Human Parvovirus B19 Nonstructural Protein 1 Inhibiting The Differentiation Of Erythrocyte Progenitor Cells

Objective To explore a new regulatory mechanism of NS1-Hes-GATA in inhibiting differentiation and maturation of erythroid progenitor cells,We transfected K652 cells with human parvovirus B19 nonstructural protein1 NS1 expressing plasmid.After transfecting,through observing the changes of expression of GATA/2 and analyzing molecular mechanisms causing the change,we demonstrated that B19 NS1 may be involved in Notch-Hes signaling pathway activation,or directly entered the nucleus of host cells and negatively regulates GATA1/2-related nuclear transcription,which consequently inhibited the differentiation and maturation of red blood cells.Methods The recombinant expression plasmid pNS1-HA of B19 NS1 was constructed artificially and transfected into human chronic myeloid leukemia cell line K652.Then the instantaneous expression model was established as recombinant plasmid group,and blank plasmid control group and blank white blood cell control group were established.The cells were collected at different time after transfection.Real-time PCR detects the expression of NS1,Notch1,Hes1/5 and GATA1/2 m RNA from each group.Western blot detects changes of protein expression.si RNA inhibiting Notch,Hes1/5,Western blot detects the expression of GATA1/2 protein.Immunofluorescence and confocal microscopy were used to observe the intracellular localization of the target protein.Results 1.pNS1-HA plasmids were digested with restriction endonucleases BamHI and Xba I,then the expected NS1 fragment was obtained with base sizes of 2031 bp.No target fragment was found in the vetor after restriction endonucleases digestion.After transfecting the p NS1-HA plasmids into permissive K562 cells,a strong band of ~78 k Da,corresponding to the size of the NS1-HA protein,was detected.There was no band of ~78 k Da in K562 cells transfected with vectors or in K562 cells without plasmid transfection.2.The expression of NS1 protein has been detected at 2nd hour after transfection,and the GATA1 m RNA has significant decreased and reached the lowest peak at 4th hour after transfection,which was only 0.1 times higher than that at 0 hour,and showed persistent low expression at 6th,8th and 24 th hour after transfection(P < 0.05).The GATA2 m RNA has significant increased at 4th hour after transfection and continued to increase at 6th,8th and reached its peak at 24 th hour after transfection,which was 8times higher than that at 0 hour(P < 0.05).Compared with blank cells,the expression of GATA1 protein decreased at 2nd hour after p NS1-HA transfection,and continued to decrease at 4th,6th,8th hour after transfection,and reaching the lowest peak at24 th hour after transfection with only 0.1 times as high as that in blank cells(P <0.05).Contrary to GATA1,GATA2 protein began to increase at 2nd hour after transfection and continued to increase at 4th,6th,8th hour after transfection,and reaching its peak at 24 th hour after transfection,which was 2.9 times as high as that in blank cells(P < 0.05).3.NICD protein was expressed at 1.6 fold higher levels in the NS1-expressing transfected cells than in the blank cells.Similarly,HES1/HES5 were higher with 1.5,1.4 fold in the NS1-expressing transfected cells than in the blank cells(P < 0.05).There was no significant change in the expression of NICD,HES1,and HES5 protins in cells transfected with empty vectors and blank cells(P > 0.05).4.Compared with the blank cells,the cells transfected with si-Notch or si-Hes5,si-Hes1 were expressed higher GATA1 and GATA2 proteins(P < 0.05).5.The expression of GATA1 protein in p NS1-HA transfecting K562 cells which pretreatment with si-Notch was significantly higher than that in cells without si-Notch pretreatment(P < 0.05).Compared with blank cells,the expression of GATA1 protein in p NS1-HA transfecting K562 cells which pretreatment with si-Notch did not change significantly(P> 0.05).p NS1-HA transfected cells pretreated with si-Hes1,the expression of GATA1 was significantly up-regulated in K562 cells transfected with p NS1-HA without si-Hes1 pretreatment,and the upregulation effect was higher than that in K562 cells pretreated with si-Notch1,si-Hes5(P < 0.05).6.Observation under confocal microscopy showed that there was obvious CY3 fluorescence signal in the cells transfecting with NS1-expressing plasmids while not in control cells.The CY3 fluorescence signal is overlapped with the nucleus(Blue),the intensity and the area of the CY3 fluorescence signal of the overlapping part was larger than that of the nonoverlapping part,indicating that the NS1 proteins were mainly expressing in the nucleus,a few in cytoplasm.Conclusion The NS1 expressing plasmid p NS1-HA of human parvovirus B19 was successfully constructed and NS1-HA protein was expressed in K562 cells transfected with p NS1-HA.After transfection with p NS1-HA,the expression of GATA2 was up-regulated and GATA1 was down-regulated in K562 cells.Of which,p NS1-HA down-regulated the expression of GATA1 by activating the Notch1-Hes1/Hes5 signaling pathway.After K562 cells were transfected with pNS1-HA,NS1-HA was expressed in both cytoplasm and nucleus,mainly in the nucleus.