The Primary Study Of Mechanisms Underlying The Regulation Of APOBEC3G Expression By Viral Macrophage Inflammatory Protein Ⅱ

Objective: viral macrophage inflammatory protein II(v MIP-II) is one of human chemokine homologues encoded by KSHV K4 gene. Our previous studies indicated that v MIP-II could increase the APOBEC3 G. The mechanisms on the modulation of APOBEC3 G expression sitll limited, the interferon and other factors have been reported to increase APOBEC3 G through JAK/STAT or ERK signaling pathways, and chemokine receptors maybe involved the signaling trasnducction. This study aims to explore the mechanisms of regulating APOBEC3 G by viral macrophage inflammatory protein II.Methods: The K4 gene was cloned to eukaryotic expression vector p EGFP- N3, and transfected 293 T cells with lipofectamine 2000, the cells transfect with p EGFP- N3 as control. The expression of v MIP-II in 293 T cells were confrimed by reverase transcription PCR and western-blot assay.The expression levels of APOBEC3 G,CCR3, CCR5, CCR6 were determined by real-time quantitative PCR(q PCR) and Western-blot assay.The expression lvels of APOBEC3 G in cells treated without or with different concentration of AG490(JAK/STAT inhibitor) and U0126(ERK inhibitor) were detected using q PCR and western-blot methods. The protein productions and phosphorylation levels of STAT1, STAT2,STAT3 and ERK1/2 were tested by western-blot. we also comapred the effect of v MIP-II and IFN-a on regulating APOBEC3 G expression. The effect of v MIP-II on APOBEC3 G Promoter was determined by Dual-Luciferase Reporter Assay System.Results:1.the m RNA and protein expression levels of APOBEC3 G were increased by 2.5 and2.2 folds in 293 T cells transfected with p EGFP-N3-K4. IFN-αinduce APOBEC3 G expression in dose-dependend manner, 1000 IU IFN-αtreated 293 T cells, the APOBEC3 G protein increased 2.6 and 2.5 folds in 293 T cells transfected with empty vectors and p EGFP-N3-K4 plasmids, respectively. v MIP-II have significantlystronger effect on upregulation APOBEC3 G expression than IFN-α(P<.0.05)2.v MIP-II increased the CCR3, CCR5, CCR6 m RNA levels and the CCR3, CCR6 protein levels. v MIP-II also elevated the STAT1,STAT2 and STAT 3 phosphorylation levels also increased in 293 T cells. The the APOBEC3 G expression upregulated by v MIP-II repress by AG490(JAK/STAT inhibitor).3.Luciferase assay demonstrated that APOBEC3 G promoter displayed higher activity in cells co-transfected with p EGFP-N3-K4 than the ones co-transfected with empty ve ctor, and its luciferase activity decreased dramatically for the p GL3-720 fragmentConclusion:1.The eukaryotic expression vector p EGFP-N3-K4 can express v MIP-II protein in293 T cells, and induce APOBEC3 G expression. v MIP-II have significantly stronger effect on upregulation APOBEC3 G expression than IFN-α2. The chemokine receptors,such as CCR3 and CCR6 may involving upregulation APOBEC3 G in 293 T cells and JAK/STAT signaling pathway is pivotal for induce APOBEC3 G by v MIP-II.3. v MIP-II induced the expression of APOBEC3 G at transcriptional level, the transcription factor binding sites are mainly at the upstream of transcriptional start site(-720bp—480bp).