The G1613A Mutation in the HBV Genome Affects HBeAg Expression and Viral Replication through Altered Core Promoter Activity

Abstract

Infection of hepatitis B virus (HBV) causes acute and chronic hepatitis and is closely associated with the development of cirrhosis and hepatocellular carcinoma (HCC). Previously, we demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A mutation significantly suppresses the secretion of e antigen (HBeAg) and enhances the synthesis of viral DNA, which is in consistence to our clinical result that the G1613A mutation associates with high viral load in chronic HBV carriers. To further investigate the molecular mechanism of the mutation, we performed the electrophoretic mobility shift assay with the recombinant RFX1 protein, a trans-activator that was shown to interact with the NRE of HBV. Intriguingly, RFX1 binds to the G1613A mutant with higher affinity than the wild-type sequence, indicating that the mutation possesses the trans-activating effect to the core promoter via NRE. The trans-activating effect was further validated by the enhancement of the core promoter activity after overexpression of RFX1 in liver cell line. In summary, our results suggest the functional consequences of the hotspot G1613A mutation found in HBV. We also provide a possible molecular mechanism of this hotspot mutation to the increased viral load of HBV carriers, which increases the risk to HCC.

A complete genomic analysis of hepatitis B virus isolated from 516 Chinese patients with different clinical manifestations

“…In addition to BCP/PC mutations, other point mutations with significance were T53C (preS2F53L), G1613A (polR841K), C1913A (coreP5T), A2159G (coreS87G), A2189C (coreI97L), and T2857C (preS1W4R). The G1613A mutation possessed the transactivating effect to the core promoter via negative regulation, and thus significantly suppressed the secretion of HBeAg and enhances the synthesis of viral DNA [Li et al, ]. Consistent with a recent study, the current analysis revealed C1913A as an independent risk factor for acute‐on‐chronic liver failure [Yan et al, ].…”

Whole genome characterization of hepatitis B virus quasispecies with massively parallel pyrosequencing

“…3B). In addition to seven mutations previously reported [4,[16][17][18], C2288A/T, C2304A, and A/G2525C/T were identified for the first time to correlate to severe liver disease. C2288A/T and C2304A lead to amino acid changes as P130T/S and P135Q in C protein, and A/G2525C/T leads to K73N/E73D of P protein.…”

In Silico Analysis of Genetic Diversity of Human Hepatitis B Virus in Southeast Asia, Australia and New Zealand

“…The core promoter is located immediately upstream of the PC/C region and consists of the basal core promoter (BCP) and the upper regulatory region (URR). URR is composed of the negative regulatory element (NRE) and the core upstream regulatory sequence (CURS), with a possible role in regulating core promoter activity [6]. The X ORF encodes for X protein, which is associated with viral regulation, cell growth and carcinogenesis by disrupting several cellular signaling pathways [7][8][9].…”

“…The two HBcAg mutations p.I/F97L and p.P130T are frequently observed concurrently in CHB patients [44], leading to the secretion of virions containing immature genomes and hypermaturations, respectively [48]. Meanwhile, double mutations c.1762A>T / c.1764G>A in the BCP region and mutation c.1613G>A within NRE, are associated with the suppressed expression of HBeAg, increased viral load in HBV carriers and increased risk of liver carcinogenesis [6,49,50]. A study on HBV X mutations reported the presence of the mutant p.V5M/L amongst Korean patients with severe liver diseases such as cirrhosis and HCC [51].…”