Construct a Model for Hepatitis B Virus Infection in Megakaryopoiesis
To elucidate the influence of HBV on mature megakaryocyte differentiation, we initiated our investigation by establishing an in vitro model that accurately recapitulates the complex process of megakaryocyte differentiation. Fresh full-term healthy maternal umbilical cord blood was used for magnetic bead positive selection to extract CD34 + hematopoietic stem cells. Under a light microscope, the cells exhibited a round shape. Megakaryocyte expansion factors were added to the culture medium, and based on morphological observations under a microscope, as well as flow cytometry analysis of the cell surface antigens CD34, CD41, and CD42, immature megakaryocytes gradually formed on days 5–7 of cell culture. During the transition from hematopoietic stem cells to immature megakaryocytes, cell density rapidly increased, and the number of cells multiplied. The majority of cells still maintained a round shape resembling lymphocytes under a light microscope. Giemsa staining revealed dividing cells. As the culture time extended, cell volume gradually increased, CD34 expression decreased, and CD41 and CD42 expression increased, indicating the formation of mature megakaryocytes. At this stage, cells started to exhibit protrusions and irregular shapes under a light microscope. Giemsa staining showed polyploidy formation, with some cells displaying clustered cytoplasmic particles. On day 11 of cell culture, the membrane of mature megakaryocytes appeared concave, and only a small number of proplatelets were visible around them. After 14 days of cell culture, proplatelet production reached its peak. Giemsa staining revealed that cell membranes often exhibited pseudopodia and platelet accumulation(Fig. 1A). Due to the challenges associated with acquiring and scarcity of umbilical cord blood stem cells, the subsequent mechanistic investigations utilized the Meg-01 megakaryocytic cell line. These cells were cultured in vitro to reach the mature megakaryocyte stage for research purposes.
Afterward, we co-cultured the pre-prepared HBV virus with umbilical cord blood stem cells of different viral loads, following the methods described earlier, to establish the HBV-infected group and the control group. During various stages of differentiation in HBV-infected megakaryocytes, we conducted observations of cell ultrastructure using transmission electron microscopy and successfully identified both Dane particles and small spherical particles (Fig. 1B). The distribution of HBsAg, indicated by red fluorescence, was detected within the cells using immunofluorescence techniques (Fig. 1C). Furthermore, at different time points, the viral load of HBV DNA in the cell supernatant was quantified using the COBAS TaqMan HBV DNA analyzer (Table 1), thus confirming the successful infection of immature megakaryocytes by HBV.
HBV inhibits the differentiation of mature megakaryocytes
To the stage of mature megakaryocytes, the mean signal intensity of CD34 expression demonstrated that the control group was lower than that in the HBVlow group, and lower than that in the HBVhigh group (the control group compared with the HBVhigh group, P = 0.0090). The mean signal intensity representing CD42 expression showed an opposite trend, with the control group was higher than that in HBVlow group, and higher than that in the HBVhigh group (the control group compared with the HBVhigh group, P<0.0001) (Fig. 2A).
HBV inhibits polyploidization and skeleton formation in mature megakaryocytes with increased potency during the late differentiation stage
During the differentiation process from immature to mature megakaryocytes, significant cellular changes occur, including increased cell size, intra-nuclear divisions leading to polyploidization9. By detecting the DNA polyploidy in mature megakaryocytes after HBV infection, further clarification can be made regarding the impact of HBV on the differentiation of mature megakaryocytes. We examined the DNA polyploidy in mature megakaryocytes through flow cytometry, the results showed that the percentage of cells with DNA ploidy ≤ 2N was higher in HBV-infected groups than in controls (P = 0.0238), while the ratio of ≥ 4N/≤2N was lower in HBV-infected groups than in the control ones (P = 0.0484) (Fig. 2B).
At the late stage of megakaryocyte maturation, the cell membranes gradually fuse and divide, resulting in proplatelet fragment release. Flow cytometry analysis for average fluorescence expression led to remarkable findings between both groups. During the mature megakaryocyte early stage, our results showed that CD34 expression was lower in the control group than that in the HBV-infected group (P = 0.015), whereas the opposite for CD42. Similar analyses conducted at the late stage showed more prominent differences in CD34 (P = 0.0003) and CD42 (P<0.00001) (Fig. 2C&D). In summary, the above findings suggest that HBV infection has inhibitory effects on the polyploidization of mature megakaryocytes, suppresses cytoskeletal formation, and hinders the process of megakaryocyte differentiation.
HBV increased the expression of UBE4B in megakaryocytes
In the preliminary experiments, we found that HBV infection can inhibit the differentiation of mature megakaryocytes, but the specific molecule and mechanism involved are unclear. Therefore, in this part of the experiment, we plan to perform a differential analysis of protein expression between the HBV-infected group and the control group using proteomics methods, to screen for differentially expressed proteins between the two groups at the mature megakaryocyte stage. We applied criteria such as fold change greater than 1.8-fold (up-regulation greater than 1.8-fold or down-regulation less than 0.56-fold) and a significance level of P < 0.0510. By proteomic analysis, we observed that the differentially expressed proteins between the two groups were primarily involved in cellular process, metabolic process, biological regulation, regulation of biological process, and cellular component organization or biogenesis (Fig. 3A&B&C). From the secondary mass spectrometry data, we identified differentially expressed proteins with a significant fold change and a P-value < 0.05. To gain insights into their functional significance, we performed bioinformatics analysis to evaluate the cellular biological processes and pathways primarily associated with these proteins. Considering the specific focus of our experiment, we narrowed down the selection of differentially expressed proteins to those involved in nucleotide excision repair processes and protein ubiquitination processes. Based on the fold change and pathway coverage of the target proteins, we prioritized intracellular trafficking-related proteins: AP-2, VPS26, and nucleotide excision repair as well as ubiquitination-related proteins: DDB2, UBE4B. We employed Western blot analysis to evaluate the protein expression levels in the two groups. The results revealed significant differences in DDB2 and UBE4B between the cell groups, with UBE4B showing a more pronounced disparity (DDB2, P = 0.04; UBE4B, P = 0.02) (Fig. 3D). The results showed no significant difference in AP-2 and VPS26 between the two groups. Furthermore, to further investigate UBE4B's involvement, we examined its nucleic acid expression, which also demonstrated statistically significant differences (P = 0.02) between the two groups (Fig. 3E). As a result, UBE4B was identified as the key protein for subsequent mechanistic experiments.
The impact of UBE4B gene knockdown on megakaryocyte differentiation in HBV infection
Due to the significant and elevated expression of UBE4B in mature megakaryocytes infected with HBV observed in preliminary experiments, this section of the study employed lentiviral transduction to knock down UBE4B expression in Meg-01 cells, to investigate the impact of UBE4B on megakaryocyte differentiation after HBV infection. Subsequently, the experiments were divided into three groups: the HBV-infected Meg-01 group (referred to as the NC group), the empty vector-infected Meg-01 group (referred to as the EV group), and the lentiviral-transduced UBE4B gene knockdown group (referred to as the shRNA-UBE4B group).
Cells were cultured to the early differentiation stage of mature megakaryocytes. We analyzed the mean fluorescence expression intensity of CD34, CD41, and CD42 in these three groups, which suggested that there was no significant difference. However, previous experiments revealed that HBV had a stronger inhibitory effect on the late stage of differentiation. Thus, when the cells were cultured to the late differentiation stage of mature megakaryocytes, a distinct distribution pattern was observed in the shRNA-UBE4B group compared to the other two groups. The cells were primarily located in the partitions of CD34dimCD41bright and CD41brightCD42bright. Further analysis of mean fluorescence expression intensity showed no difference in CD34 expression between the NC and EV groups. In contrast, the expression of CD34 was lower in the shRNA-UBE4B group than in the NC group (P < 0.00001). Additionally, there were no differences in CD41 and CD42 expression between the NC and EV groups. However, in the shRNA-UBE4B group compared to the NC group, the expression of CD41 and CD42 was higher (P < 0.00001). It indicated that the maturation of HBV-infected megakaryocytes was enhanced during the late stage after UBE4B gene knockdown (Fig. 4).
Inhibition of megakaryocyte differentiation by HBV-mediated regulation of p53, ERK1/2, and phosphorylated proteins via UBE4B
UBE4B can regulate multiple substrate proteins, such as p53 family proteins and MAPK family proteins. In somatic cells, UBE4B can interact with substrate protein MDM2 to hinder the activation of p5311. Previous research has indicated that p53 activity increases during megakaryocyte terminal differentiation, and this increase is functionally associated with the cessation of mitosis and promotion of apoptosis 12. Likewise, MAPK proteins are also closely linked to the differentiation and development of megakaryocytes13. Consequently, the objective of this experimental section is to investigate whether mature megakaryocytes infected with HBV impede cell differentiation by upregulating UBE4B protein, thereby affecting the expression and activation of p53 and MAPK proteins.
Meg-01 cells were cultured to mature megakaryocytes and then divided into two groups: the HBV-infected group and the control group. We detected the expression of UBE4B, UBE4B-regulated protein p53, phosphorylated p53(p-p53), as well as ERK1/2 and phosphorylated ERK1/2(p-ERK1/2). The results indicated that the expression of UBE4B was lower in the control group than in the HBV-infected group (P = 0.025). The expression of both p53 and p-p53 was higher in the control group compared to the HBV-infected group (P = 0.031 and P = 0.0003, respectively). Comparing the ratio of p-p53 to total p53, the control group showed higher levels of p53 phosphorylation than the HBV-infected group (P < 0.00001). There was no statistically significant difference in the expression of ERK1/2 between the control and the HBV-infected group, but the expression of p-ERK1/2 was lower in the control group than in the HBV-infected group (P = 0.0015). Similarly, when comparing the ratio of p-ERK1/2 to total ERK1/2, the control group exhibited lower levels of ERK1/2 phosphorylation compared to the HBV-infected group (P = 0.0009). These results suggest that HBV upregulates UBE4B while decreasing the phosphorylation level of p53 and increasing the phosphorylation level of ERK1/2. It is suggested that HBV may inhibit the differentiation of mature megakaryocytes by modulating p53 and ERK1/2 (Fig. 5A).
To elucidate the regulatory role of UBE4B on p53 and ERK1/2 in megakaryocytes following HBV infection, we investigated the expression levels of p53, ERK1/2, and their phosphorylated proteins after UBE4B gene knockdown. During the early stage of mature megakaryocytes, the shRNA-UBE4B group exhibited higher expression levels of p-p53 and p53 compared to the NC group (P = 0.0032 and P = 0.0041, respectively). However, the difference in p-ERK1/2 between the two groups did not reach statistical significance (P = 0.0569), while the expression of ERK1/2 was higher in the shRNA-UBE4B group than in the NC group (P = 0.0020). Further analysis of the ratios of p-p53 to p53 and p-ERK1/2 to ERK1/2 revealed that there was no statistically significant difference in the phosphorylation level of p53 between the two groups. However, the phosphorylation level of ERK1/2 was lower in the shRNA-UBE4B group compared to the NC group (P = 0.0216). During the late stage of differentiation of mature megakaryocytes, the shRNA-UBE4B group showed higher expression levels of p-p53 and p53 compared to the NC group (P = 0.0002 and P < 0.0001, respectively), while the expression levels of p-ERK1/2 and ERK1/2 were lower in the shRNA-UBE4B group compared to the NC group (P = 0.0028 and P = 0.0020, respectively). Further analysis of the ratios of p-p53 to p53 and p-ERK1/2 to ERK1/2 indicated that the phosphorylation level of p53 was higher in the shRNA-UBE4B group than in the NC group (P = 0.0168), while the phosphorylation level of ERK1/2 was lower in the shRNA-UBE4B group compared to the NC group (P = 0.0017). No significant differences were observed between the EV and NC groups. These results suggest that UBE4B has a more significant regulatory effect on the expression and phosphorylation of p53 and ERK1/2 during the late stage of mature megakaryocyte differentiation (Fig. 5B).