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CHAPTER 3.

Reason: Content of chapter 3 contains unpublished data and is currently in preparation to submit in a peer reviewed journal

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IDENTIFICATION OF HOST PROTEIN COMPLEXES INVOLVED IN HEPATITIS B VIRUS (HBV) BIOSYNTHESIS

thesis
posted on 2023-03-14, 15:08 authored by Naimur RahmanNaimur Rahman

The goal of the studies I describe in this thesis is to better understand how the cellular proteome responds to Hepatitis B Virus (HBV) infection and identify potential therapeutic targets. 

I used a cellular HBV replication model and performed proteomic analyses to understand how HBV evades innate immunity as a function of cell cycle progression. I identified that the conserved LSm (Like-Sm1-8) proteins were differentially regulated in HBV replicating cells treated with IFN-α. Specifically, in the G2/M phase, IFN-α increased protein level of LSm1, the unique subunit of the cytoplasmic LSm1-7 complex involved in mRNA decay. By contrast, IFN-α decreased LSm8, the unique subunit of the nuclear LSm2-8 complex, a chaperone of U6 spliceosomal RNA. These studies determined that the cytoplasmic LSm1-7 complex is antiviral, whereas the nuclear LSm2-8 complex is pro-viral. Mechanistic studies also led to the identification of the splicing inhibitor Cp028, acting upstream of the LSm2-8 complex, as an inhibitor of viral RNA levels and a potential anti-HBV compound.

In the second part of my dissertation, I aimed to identify alternate approaches to silence viral transcription in chronic HBV infection. Despite the availability of potent antivirals, complete elimination of the viral minichromosome (cccDNA), the template of viral transcription, is unsuccessful resulting in viral persistence.  Earlier studies showed that the RNA helicase DDX5 restricts HBV biosynthesis by an unknown mechanism. I performed proteomic studies to search for DDX5 interacting proteins. I identified Interferon gamma inducible protein 16 (IFI16), as the most prominent DDX5-interacting protein. Employing size exclusion chromatography (SEC) of native nuclear extracts in combination with LC-MS/MS of SEC fractions and various functional assays, I demonstrated that DDX5 and IFI16 form a large (750-350 KDa), RNA-dependent epigenetic silencing complex. Although the identity of this RNA remains to be determined, my studies conclusively demonstrate that DDX5 represses cccDNA-driven viral transcription by forming this RNA-dependent complex with IFI16, in association with the epigenetic silencing Polycomb Repressive Complex 2(PRC2).  Importantly, this DDX5/IFI16/PRC2/RNA complex mediates the transcription silencing modifications of the HBV minichromosome in response to Interferon-α.  I conclude that DDX5 downregulation is a pro-viral factor in HBV biosynthesis. 

Overall, this dissertation work provides new insights into the interaction between HBV and the host proteome and identifies potential targets for the development of more effective therapies for the treatment of HBV infection.

History

Degree Type

  • Doctor of Philosophy

Department

  • Basic Medical Sciences

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Ourania M. Andrisani

Additional Committee Member 2

Shihuan Kuang

Additional Committee Member 3

Susan M. Mendrysa

Additional Committee Member 4

Uma K. Aryal