Hepatitis B Virus X Protein Mutation T118N Reshapes Cell Cycle and Apoptosis in Liver Cells

A single amino acid change in one of the hepatitis B virus’s most notorious proteins may be enough to alter how infected liver cells divide and die, according to new research from scientists at Institut Teknologi Bandung in Indonesia. The study, published in Molecular Biology Reports, examined the hepatitis B virus X protein mutant known as T118N and compared its effects on cell cycle progression and programmed cell death with those of the wild-type protein and another well-characterized mutant, K130M/V131I. The findings suggest that this mutation, which circulates in Indonesian patients at a frequency comparable to the internationally recognized K130M/V131I variant, operates through a subtly different mechanism and deserves closer scrutiny as a potential contributor to chronic liver disease and hepatocellular carcinoma.

Hepatitis B virus, a member of the Hepadnaviridae family, remains one of the leading causes of liver infection worldwide, and chronic infection can progress to hepatocellular carcinoma, one of the most lethal malignancies globally. Among the viral factors implicated in this malignant transformation, the X protein, commonly abbreviated HBx, occupies a central position. HBx is a multifunctional regulatory protein that does not directly bind DNA but instead manipulates host cell signaling pathways, transcriptional programs, and survival machinery. Decades of research have established that HBx can promote cell cycle entry, interfere with DNA repair, modulate p53-dependent responses, and tilt the balance between pro-survival and pro-death signals. Because of this pleiotropic activity, mutations accumulating in the X gene during the long course of chronic infection are thought to fine-tune the virus’s oncogenic potential, and several HBx variants have been linked epidemiologically to more aggressive liver disease.

The Indonesian team focused on the T118N substitution, in which the amino acid threonine at position 118 of HBx is replaced by asparagine. This variant had previously been detected in local clinical samples at a frequency similar to the K130M/V131I double mutant, referred to throughout the study as HBx_KV, which has been associated in multiple Asian cohorts with the development of liver carcinoma. The researchers’ earlier work had shown that the T118N mutant, designated HBx_T, actually reduced colony formation by HepG2 liver carcinoma cells, hinting that its biological effects might not simply mirror those of the better-known oncogenic mutants. To dissect what HBx_T actually does inside cells, the group designed a systematic comparison of gene expression, cell cycle distribution, and apoptotic cell death in HepG2 cells expressing either the wild-type HBx gene, the HBx_KV mutant, or the HBx_T mutant.

The experimental strategy combined three complementary readouts. First, the team used reverse transcription quantitative polymerase chain reaction, RT-qPCR, to measure the messenger RNA levels of four genes that sit at the crossroads of proliferation and apoptosis: Cyclin E, a driver of the G1-to-S phase transition; p21, a cyclin-dependent kinase inhibitor that restrains cell cycle progression; Bax, a pro-apoptotic member of the Bcl-2 family; and Bcl-2 itself, the archetypal anti-apoptotic protein. Second, they quantified cell cycle profiles by flow cytometry using propidium iodide staining, which allows the DNA content of individual cells to be measured and the proportions of cells in different cell cycle phases to be estimated. Third, they assessed apoptosis by Annexin V and propidium iodide co-staining, a standard flow cytometric approach that distinguishes early apoptotic cells, which expose phosphatidylserine on their outer membrane, from late apoptotic or necrotic cells, which lose membrane integrity altogether. As an independent confirmation of apoptotic execution, the researchers measured caspase 3 activity using a colorimetric assay kit.

The expression data revealed a clear and time-dependent pattern. At 72 hours after transfection, cells expressing the HBx_T mutant showed increased Cyclin E expression, an effect that paralleled what was observed with the HBx_KV mutant. Cyclin E drives cells through the restriction point of the G1 phase, and its upregulation is a classic hallmark of HBx-mediated proliferative signaling, so the finding places HBx_T squarely within the family of X protein variants capable of pushing hepatocytes toward DNA replication. More intriguing, however, was the behavior of the apoptotic regulators. Both Bax and Bcl-2 messenger RNA levels were elevated in HepG2 cells expressing HBx_T compared with cells expressing HBx_KV at the 72-hour time point. This simultaneous rise of a pro-apoptotic and an anti-apoptotic transcript suggests that HBx_T provokes a stronger perturbation of the Bcl-2 family rheostat, rather than a simple one-directional push toward either survival or death.

The flow cytometric apoptosis analysis sharpened this picture. At 48 hours after transfection, cells expressing HBx_T displayed increased populations of both Annexin V-positive/propidium iodide-negative cells, which represent early apoptosis, and Annexin V-positive/propidium iodide-positive cells, which represent late apoptosis or secondary necrosis, when compared with cells expressing the wild-type HBx protein. In other words, the T118N mutant appears to be a more potent inducer of apoptotic cell death than the unmutated viral protein at this time point. This observation is consistent with the team’s earlier finding that HBx_T reduces colony formation in HepG2 cells, and it aligns with a growing body of literature showing that HBx can have context-dependent, sometimes pro-apoptotic effects. Prior studies have demonstrated that HBx sensitizes hepatocytes to death receptor-mediated apoptosis through Bax, that the protein contains a BH3-like motif enabling direct interaction with Bcl-2 family members, and that persistent hepatocyte apoptosis can paradoxically promote tumorigenesis by generating oxidative stress and compensatory regenerative pressure in liver tissue.

The caspase 3 activity assay provided a biochemical anchor for these cytometric observations. Caspase 3 is the principal executioner caspase, the enzyme whose activation commits a cell irreversibly to the apoptotic program, cleaving structural proteins and dismantling the cell from within. By measuring its activity colorimetrically, the researchers could verify that the Annexin V-positive populations reflected genuine activation of the apoptotic machinery rather than artifacts of staining. Taken together with the elevated Bax transcript levels, the data support a model in which HBx_T tips the mitochondrial apoptotic pathway toward activation, even as the concurrent rise in Bcl-2 suggests a counterbalancing survival response mounted by the cells. The net outcome at any given moment in an infected liver would therefore depend on the dynamic interplay between these opposing signals, a nuance that static endpoint measurements can only partially capture.

What emerges from the study is that HBx_T influences cell cycle and apoptosis regulation through a mechanism that is similar to, but measurably distinct from, that of HBx_KV. Both mutants enhance Cyclin E expression, pointing to shared proliferative activity, but HBx_T elicits a stronger transcriptional response in the Bcl-2 family axis and a more pronounced apoptotic phenotype relative to wild-type HBx. The authors emphasize that these differences could matter for disease progression in regions where the T118N variant circulates. Chronic hepatitis B is a decades-long negotiation between virus and host, and variants that modulate the rate of hepatocyte death and proliferation can shape the inflammatory and regenerative landscape in which cancer eventually arises. Persistent apoptosis, as several studies cited by the team have shown, can fuel tumorigenesis through oxidative stress independent of compensatory liver regeneration, making an apoptosis-inducing HBx variant a candidate worth tracking in longitudinal cohort studies.

The researchers are careful to frame their conclusions as preliminary. The work was conducted in a single hepatoblastoma-derived cell line, HepG2, using transient transfection rather than authentic viral infection, and the measurements capture snapshots at 48 and 72 hours rather than the protracted timescales relevant to human chronic infection. The authors state explicitly that further investigations are still required to understand the involvement of the HBx_T mutant in the development of chronic liver disease and liver cirrhosis. Future work will likely need to address how the T118N substitution alters the protein’s structure and its interactions with host factors, whether the same phenotypes appear in primary hepatocytes or in vivo models, and how HBx_T behaves in combination with other common HBV mutations, such as those in the basal core promoter or precore regions, that frequently co-occur in patients with advanced disease.

Nevertheless, the study adds a meaningful piece to the puzzle of HBV-associated carcinogenesis. By showing that a naturally circulating Indonesian HBx variant modulates the Cyclin E, p21, Bax, and Bcl-2 network in a distinguishable way from the canonical K130M/V131I mutant, the Bandung team underscores a broader principle in viral oncology: the oncogenic potential of hepatitis B virus is not a fixed property of the virus but a moving target shaped by the mutations that accumulate in individual patients. Mapping the functional spectrum of these variants, particularly those prevalent in under-studied regions and genotypes, could ultimately improve risk stratification for hepatocellular carcinoma and inform surveillance strategies for the hundreds of millions of people living with chronic hepatitis B worldwide.

Subject of Research: Effect of the hepatitis B virus X protein T118N mutation on cell cycle and apoptosis regulation in HepG2 liver cells

Article Title: Effect of Hepatitis B virus X T118N mutant protein in HepG2 cell cycle and apoptosis regulation

Article References: Artarini, A., Seniadi, K., Mahaputra, A. R. G., Ridwani, D. S. K., & Prasetyo, V. N. A. (2026). Effect of Hepatitis B virus X T118N mutant protein in HepG2 cell cycle and apoptosis regulation. Molecular Biology Reports, 53(1), Article 1708. https://doi.org/10.1007/s11033-026-12923-3

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12923-3

Keywords: hepatitis B virus, HBx, T118N mutation, HepG2 cells, cell cycle, apoptosis, Cyclin E, Bax, Bcl-2, hepatocellular carcinoma, flow cytometry, RT-qPCR