Kira Sviderskaia, University of Alberta

Kira Sviderskaia1, Rohith Vedhthaanth Sekar2, Jessica Skoreyko, Mary Hernando1, Andrea Visnjevac4, Mohaddeseh Pakzamir1, Dhruvi Patel3, Archee Panwar1, Shu Yue Luo4, Maulik Badmalia3, Olivier Julien4, Vanessa Meier-Stephenson1,3,5 1 Department of Medicine, Division of Infectious Diseases, University of Alberta, Edmonton, AB, Canada 2 Department of Physics, University of Alberta, Edmonton, AB , Canada 3 Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB , Canada 4 Department of Biochemistry, University of Alberta, Edmonton, AB , Canada 5 Li Ka Shing Institute of Virology, University of Alberta, Edmonton, AB , Canada

Expressing & Multiplying

Chronic Hepatitis B virus (HBV) affects over 296 million people worldwide. Our laboratory aims to develop currently unattainable cccDNA-targeting therapeutics by anchoring at a highly conserved secondary DNA structure, a G4-quadruplex (G4Q). Here, we characterize the G4Q structure and identify its protein binders contributing to transcription activity to further define the interaction and biological significance, opening avenues for drug development. We showed that G4Q is an integral part of the promoter, as expression was significantly impacted upon deletion or misfolding, and dose-dependently decreased with mithralog drugs (luciferase plasmid under control of HBV promoter transfected into HepG2 cells; drug binding confirmed using melting CD and localization to the nucleus using microscopy). Similarly, transcriptional activity was impacted by point mutations, insertions and deletions. The 30 mutants tested hint at rigidly conserved structure of G1734-G1739 nucleotides, we hypothesize that rest of highly conserved sequence employs several confirmations to finetune viral expression. We used secondary structure predictors and MD stimulation to further define the structure. To discover novel proteins involved in G4Q transcription regulation through DIA proteomic analysis, we compared protein binders of folded ssG4Q structure and its linear dsDNA counterpart (FPLC and CD used to purify and test folding). Significant candidates were chosen based on SAINT and ANOVA with Turkey's HSD, including transcriptional and epigenetic factors. Studies confirming protein-G4Q binding through bead pulldown followed by western blot are on-going. We pinpoint functionally important G4Q sites and define its protein interactors. These findings could be used to rationally design therapeutics against HBV infection.