融合肽抑制剂T20抵抗株包膜蛋白热变异位点547对人类免疫缺陷病毒-1生物学功能的影响

目的 分析人类免疫缺陷病毒(HIV)的融合肽抑制剂T20抵抗突变株热变异位点547对病毒生物学功能的影响.方法 采用PCR和定点突变方法构建HIV包膜蛋白(Env)突变体pSM-HXB2-DIV/DIM/SIM和PSM-LAI-DIV四种表达质粒,并构建了含有547D突变并置换HXB2或LAI株gp120部分的Env质粒,pSM-LAI gp120/HXB2 gp41-DIV (L/H-DIV)和pSM-HXB2 gp120/LAI gp41-DIV(H/L-DIV).假病毒感染实验和细胞融合实验分析Env的功能;Western blot检测Env表达.圆二色谱(CD)分析6螺旋束(6HB)的稳定性;PyMOL软件分析含有547D的6HB结构.结果 HXB2为骨架的547DIV、DIM及SIM和LAI为骨架的DIV假病毒的感染性低于其野生型(wt),前者的各变异Env的融合活性也明显降低;L/H-DIV假病毒感染性与HXB2 wt相似,但融合活性明显低于wt;H/L-DIV假病毒的感染性低于两种野生型,融合活性与LAI wt相似,但低于HXB2 wt.CD结果证明547D突变降低6HB的稳定性;结构分析显示,547D突变没有改变NHR中该位点与CHR中Q652位点之间的氢键,但可能通过增加NHR的负电荷从而减少病毒与T20的结合.结论 547D突变降低LAI与HXB2株的感染性和HXB2株的融合活性;该突变通过降低6HB稳定性和增加gp41所带负电荷来减少病毒与T20的结合.

Effects of hot spot 547 in the envelope of resistance strain to fusion peptide inhibitor,T20, on biological functions of human immunodeficiency virus-1

Objective To investigate the effects of hot spot 547 in the envelope (Env) of resistance strain to peptide inhibitor,T20,on the biological functions of human immunodeficiency virus (HIV).Methods The plasmids pSM-HXB2-DIV/DIM/SIM and pSM-LAI-DIV containing gp160 were constructed using PCR and site direct mutagenesis.The plasmids containing LAI gp120/HXB2 gp41-DIV (L/H-DIV) and HXB2 gp120/LAI gp41-DIV (H/L-DIV) Env were also constructed.We analyzed the biological functions of these Envs using pseudovirus assay and cell-cell fusion assay and detected the Env expressions by western blot.The six helix bundle (6HB) was analyzed using circular dichroism spectroscopy (CD).The effect of mutation 547D on the molecular structures of 6HB was observed using PyMOL software.Results The infectivity of HXB2 pseudoviruses carrying 547DIV,DIM,SIM and LAI pseudovirus carrying 547DIV mutation is lower than those of its wild type (wt) pseudoviruses.The fusion activity of HXB2 Env mutants is lower than those of HXB2 wt Env.The infectivity of the pseudovirus carrying L/H-DIV Env is similar as that of HXB2 wt Env,but its fusion activity is lower than that of HXB2 wt Env.The infectivity of H/L-DIV is lower than that of both LAI wt and HXB2 wt,but its fusion activity is similar as that of LAI wt and lower than that of HXB2 wt.The results of CD analysis indicated that 547D mutation destabilized the 6 HB.Modeling resistance mutation on 6HB revealed that the hydrogen bound between 547 and Q652 was not interrupted by substitution and 547D mutation decreased the binding of T20 probably via the increasing negative charge.Conclusions Hot spot mutation 547D reduced the infectivity of LAI and HXB2 Envs and fusion activity of HXB2 Env.The 547D substitution destabilized the 6HB and increased the negative charge to reduce inhibitor,T20,binding.