Deletion and Functional Analysis of Hepatitis B Virus X Protein: Evidence for an Effect on Cell Cycle Regulators

Abstract

Background/Aims: The hepatitis B virus X protein (HBx) is a viral trans-activator that plays a crucial role in pathogenesis of hepatocellular carcinoma (HCC) via an unknown mechanism. The role of HBx in modulating cell proliferation and programmed cell death is replete with controversies. Thus, the goal of this study was to elucidate the effect of HBx and its deletion mutants on cell cycle progression in human hepatoma cells. Methods: Huh7 cells transfected with either full-length or truncated HBx were tested for their mitogenic potential based on their effect on the expression of key cell cycle-related proteins (p27, cyclin D1, p21, and p53) and pro-apoptotic proteins such as cleaved poly (ADP-ribose) polymerase (PARP) and Bax. Western blotting and immunofluorescence techniques were applied to detect changes in the expression levels and intracellular localization, respectively, of the investigated proteins. Also, Quantitative real-time PCR (qRT-PCR) was used to detect changes in RNA levels. Results: An increased anchorage-independent growth of cells transfected with HBx-WT and its deletion mutants was observed. The cell cycle regulatory molecules were differentially modulated by full-length HBx (1-154) and its different N- and C-terminal truncated forms (HBx (31-154), HBx (61-154), HBx (1-94), and HBx (61-124)). An enhanced modulation of p27, p21, and cyclin D1 was associated with HBx (1-154), whereas p53 expression was significantly inhibited by HBx (61-124). Similarly, the expression of cleaved PARP and Bax was efficiently suppressed by HBx (1-94) and HBx (61-154). Conclusion: The HBx-WT and its mutants play a critical role in the pathogenesis and progression of HCC by modulating cell cycle regulatory proteins.

Investigating Core Signaling Pathways of Hepatitis B Virus Pathogenesis for Biomarkers Identification and Drug Discovery via Systems Biology and Deep Learning Method

“…Negative-expression of Fas might impede the sustainable anti-viral response of death ligand stimulus-specific apoptotic pathway. In contrast, as a target gene relevant to execution and completion of apoptosis, the overexpressed CDKN1A triggered by HBx-induced HDAC4 inhibition was concordant with the earlier research that HBx can relieve a block on CDKN1A expression and prolong G1→S transition in human hepatoma cells [156,157]. Even though p21, the protein encoded by CDKN1A, is treated as a cyclin-dependent kinase inhibitor capable of inhibiting all cyclin/CDK complexes, it often promotes the assembly of type-D cyclins with CDK4 and CDK6 and arrests cell cycle progression in the G1 phase [156,158], which not only protects cells against apoptosis but also enhances the chance of tumorigenesis.…”

Inflammatory Gene Expression Associates with Hepatitis B Virus cccDNA- but Not Integrant-Derived Transcripts in HBeAg Negative Disease

“…Primers targeting the 3′ end of all transcripts (3′T) will amplify both cccDNA- and iDNA-derived RNAs, whereas those targeting the repeat region (RR), which is absent in the majority of id-RNAs, will only amplify cccDNA-derived RNAs [ 14 , 29 ] ( Figure 3 A). The 3′T primers will amplify pC/pg splice variants [ 11 ] and two of the three known HBx truncated transcripts [ 30 ]. Both 3′T and RR primer pairs target regions conserved among diverse genotypes and showed a linear amplification of HBV genotype D over a 5-log range ( Supplementary Figure S4A,B ).…”

Cellular senescence and hepatitis B‐related hepatocellular carcinoma: An intriguing link

“…During the past few years, there has been a shift towards the study of truncated HBx proteins, whose functional mode presents differences as opposed to the wild type 50 . Transfection of human hepatoma cell line Huh7 with full‐length or truncated HBx was tested in a series of cell cycle‐related proteins and the results showed an enhanced modulation in both cases 51 . In another study, the effect of HBx on G1/S checkpoint control, based upon the status of p53, was investigated in NIH3T3 cells and it was shown that when p53 was present, transcription of p21waf1/cip1 was activated in a dose‐dependent manner 52…”