Department of Microbiology, A Leader in Microbiology and Microbial Pathogenesis Research and Training.

Career goals- My goal is to become an independent investigator of infectious viral diseases. I am interested in using interdisciplinary approaches to develop novel strategies and therapeutics for preventing and controlling viral transmission that will be applied clinically.

Thesis proposal- Human cytomegalovirus (HCMV) is the leading cause of virus-induced birth defects and is a major opportunistic pathogen for the immunocompromised^1,2. The clinical recognition of primary HCMV infection is complicated by the fact that acute replication is typically asymptomatic in immunocompetent individuals. A r​obust immune response limits viral replication to sites of persistence in the salivary glands, mammary glands, and kidneys. At these sites, HCMV establishes a long-lived, persistent infection that promotes transmission via saliva, breast milk, and urine to naive individuals. A major challenge to controlling HCMV infection is to define the mechanisms that control viral persistence and transmission. Our lab studies a large, stable non-coding RNA encoded by HCMV that has been linked to viral persistence³. This intron is conserved across all species of cytomegaloviruses, suggesting it may play a significant role in pathogenesis⁴. We have used Murine Cytomegalovirus (MCMV) as a model system to study the function of the intron in viral replication in vitro and in vivo. MCMV mutants lacking the stable intron replicate normally during the acute phase of infection, yet fail to establish persistent replication in the salivary gland of the mouse⁴. Therefore, we hypothesize that the MCMV intron locus is a key determinant for viral persistence. The objective of my thesis work is to investigate transcriptional regulation of the MCMV intron locus in vitro and in vivo to understand the molecular mechanisms that control intron locus temporal expression and ultimately function.

Aim 1: Fully define the MCMV intron locus. In addition to the 7.2-kb intron, multiple RNAs have been detected from the MCMV intron locus. To determine whether all RNAs identified are derived from a common primary transcript, I am currently resolving the exon and intron boundaries of the precursor RNA using 5’RACE, RNase protection and northern blot analysis.

Aim 2: Identify regulatory elements that drive transcription of the MCMV 7.2-kb intron locus. The DNA sequence proximal to the intron locus lacks identifiable transcriptional control elements such as a TATA box. To investigate the promoter elements that temporally regulate expression of the intron locus over the course of infection, I am using a luciferase reporter system to identify regions that confer transcriptional control in vitro.

Aim 3: Identify viral factors that regulate MCMV intron locus transcription. The intron locus relies on viral replication to occur at late times during infection for expression. To demonstrate that the intron locus is highly regulated by viral transcriptional repressors and activators over the course of infection, I will identify viral factors that bind to DNA regions upstream and/or downstream of the transcriptional start site by employing EMSA, ChIP, and reporter assays.​

1.        Landolfo, S. et al. The human cytomegalovirus. Pharmacology & therapeutics 98, 269-97(2003).

2.        CDC - CMV: Congenital CMV Infection Trends and Statistics. at <http://www.cdc.gov/cmv/trends-stats.html>

3.        Kulesza, C.A. & Shenk, T. Human cytomegalovirus 5-kilobase immediate-early RNA is a stable intron. Journal of virology 78, 13182-9(2004).

4.        Kulesza, C.A. & Shenk, T. Murine cytomegalovirus encodes a stable intron that facilitates persistent replication in the mouse. Proceedings of the National Academy of Sciences of the United States of America 103, 18302-7(2006).

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