Hepatitis B Virus Polymerase-Envelope Fusion Protein: Regulation of Translation, Stability, and DistributiondissertationsWang, YuzhoucreatorTong, ShupingAdvisorLi, JisuAdvisorSobieraj, AndreaReaderBrown University. Biology and Medicine: Biotechnologysponsor2023, None p.born digitalThesis (Sc. M.)--Brown University, 2023thesesIn the circular 3.2-kb genome of hepatitis B virus (HBV), the polymerase (P) gene overlaps its 5’ end with 3’ precore/core gene and its central part with the envelope (preS1/preS2/S) gene. Alternative translation initiation from the precore or core AUG codon generates hepatitis B e antigen (HBeAg), a secreted protein or core protein, which assembles into core particles inside cells. Similarly, translation initiated from preS1, preS2, and S region AUGs generates large (L), middle (M), and small (S) envelope proteins, with efficient secretion of S protein inhibited by L protein (containing extra preS1/preS2 domains). At the mRNA level, HBeAg and core protein are respectively translated from 3.5-kb pcRNA and slightly shorter pgRNA. pgRNA also produces low level of P protein through ribosomal leaky scanning or translational termination-reinitiation. All seven HBV proteins are translated from unspliced mRNAs. Splicing of 3.5-kb RNA enables expression of a P-L fusion protein (p43), with N-terminal 47aa from P protein joined by L protein lacking first 18aa. We found that 5’ preS1 mutations to prevent L protein expression from pgRNA construct markedly increased extracellular p43, which could be further increased by a C48* nonsense mutation in the core gene. The present study further examined regulation of p43 expression, stability, and distribution. L-minus pcRNA construct produced little p43, which could be overcome by adding C48* core-minus mutation. p43 level could also be increased by other nonsense mutations in the core gene or precore region, and even by a nonsense mutation in the P gene. However, providing core protein or HBeAg in trans did not reduce p43 level. Spliced pgRNA or pcRNA construct produced much less S protein. Co-transfection with an expression construct for S protein markedly increased extracellular p43 from such constructs. In conclusion, p43 expression can be promoted by translational termination-reinitiation including from a naturally 1 occurring precore mutation. S protein stabilizes p43 and promotes its secretion, while L and core proteins retain p43. RNA splicing to convert P protein into P-L fusion protein (p43) also converts core protein into a core-P fusion protein, which is very likely a destabilizer of p43 and possibly also S protein.Hepatitis B VirusEnglishRPB20230602