My name is Razia Tajwar, and I am a Postdoctoral Fellow in the lab of Dr. John Tavis at the School of Medical Sciences, Saint Louis University, MO, USA. By training I am a Biochemist, and my project is characterizing HBV polymerase (HBV P) using biochemical and biophysical methods to understand its functions. This work will lead to development of biochemical high-throughput assay to screen inhibitors against functions of HBV P that are not currently targets of drugs. I am using protein engineering methods to produce functional HBV P derivatives.
I want to give a brief introduction about HBV P which will help you understand my work better. HBV replicates via protein-primed reverse transcription reaction catalyzed by the HBV P, a multidomain protein consists of terminal protein (TP), spacer (SP), reverse transcriptase (RT) and RNase H (RH) domains. Nucleotide analogs (NA) available as a treatment for the HBV infection which targets polymerase activity of RT domain and can suppress the infection but fail to eliminate it completely plus the treatment is expensive and lifelong. The aim of our biochemical and structural characterization of HBV P is to find new targets for drug development. The TP domain catalyzed initial reaction of reverse transcription called protein priming making it a very important drug target, but that reaction is poorly understood. A second important drug target is the RNase H domain which degrades the RNA from the RNA/DNA heteroduplex formed after the synthesis of the minus polarity strand by RT domain. Then the second DNA strand (plus polarity strand) is synthesized by the polymerase activity using minus polarity strand as a template. Inhibiting the activity of either TP or RNase H or both can block the reverse transcription completely which could contribute to combination therapies leading to functional cure of HBV infection. There is not much biochemical and structural information available for protein-priming and RNase H activity reactions due to difficulty in producing active recombinant HBV P or its derivatives.
In 2022, I predicted the structure of HBV polymerase using Alphafold and our lab validated it with already available experimental data for HBV P. Now, I am using that predicted structure of HBV P for protein engineering project and produced at least two active derivatives of HBV P which can bind to epsilon RNA (an important part of the viral pregenomic RNA that is copied into DNA) in in vitro assay, plus at least four active variants of the RNase H. The TP domain of HBV P binds specifically to human epsilon but one of our initial engineered constructs of HBV P showed binding to both human and duck epsilon and even catalyzed a version of the protein priming reaction in the absence of any epsilon. Taking in account the structural features from our initial HBV P variant, I designed four more derivatives of HBV P keeping the structural features of HBV P that may be important in defining the specificity for protein-priming reaction. I also developed a fluoresce polarization assay to analyze the binding of epsilon with HBV P derivatives. This assay is sensitive and real time binding assay. These efforts will help us to understand protein-priming reaction, the regions and residues of HBV P which are playing crucial roles in the binding to epsilon and initiating reverse transcription. Understanding of such structural information help us to identify important binding pockets which we can use to design novel and effective inhibitors.
Similarly, I am making good progress in my efforts to produce active RNase H domain which can lead to develop high throughput biochemical assay for screening of inhibitors. I worked on the hypothesis that inconsistency in producing active RNase H is due to unstable regions in the RNase H domain. Again, our predicted structure of HBV P help me to identify those regions and I used protein engineering methods of mutations and truncation to produce stable and functional RNase H variants. Now, I have produced four stable RNase H variants which I am characterizing for their activity and substrate binding. I hope soon we will be able to develop biochemical assay for the screening of inhibitors against RNase H activity.
Our predicted structure of HBV P guided protein engineering efforts enables me to make this progress in the field and I am very excited about my future experiments.