Replication efficiency and sequence analysis of full‐length hepatitis B virus isolates from hepatocellular carcinoma tissues

Abstract

Prolonged replication of hepatitis B virus (HBV) in liver tissues of hepatitis B patients has been considered as an important risk factor for the development of malignancy. Few studies on full-length HBV sequencing in association with the replication efficiency of isolates from HCC tissues have been reported. To study the structural and functional genomics of HBV isolates from Chinese hepatocellular carcinoma (HCC) patients, full-length HBV genomes were amplified from 6 HBV-marker positive HCC tissues and used to transfect HepG2 cells. Five of 6 isolates showed high replicative efficiency. All isolates were of genotype C and "hotspots" mutations were detected in the B cell and T helper (Th) cell epitopes of the envelope and the core region. In addition, the X region of 2 isolates contained a stop-codon mutation that was predicted to result in a truncated X protein. High replicative HBV immune escape mutants that persist in infected hepatocytes could be 1 of the important factors to initiate pathological processes for the development of HCC in Chinese patients. © 2002 Wiley-Liss, Inc. Key words: hepatitis B virus; hepatocellular carcinoma; replicationHepatitis B virus (HBV) belongs to the Hepadnaviridae family, which is unique in possessing a partially double-stranded DNA genome. This genome consists of 4 open reading frames encoding the envelope antigen (S/Pre-S), the core antigen (C/Pre-C), the viral polymerase (P) and a multifunctional transcriptional transactivator, the X protein. In HBV endemic areas, chronic HBV infection is closely associated with hepatocellular carcinoma as more than 90% of Chinese hepatocellular carcinoma (HCC) patients have been found positive for HBV-markers. 1 Although it is generally accepted that carcinogenesis is a multi-step event, the functional properties of HBV strains involved in the early stage of carcinogenesis have not been fully explored. In a previous study, we compared the nucleotide sequence and replicative efficiency of sequential HBV isolates from a Chinese patient who progressed from HBV asymptomatic carrier to hepatocellular carcinoma and showed that post-HCC HBV isolates possessed enhanced replicative efficiency. 2 High replicative HBV strains might be associated with HCC if they led to increased number of infected hepatocytes, resulting in severe liver injury mediated by host immune responses; to emergence of mutants that would escape from host immune surveillance; 3 or to increased rate of integration of viral DNA. To further investigate the incidence and possible roles of high replicative immune escape mutants derived from HCC tissues, we describe the structural and functional analysis of fulllength HBV genomes from 6 independent Chinese HCC tissues. MATERIAL AND METHODS Liver cancer tissue samplesLiver cancer tissues from serum HBsAg-positive hepatocellular carcinoma patients were collected after resection of the tumor and the diagnoses were confirmed by histopathological examination. Sample collection was in accordance with Chinese State Ethic Regulations. The...

Coexistence of Two Distinct Secretion Mutations (P5T and I97L) in Hepatitis B Virus Core Produces a Wild-Type Pattern of Secretion

“…the wild-type HBV, to our knowledge, this was the first study of the functional significance of HBV core mutations using the genetic context of a naturally occurring variant. Our study demonstrates that not only viral replication, but also the immature secretion phenotype, can occur in Huh7 cells independent from the expression of an M envelope and full-length wild-type X proteins (18).…”

“…2). The diminished phenotype of immature secretion of mutant T5P/ I97L in the Shanghai adr background in HepG2 cells could be related to both host factors (HepG2) and viral factors (e.g., X and M protein deficiency) (18). Previously, we noted that both the extracellular immature secretion phenotype and the intracellular replication advantage phenotype tend to be more pronounced in Huh7 cells than in HepG2 cells (23,33).…”

“…In addition to the hot spot mutation I97L, it contains multiple frequent mutations, including core mutations P5T and S87G, enhancer II mutations at nucleotides 1762 and 1764, and mutations truncating the X protein and abrogating the production of the pre-S2-containing M envelope protein (18). The 3.2-kb monomeric HBV genome in clone 14 was released from the pUC18 vector by SapI digestion, religated, and linearized by BamHI before being cloned into plasmid pBluescript.…”

Activating mechanism of transcriptor NF-kappaB regulated by hepatitis B virus X protein in hepatocellular carcinoma

“…It has been found in some studies that HBx mutation was common in HCC compared with corresponding liver tissues [28] . Frequent types of HBx mutation were COOHterminal truncation and hotspot mutations in certain amino acids [29][30][31] . COOH-terminally truncated HBx is encoded by truncated X gene, which usually derives from HBV integrated into host genome.…”

Global proteome analysis of hepatitis B virus expressing human hepatoblastoma cell line HepG2

“…Twenty-five nanograms of cloned full-length HBV DNA (adw subtype) [Lin et al, 2002] were labeled using random primers with [ 32 P]dCTP (YaHui Co., Beijing, China), and used as the probe for hybridization. Signals were detected by autoradiography, and the blots were scanned using densitometry.…”