Introduction: Over 296 million people are currently living with chronic hepatitis B which can lead to the development of liver cirrhosis and hepatocellular carcinoma (HCC). Prior to nuclear export, the pregenomic RNA (pgRNA) can be spliced by the host cell spliceosome to form shorter RNA sequences known as splice variants. Splice variants retain the encapsidation signal and so are packaged into core particles, reverse transcribed into shorter DNA genomes and secreted from the cell as defective viral particles. 17 splice variants have been characterised thus far, and whilst the role of splice variants currently remains unknown, an increased proportion of splice variants in patient sera has been associated with the development of HCC. Furthermore, different splice variants have been shown to have different effects on wildtype HBV replication. Due to the deletions in the open reading frames, splice variants can encode novel fusion proteins, and some novel fusion proteins have been found to play a role in their respective splice variant’s effect on wildtype HBV replication. It remains unknown how the second most common splice variant, Sp9 and its novel fusion proteins, affects wildtype HBV replication.
Methods: A greater than genome length (1.3mer) Sp9 clone was co-transfected with a replication competent wildtype HBV clone and the replication phenotype of wildtype HBV was analysed five days post-transfection. Sp9 novel fusion protein overexpression plasmids and mutant Sp9 clones that knocked out Sp9’s novel fusion proteins were also co-transfected with WT HBV and the replication phenotype of wildtype HBV was analysed.
Results: Co-transfection of Sp9 with WT HBV markedly reduced HBV DNA, pgRNA and S mRNA production, as well as intracellular HBV core and S protein production. Intracellular and secreted HBV E and S antigen levels were also markedly reduced by Sp9. Cytotoxicity assays confirmed that Sp9 is not cytotoxic to the cells. Co-transfection of WT HBV with Sp9 novel protein knockout clones partially restored HBV replication.
Conclusion: The role of HBV splice variants in HBV replication and pathogenesis remains unclear. This study shows that Sp9 significantly reduces HBV replication, which may be due to the novel fusion proteins expressed by Sp9.