HBV-mediated Suppression of Megakaryocyte Differentiation through UBE4B Upregulation and Modulation of p53 and ERK1/2

Background: Hepatitis B virus (HBV) infection can cause thrombocytopenia through its effects on hematopoiesis, but the mechanisms underlying this process are still unclear. The process of platelet generation encompasses multiple stages, among these stages, the differentiation of mature megakaryocytes plays a pivotal role in platelet production. Objective: In this study, we investigated the impact of HBV on the differentiation of mature megakaryocytes and its molecular mechanism. Methods: Different doses of HBV virus solution extracted from HepAD38 cells were co-cultured with hematopoietic stem cells isolated from fresh, full-term healthy maternal cord blood through magnetic bead sorting. The study aimed to assess the variances in megakaryocyte differentiation and maturation after HBV infection. Furthermore, Label-free quantitative proteomics was employed to analyze the differential proteins during the mature megakaryocyte stages pre- and post-HBV infection, with a focus on elucidating their respective molecular mechanisms. Results: Through comparison, it was discovered that HBV could impede the differentiation process of fully developed megakaryocytes. Specifically, during the maturation stage of megakaryocytes, HBV had the ability to hinder cell DNA polyploidization as well as the formation of cytoskeletal proteins. Furthermore, its suppressive effect was more pronounced in the later stages of differentiation, consequently leading to impaired platelet production. Proteomic analysis revealed noteworthy disparities in UBE4B protein levels within mature megakaryocytes after HBV infection. Upon transfection with lentivirus and subsequent knockdown of UBE4B in mature megakaryocytes, a noticeable alleviation of HBV's inhibitory impact on mature megakaryocytes was observed, accompanied by regulation of p53 and ERK1/2 expression and phosphorylation in the cells. Conclusion: HBV can upregulate the expression of UBE4B, inhibit the expression and phosphorylation of p53, enhance the expression and phosphorylation of ERK1/2, suppress the differentiation of mature megakaryocytes, thereby leading to platelet production disorders.