Differences in HBV Replication, APOBEC3 Family Expression, and Inflammatory Cytokine Levels Between Wild-Type HBV and Pre-core (G1896A) or Basal Core Promoter (A1762T/G1764A) Mutants

Abstract

Background: Chronic hepatitis B virus (HBV) infection is the leading cause of hepatocellular carcinoma (HCC) worldwide. HBV variants, particularly the G1896A precore (PC) and A1762T/G1764A basal core promoter (BCP) mutations, are established risk factors for cirrhosis and HCC, but the molecular biological basis is unclear. We hypothesized that these variants result in differential HBV replication, APOBEC3 family expression, and cytokine/chemokine expression. Methods: HepG2 cells were transfected with monomeric full-length containing wildtype, PC, or BCP HBV. Cells and supernatant were collected to analyze viral infection markers (i.e., HBsAg, HBeAg, HBV DNA, and RNA). Cellular APOBEC3 expression and activity was assessed by quantitative real-time (qRT)-PCR, immunoblot, differential DNA denaturation PCR, and sequencing. Cytokine/chemokines in the supernatant and in serum from 11 CHB carriers (4 non-cirrhotic; 7 cirrhotic and/or HCC) with predominantly wild-type, PC, or BCP variants were evaluated by Luminex. Results: HBeAg expression was reduced in PC and BCP variants, and higher supernatant HBV DNA and HBV RNA levels were found with A1762T/G1764A vs. G1896A mutant (p < 0.05). Increased APOBEC3G protein levels in wild-type vs. mutant were not associated with HBV covalently closed circular DNA G-to-A hypermutations. Differences in cytokine/chemokine expression in culture supernatants, especially IL-13 were observed amongst the variants analyzed. Noticeable increases of numerous

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“…Moreover, HBxAg stimulates virus gene expression and replication and is crucial for the establishment and maintenance of chronic carrier state. Intrahepatic inflammatory reactions [ 16 , 17 , 18 ] lead to the induction of several suppressive pathways and subsequent recruitment of regulatory cells that drive functional demolition of T cells [ 19 , 20 , 21 , 22 , 23 ]. These cells start overexpressing inhibitory receptors [ 9 , 24 , 25 , 26 , 27 ] that further dampen their functional status causing immune exhaustion, resulting in viral persistence and further disease progression [ 28 ].…”

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