Takayuki Murata

UNLABELLED: Hepatitis B virus (HBV) infects hepatocytes by using sodium taurocholate cotransporting polypeptide (NTCP) as a receptor. NTCP can render non-permissive hepatocyte cell lines such as HepG2 permissive to HBV infection. However, only a few reports investigate the NTCP function in HBV infection beyond virus binding. Here, we performed immunopurification followed by liquid chromatography-tandem mass spectrometry to identify ZO-2 (aka TJP2) as a novel NTCP-binding protein. Knockdown or knockout of ZO-2 in NTCP-transduced HepG2 cells decreased the amount of NTCP at the cell surface, leading to reductions in cellular attachment and infection by HBV. Incubation of cells with HBV surface molecules preS1 resulted in the dissociation of NTCP from ZO-2 and in the formation of NTCP-preS1-actin complexes that were internalized into the cell. Latrunculin A, an inhibitor of actin polymerization, suppressed the preS1 internalization into hepatocytes and HBV infection. In conclusion, ZO-2, together with actin, regulates the function of NTCP as a receptor of HBV, providing a new model for HBV-cell interactions and virus internalization. IMPORTANCE: Although a number of candidates have been reported to bind to the hepatitis B virus (HBV) envelope, accumulating evidence indicates that NTCP is accepted as a functional receptor for HBV infection. Thus, NTCP is an attractive target for antiviral therapies. Here, we showed that ZO-2 interacts with NTCP. The silencing of ZO-2 decreased HBV infection, whereas ZO-1 and ZO-3 knockdown had no effect on HBV infection. Moreover, knockout of ZO-2 induced the downregulation of NTCP from the cell surface. This aberrant NTCP localization causes the reduction of the half-life of NTCP in ZO-2 knockout cells. PreS1 treatment or HBV infection disrupted the NTCP/ZO-2 complex through the dissociation of the actin-binding domain of ZO-2, leading to internalization of a newly formed preS1/NTCP/actin complex into the cell. The actin polymerization inhibitor latrunculin A suppressed HBV infection. These results suggest that ZO-2 regulates cell surface localization of NTCP.

Background/Objectives: Epstein-Barr virus (EBV) is associated with a subset of gastric carcinomas characterized by latency programs that promote survival of infected cells. EBV-encoded BamH I A rightward transcript (BART) microRNAs contribute to apoptosis resistance in infected epithelial cells. This study investigated whether dasatinib, a Src family kinase (SFK) inhibitor, selectively targets EBV-positive gastric epithelial cells and examined the molecular mechanisms underlying this effect. Methods: EBV-positive and EBV-negative gastric epithelial cell models were analyzed to evaluate cell viability, apoptosis induction, signaling pathways, and viral gene regulation. BART miRNA expression was quantified by RT-qPCR, and promoter activity was examined using luciferase reporter assays. Downstream target gene expression was analyzed at both the transcript and protein levels. Recombinant EBV lacking BZLF1 or LMP2A was used to assess the contributions of lytic activation and LMP2A-associated signaling. Results: Dasatinib preferentially reduced viability and induced apoptosis in EBV-positive gastric epithelial cells compared with EBV-negative counterparts. Treatment suppressed phosphorylation of Src and ERK and reduced expression of the anti-apoptotic proteins BCL-xL and MCL1. Apoptosis was also observed in cells infected with LMP2A-deficient EBV, suggesting that the effect cannot be fully explained by inhibition of LMP2A-associated signaling. Dasatinib inhibited BART miRNA promoter activity and reduced pri-, pre-, and mature miR-BART levels, accompanied by increased expression of pro-apoptotic target genes including CASZ1a, OCT1, ARID2, TP53INP1, and DAB2. In parallel, dasatinib suppressed BZLF1 promoter activity without evidence of lytic reactivation. Conclusions: Dasatinib promotes apoptosis in EBV-positive gastric epithelial cells in association with coordinated suppression of SFK signaling and EBV-encoded BART miRNA expression, accompanied by derepression of pro-apoptotic cellular genes. These findings reveal a previously underappreciated vulnerability of EBV-positive epithelial cells and suggest that targeting host kinase signaling pathways that regulate viral microRNAs may represent a potential therapeutic strategy for EBV-associated malignancies.

Epstein-Barr virus (EBV)-associated T- and natural killer (NK)-cell neoplasms encompass a heterogeneous spectrum, ranging from persistent lymphoproliferative disorders (e.g., severe mosquito bite allergy, systemic chronic active EBV disease) to highly aggressive malignancies (e.g., extranodal NK/T-cell lymphoma [ENKTL], aggressive NK-cell leukemia). Genomic and epigenetic studies have revealed shared host genetic alterations-most notably in JAK-STAT signaling, epigenetic regulators, TP53, and DDX3X-supporting a pathogenic and clinicobiological continuum across these disorders. Defective EBV further reshapes viral gene expression programs and contributes to oncogenesis. l-asparaginase-based chemoradiotherapy has improved outcomes in early-stage ENKTL; however, effective treatments for advanced-stage disease and other EBV-associated T/NK-cell neoplasms remain limited. Emerging molecular subclassifications and large-scale prospective cohorts can help clarify disease heterogeneity and accelerate the development of precision therapeutic strategies.

Epstein-Barr virus (EBV) infects over 90% of humans and is associated with both hematological and epithelial malignancies. Here, we analyzed 990 EBV genomes (319 newly sequenced and 671 from public databases) from patients with various diseases to comprehensively characterize genomic variations, including single nucleotide variations (SNVs) and structural variations (SVs). While most SNVs were a result of conservative evolution and reflected the geographical origins of the viral genomes, we identified several convergent SNV hotspots within the central homology domain of EBNA3B, the transactivation domain of EBNA2, and the second transmembrane domain of LMP1. These convergent SNVs appear to fine-tune viral protein functionality and immunogenicity. SVs, particularly large deletions, were frequently observed in chronic active EBV disease (28%), EBV-positive diffuse large B-cell lymphoma (48%), extranodal NK/T-cell lymphoma (41%), and Burkitt lymphoma (25%), but were less common in infectious mononucleosis (11%), post-transplant lymphoproliferative disorder (7%), and epithelial malignancies (5%). In hematological malignancies, deletions often targeted viral microRNA clusters, potentially promoting viral reactivation and lymphomagenesis. Non-deletion SVs, such as inversions, were also prevalent, with several inversions disrupting the C promoter to suppress latent gene expression, thereby maintaining viral dormancy. Furthermore, recurrent EBNA3B deletions suggested that this viral transcription factor functions as a tumor suppressor. EBNA3B knockout experiments in vitro revealed downregulation of human tumor suppressors, including PTEN and RB1, which could explain the enhanced lymphomagenesis observed in EBNA3B-deficient lymphoblastoid cell line xenografts. Our findings highlight both disease-specific and general contributions of EBV genomic alterations to human cancers, particularly in hematological malignancies.

BACKGROUND: Rotavirus infections are a major cause of severe gastroenteritis in children. Human rotavirus strains with the unconventional G8P[14] genotype have sporadically been detected in diarrheic patients in different parts of the world. However, full genomes of only two human G8P[14] strains from Africa (North Africa) have been sequenced, and the origin and evolutionary patterns of African G8P[14] strains remain to be elucidated. METHODS: In this study, we sequenced the full genome of an African G8P[14] strain (RVA/Human-wt/KEN/A75/2000/G8P[14]) identified in archival stool samples from a diarrheic child in Kenya. RESULTS: Full genome-based analysis of strain A75 revealed a unique genogroup constellation, G8-P[14]-I2-R2-C2-M2-A11-N2-T6-E2-H3, with the I2-R2-C2-M2-A11-N2-T6-E2-H3 part being common among rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis showed that all the 11 genomic segments of strain A75 are closely related to segments found in artiodactyl rotavirus strains, and likely strain A75 derived from spillover transmission of an artiodactyl rotavirus strain to humans. CONCLUSION: This is the first report on a full genome-based characterization of a human G8P[14] strain from East Africa. This study demonstrates the diversity of human G8P[14] strains in Africa and contributes to the elucidation of their spreading and evolution, which includes zoonotic transmission from artiodactyls.