HIV-1 B、C亚型Tat第41位赖氨酸对HIV-1转录活化的影响

Differential transactivation of HIV-1 subtypes B and C by Tat lysine 41

目的对反式转录激活因子Tat第41位赖氨酸(K41)在HIV-1转录活化过程中的作用进行探讨,并对B、C亚型HIV-1中Tat K41的作用进行比较。方法通过荧光素酶活性实验检测野生型Tat、突变型Tat K41A、Tat K41R对HIV-1转录活性的影响,并比较B、C亚型HIV-1中Tat K41作用的差异;通过免疫沉淀(immunoprecipitation,IP)实验检测B、C亚型HIV-1中Tat K41是否影响Tat与SIRT1的结合;通过Western blot检测细胞核内B、C亚型HIV-1的Tat K41是否影响p65 K310的乙酰化水平。结果与B亚型HIV-1不同,C亚型HIV-1的Tat K41突变后显著降低荧光素酶活性,减弱C亚型Tat与SIRT1的结合,降低细胞核内p65 K310ac的水平。结论C亚型Tat K41在HIV-1转录中发挥重要作用。

Objective To investigate the effect of trans-activation of transcription(Tat)lysine 41 on HIV-1 transcription and compare the effect of HIV-1 subtypes B and C.Methods The effects of wildtype Tat,Tat K41A and Tat K41R mutants on HIV-1 transcription were assessed by luciferase assay,and the differences of Tat K41 effect were compared in subtypes B and C viruses.Next,immunoprecipitation(IP)assay was used to detect whether Tat K41 of HIV-1 clades B and C affects the interaction between Tat and SIRT1.Finally,Western blot was used to detect whether K41 of Tat B and Tat C affects the acetylation level of p65 K310 in the nucleus.Results Mutants of subtype C Tat K41 significantly inhibited Tat transactivation,reduced the interaction between SIRT1 and Tat C and decreased the level of p65 K310ac in the nucleus compared with wildtype of subtype C Tat,but there were no significant changes in subtype B Tat.Conclusions Subtype C Tat K41 plays a vital role in HIV-1 transcription.