| Introduction:Human apolipoprotein B mRNA editing enzyme catalytic polypeptide 3A,one of the antiviral functional factors of the innate immunity of cells,which can convert cytosine into uracil in the single strand DNA or RNA,recoding virus gene and inhibiting replication of multiple viruses,such as hepatitis B virus(HBV),the human Immunedeficiency virus(HIV),and human papillomavirus(HPV).Previous studies reported that alpha interferon can upregulate expression of A3 A in hepatocytes,and convert cytosine into uracil in the covalently closed circular DNA,thus resulting cccDNA degradation by editing excision repair mechanism.This may bring people hope for curing HBV.However,there are still some problems in the deamination of A3 A,which not only plays a role in deamination of exogenous DNA such as viruses,but also edits the genome of cells,causing DNA damage and genomic mutations in host cells,therefore affecting its antiviral effect and safety.Hepatitis B virus core protein has the characteristics of specific binding to cccDNA and mRNA.In this study,to further improve the effects of A3 A in inhibiting HBV replication,we plan to construct fusion protein of A3A-HBc to enhance the ability of A3 A specifically binding to cccDNA and RNA,thereby enhancing its antiviral effect.Objective:In order to improve the effects of A3 A on inhibiting HBV replication,fusion protein expression vectors containing the apolipoprotain B mRNA editing enzyme catalytic polypeptide-like3A(APOBEC3A,A3A)and full-length or truncated hepatitis B virus core protein(HBc)were constructed.To study the expression and localization of fusion proteins in cells,and identify their cytosine deaminase activity,we preliminarily studied the antiviral effects of fusion proteins.Methods:1.Using in-Fusion cloning method,the A3 A gene and the coding sequence of HBc or four kinds of truncated HBc respectively were cloned into a eukaryotic expression vector pcDNA3.0,and the eukaryotic expression vectors pcA3A-HBc,pcA3A-HBc144 S,pcA3A-HBc144 E,pcA3A-HBc144 AAA,and pcA3A-HBc144 A were successfully constructed.Following that,five kinds of recombinants were transfected into the commonly used exogenous gene high expression cell line HEK293 T respectively after confirmed by DNA sequencing.Immunofluorescence cytochemistry was used to detect the localization and distribution of fusion proteins in HEK293 T cells.The expression of fusion proteins were detected by Western blot.The deaminase activity analysis of fusion proteins was performed by electrophoresis on a urea denaturing polyacrylamide gel.2.HBV replication expression plasmid pCH-3091 and five recombinant plasmids or pcA3 A plasmid were co-transfected into the hepatoma cell Huh7 cells.Then,immunofluorescence cytochemistry was used to detect the expression and localization of fusion proteins in cells;The influence of fusion proteins on Huh7 cell proliferation and cell cycle were detected by CCK8 analysis and flow cytometry;The levels of HBsAg and HBeAg in the culture supernatant were measured by ELISA;Real-time quantitative PCR was used to detect the level of HBV DNA in Huh7 cells.1.The five recombinant plasmids were verified by DNA sequencing;Five recombinant plasmids were expressed in HEK293 T cells successfully;The expression levels of fusion proteins containing the truncated HBc were higher than those of fusion protein containing the full length HBc.A3A-HBc was mainly distributed in the cytoplasm of HEK293 T cells,and A3A-HBc144 S,A3A-HBc144 E,A3A-HBc144 A,A3A-HBc144 AAA were all localized in the nucleus of HEK293 T cells.There was no significant difference between the cytosine deamination activity of the fusion proteins containing the truncated HBc and the control group A3 A,which was higher than that of the fusion protein with full-length HBc(A3A-HBc).2.In the Huh7 cell model for antiviral experiments,the fusion protein containing the full length HBc was mainly localized in cytoplasm,while those proteins containing the truncated HBc were all localized in the nucleus.The overexpression of five fusion proteins was not significantly toxic to Huh7 cells and cell cycle.3.In the Huh7 cell model for antiviral experiments,at three days,the level of HBsAg in the culture supernatant of A3A-HBc144 S,A3A-HBc144 E,A3A-HBc144 AAA,and A3A-HBc144 A group were respectively 31.44±1.90%?11.42±2.74%?18.36±6.06%?43.76±0.66%,(p < 0.05)of the empty vector pcDNA control;The level of HBeAg in the culture supernatant in each group was respectively 59.51±2.61%(p < 0.05),32.95±2.47%(p < 0.05),45.86±2.61%(p < 0.05),and 84.18±11.03%(p > 0.05);The level of HBV DNA in cell lysis in each group was respectively(75.65±2.68%?46.80±3.39%?58.04±9.21%?54.10±6.65%,p < 0.05).Among them,the HBsAg,HBeAg,and HBV DNA decreased most significantly in the fusion protein A3A-HBc144 E and A3A-HBc144 AAA groups.At seven days,the level of HBsAg in the culture supernatant in each group was respectively(56.85±9.79%?12.15± 2.12%?21.32±7.24%?56.50±2.93%,p < 0.05);The level of HBeAg in the culture supernatant in each group was respectively(64.91±12.14%?38.13±4.75%?42.28±0.06%,58.71±0.92%,p < 0.05);The level of HBV DNA in cell lysis in each group was respectively 80.61±3.65%(p < 0.05),87.07±9.13%(p > 0.05),54.52±6.21%(p < 0.05),52.60±3.21%(p < 0.05).At 7 days,the levels of HBsAg and HBeAg in A3A-HBc144 E Results: and A3A-HBc144 AAA groups decreased most significantly,but the level of HBV DNA in A3A-HBc144 E group did not decrease significantly.Conclusions:1.The eukaryotic expression vectors of the fusion protein of A3 A with full-length or truncated HBc were successfully constructed and expressed in HEK293 T cells and Huh7 cells.2.The cytosine deamination activity of fusion proteins containing the truncated HBc was equivalent to that of A3 A,while the cytosine deamination activity.of fusion protein containing the full-length HBc was lower than that of A3 A..3.The fusion protein containing the full length HBc was mainly localized in cytoplasm,the fusion proteins containing the truncated HBc were all localized in the nucleus.4.In vitro experiments,the fusion proteins containing the truncated HBc all had the potential to inhibit the replication and expression of HBV.Among them,two kinds of fusion proteins(A3A-HBc144 E and A3A-HBc144AAA)had stronger potential inhibition effect on HBV replication,which lay a foundation for further research on antiviral effect. |
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