsin
Research Focus: Viral manipulation of cellular processes
PhD: Northwestern University, Chicago, IL (2000) Cancer Biology
Postdoctoral Fellow: Princeton University, Princeton, NJ (2007) Proteomics/Cytomegalovirus Biology
Uncovering molecular mechanisms of viral protein function
My laboratory is interested in determining the underlying molecular mechanisms of human cytomegalovirus protein function during infection. Our current projects focus on defining how viral proteins manipulate cellular processes early during infection to construct a permissive cellular environment for replication. We accomplish our goal by combining targeted proteomics and viral genetic manipulations with basic approaches in cellular and molecular biology. Human cytomegalovirus (HCMV) is a member of the beta-herpesvirus family. Infections in healthy children and adults are generally asymptomatic. However, primary infection, reinfection or reactivation of latent virus in immunologically immature or compromised individuals results in life threatening disease. HCMV is also the leading viral cause of congenital birth defects.
The virus expresses a large repertoire of proteins during lytic replication. We have constructed a library of viruses containing different epitope tagged open reading frames (Fig. 1). These tags include the TAP (tandem affinity purification), YFP (yellow fluorescence protein), and FLAG epitope. Viral and cellular interacting proteins are isolated from infected cells using rapid one-step immuno-affinity purification and are then identified by mass spectrometry (Fig. 2). We use the information in conjunction with phenotypic data to formulate testable hypotheses. Follow-up studies include tagging the targeted cellular and viral proteins to further define the cellular pathways involved.
Figures 1 and 2
Cytomegalovirus manipulation of cellular stress response pathways
One of our primary interests is to understand how HCMV proteins manipulate the cellular tumor suppressor protein, p53 and affiliated cellular stress response pathways. HCMV infection induces p53 yet alters its transcriptional activity. We have demonstrated that the HCMV protein pUL29/28 with pUL38 binds and stabilizes a population of p53 that is transcriptionally inactive at a subset of promoters including p21Cip1 (Fig. 3). Proteomic studies have shown that pUL29/28 also bind a deacetylase and chromatin remodeling complex. Current studies are focusing on defining the underlying molecular mechanism and functional role of p53 regulation during infection. HCMV pUL38, independent of pUL29/28, is also necessary and sufficient to antagonize the tumor suppressor, TSC2. We are interested in determining the functional relationship between these two events.
Coordinated manipulation of cell cycle to support viral replication
Our laboratory and others have observed that the antiviral activity of maribavir is influenced by the mitotic cell cycle and can select for antiviral resistant variants. Combining proteomics with viral genetics, we defined the mechanism of antiviral resistance associated with the HCMV gene, UL27. We determined that inhibition of replication by maribavir involves pUL27-dependent degradation of Tip60 acetyltransferase and induction of the Cdk inhibitor, p21Cip1. In addition, we observed that inhibiting cellular signaling pathways during infection resulting in increased p21Cip1 levels synergizes with maribavir to block infection. This includes STAT3 signaling and we have defined mechanisms of HCMV manipulation of STAT3 during infection. Current studies are focusing on the role of these viral and cellular proteins in regulating HCMV replication and antiviral activity.
Use of advanced tools to define host-pathogen interactions
Our research applies advanced methods in proteomics and other technologies developed in the Biotechnology and Bioengineering Center to study cytomegalovirus and other pathogens through collaborative projects. These include: Defining ubiquitin and ubiquitinated proteins as activators of the Pseudomonas aeruginosa toxin, ExoU (Frank Lab, MCW); Identifying substrates of the Mycobacterium tuberculosis protease involved in latency, PepD (Zahrt Lab, MCW); Defining the impact of HCMV infection on connective tissue mechanics (Wakatsuki Lab, InvivoSciences); Determining the nuclear proteome of HCMV-infected cells at early times of infection (MCW Proteomics Core) and computational modeling of HCMV infection (Dash Lab, MCW). Our research is at the forefront of the HCMV field, and through our collaborations we are expanding our scientific understanding of our favorite pathogen. Our collaborative efforts continue to yield new insight regarding host-pathogen interactions.