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Mariani, Michael

Cellular, Molecular, and Biomedical Sciences Graduate Program

2021

Ph. D.

Human herpesviruses are ubiquitous pathogens worldwide with 90% of the global population infected with one or more Human herpesviruses (HHV's) by adulthood. All herpesviruses have three unique life cycle stages. Upon resolution of a primary acute stage infection, they can establish a latent stage infection within the host cell nucleus. This stage is characterized primarily by transcriptional quiescence of the viral genome. Specific physiological conditions (e.g., cell stress) can cause the latent virus to enter the reactivation stage, often many years after resolution of the acute infection, in which the virus becomes replicationally active again. HHV's are known to cause disease in humans in all three stages of their lifecycle and chronic infection is becoming increasingly associated with a wide range of human morbidities. The field has been met with challenges in establishing tractable cell models to study HHV infection in vitro and the exact mechanisms regulating the maintenance of, and transition between, the life cycle stages are largely undefined; however, chromatin structure and function are known to play a role. In this dissertation I employ both molecular and computational approaches to study the higher order chromatin structures and chromatin states in cell models of infection for two Human herpesviruses at specific stages in their life cycles. In the first part of this dissertation, I successfully demonstrate the establishment of a latent Human betaherpesvirus 6A (HHV-6A) infection, which requires integration of its genome into the host cell genome, in an in vitro-derived HEK-293 cell model. I then employ unbiased epigenomic methods along with bioinformatics techniques to identify three-dimensional virus-host contact regions and characterize the chromatin states of the latent virus. I then demonstrate the above in both a patient-derived cell model and in infected primary cells. Finally, I developed a novel and innovative computational approach to identify the sites of HHV-6A integration in host cell chromosomes using next generation sequencing data: to my knowledge, a first in the field. In the second part of this work, I employ a human fetal lung fibroblast (HFL) cell model of acute infection to study Varicella Zoster Virus (VZV) chromatin biology -- specifically with regards to the transcription factor CTCF. CTCF is a key organizer of chromatin three-dimensional structure and plays a role in chromatin organization and transcriptional regulation in certain HHV's. Herein, I present evidence that CTCF is likely involved in virus-host structural interactions -- an important finding that is countervailing to the current suppositions of the Human alphaherpesvirus field. Overall, the work presented herein offers novel insight into the complex and dynamic relationship between HHV's and host cell chromatin. With a more comprehensive characterization of the higher order chromatin structures and chromatin states that define this relationship, we can better define the HHV life cycle. Such knowledge will allow for the development of improved treatments against these insidious infections.