Host and viral determinants of hepatitis B virus transcription

About This Grant

Project Summary The long-term goal of this MOSAIC Pathway to Independence proposal is to understand and characterize the mechanisms by which episomal DNA transcription is silenced and promoted through the lens of hepatitis B virus (HBV). HBV establishes and maintains chronic infection via the persistence of its covalently closed circular DNA genome (cccDNA), and cccDNA serves as the template for all viral mRNAs. Many studies of HBV replication have identified HBV’s regulatory X protein (HBx) as a key viral protein involved in the promotion of viral transcription and numerous other steps in HBV’s life cycle. However, how HBx, the smallest HBV protein, achieves its many functions necessary for HBV replication remains largely unknown. During my postdoctoral training, I have applied and expanded the capabilities of a new RNA-based method for the study of HBV in cell culture, identified host restriction factors that are antagonized by HBx, and developed technologies that could enable comprehensive, low-biased studies of HBx variants. To this end, in the mentored phase of this K99 award, I will obtain a mechanistic understanding of how cccDNA transcription is regulated by a complex network of host proteins and interactions with HBx. This will be achieved through detailed functional characterization of non- SMC element 3 (NSE3) and mitochondrial topoisomerase 1 (TOP1MT), host factors identified as specifically disrupted by HBx. In addition, I will receive training in deep mutational scanning (DMS) to uncover the genetic basis for HBx’s promotion of viral RNA expression and CRISPR interference (CRISPRi) methods to uncover new host factors involved in episomal circular DNA transcription that were previously missed by gene knock-out approaches. Additional aspects of my comprehensive career development plan include attending workshops and seminars on the topics of project management, mentorship, and grant writing. The training phase will be carried out in the laboratory of Dr. Charles Rice at The Rockefeller University (RU), one of the world’s experts in virology and host-pathogen interactions. A critical component of career development will be the close counsel of a highly experienced Advisory Committee, comprised of Drs. Charles Rice, Robert Roeder, and Jeremy Rock (all RU), and Xiaolan Zhao (Memorial Sloan Kettering Cancer Center). The innovative techniques and comprehensive datasets generated in the K99 training phase will establish a detailed understanding of HBx and the host gene network that promote or silence HBV cccDNA and more importantly contribute to viral persistence. This award will be key to my success as a young independent investigator in a highly competitive field. Altogether, the training will help me achieve both my short-term goals of expanding my scientific expertise and systems-biology experimental toolkit and my long-term goal to become an independent investigator at the intersection of systems biology, viral genomics, and host-virus interactions. This proposal will contribute significant insights into our basic understanding of HBV transcription and maintenance of chronic infection and could ultimately serve as the basis for identifying new curative therapies for HBV.