HBx, a multifunctional regulatory protein, plays an essential role in the replication and pathogenesis of the hepatitis B virus (HBV). In this study, we found that in human hepatoma cells, the tumor suppressor p53 downregulates HBx via ubiquitin-dependent proteasomal degradation. p53 transcriptional activity that results from HBV infection was not essential for this effect. This was shown by treatment with a potent p53 inhibitor, pifithrin-α. Instead, we found that p53 facilitated the binding of E6-associated protein (E6AP), which is an E3 ligase, to HBx and induced E6AP-mediated HBx ubiquitination in a ternary complex of p53, E6AP, and HBx. The ability of p53 to induce E6AP-mediated downregulation of HBx and inhibit HBV replication was demonstrated in an in vitro HBV infection system. This study may provide insights into the regulation of HBx and HBV replication, especially with respect to p53 status, which may also help in understanding HBV-associated tumorigenesis in patients.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H₂O₂, during infection in hepatocytes of patients. In this study, we demonstrated that H₂O₂ inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H₂O₂ failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H₂O₂ upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H₂O₂ was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H₂O₂. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

Hepatitis C virus (HCV) is constantly exposed to considerable oxidative stress, characterized by elevated levels of reactive oxygen species, including hydrogen peroxide (H₂O₂), during acute and chronic infection in the hepatocytes of patients. However, the effect of oxidative stress on HCV replication is largely unknown. In the present study, we demonstrated that H₂O₂ downregulated HCV Core levels to inhibit HCV replication. For this purpose, H₂O₂ upregulated p53 levels, resulting in the downregulation of both the protein and enzyme activity levels of DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b, and activated the expression of E6-associated protein (E6AP) through promoter hypomethylation in the presence of HCV Core. E6AP, an E3 ligase, induced the ubiquitin-dependent proteasomal degradation of HCV Core in a p53-dependent manner. The inhibitory effect of H₂O₂ on HCV replication was almost completely nullified either by treatment with a representative antioxidant, N-acetyl-L-cysteine, or by knockdown of p53 or E6AP using a specific short hairpin RNA, confirming the roles of p53 and E6AP in the inhibition of HCV replication by H₂O₂. This study provides insights into the mechanisms that regulate HCV replication under conditions of oxidative stress in patients.

Orthohantaviruses are zoonotic pathogens that cause severe diseases in humans primarily through inhalation of aerosols from rodent excreta. Recent studies suggest that ectoparasites may be potential vectors for Orthohantaviruses. This study aimed to obtain molecular evidence of Orthohantavirus in trombiculid mites collected from wild rodents. In April 2025, 4963 trombiculid mites were collected from 128 wild rodents captured in 17 regions of the Republic of Korea (ROK). Among them, 1660 mites were grouped into 204 pools by collection sites and tested for the Orthohantavirus RNA using quantitative real-time polymerase chain reaction (PCR) and reverse transcription-nested PCR. OrthoHantavirus RNA was detected in seven trombiculid mite pools, with a minimum infection rate of 0.4/100 mites. Orthohantavirus hantanense was identified in trombiculid mites from Cheongju, Gimcheon, and Yeongdeok, while O. jejuense was identified in trombiculid mites from Boryeong and Jeongeup. Notably, Orthohantaviruses were not detected in the host rodents of the Orthohantavirus-positive trombiculid mites from Cheongju, Jeongeup, and Gimcheon, indicating host-independent infection. This study is the first to report detection of Orthohantaviruses, O. hantanense and O. jejuense, from trombiculid mites in the ROK. The host-independent infection suggests that these mites could serve as independent vectors/reservoirs for Orthohantaviruses, distinct from previously known transmission routes.

The hepatitis C virus (HCV) Core activates the ATM-Chk2 pathway, leading to phosphorylation of p53 at Ser-15, which inhibits mouse double minute 2 (MDM2)-mediated proteasomal degradation. This study reveals that HCV Core also promotes E6-associated protein (E6AP)-mediated degradation of p53 during HCV replication. In the presence of HCV Core, E6AP expression induced p53 ubiquitination, reduced its stability, and decreased p53 levels, whereas E6AP knockdown increased p53 levels. The E3 ubiquitin ligase activity of E6AP was critical for this process, as demonstrated using the E6AP C833A mutant and the E3 ligase inhibitor Heclin. Proteasomal inhibition with MG132 confirmed that HCV Core and E6AP act together to regulate p53 levels via the proteasome. Importantly, HCV Core-induced p53 phosphorylation was essential for E6AP-mediated degradation, as shown by the impairment of degradation in the presence of the ATM inhibitor KU-55933. E6AP also targeted p53 phosphorylated at Ser-15 by etoposide, as well as phosphomimetic mutants such as p53 S15D, but not non-phosphorylatable mutants such as p53 S15A. These findings suggest that HCV Core-induced p53 phosphorylation enhances E6AP-mediated degradation while preventing MDM2 from targeting p53, thereby maintaining p53 levels that support cell survival, viral replication, and potentially oncogenesis in human hepatocytes.