摘要
目的分析整合酶(IN)区主要耐药突变对HIV-1 CRF01_AE毒株耐药的影响,并比较与B亚型毒株的差异。方法根据美国斯坦福大学HIV耐药数据库选择7个IN区突变或联合突变(T66K、F121Y、Q148K、N155H、G118R、R263K、Q148K/N155H),通过无缝克隆同源重组及点突变的方法引入到HIV-1 B亚型感染性克隆pNL4-3和CRF01_AE感染性克隆pGX002的IN区,转染293T细胞包装病毒,在MT2细胞上扩大培养并测定感染性滴度。检测4种整合酶链转移抑制剂(INSTIs),拉替拉韦(RAL)、埃替拉韦(EVG)、多替拉韦(DTG)、比昔格韦(BIC)对14株突变病毒的半抑制浓度(IC_(50))及其与野生型病毒相比提高的倍数。结果成功构建携带7个IN区突变或联合突变的B亚型和CRF01_AE质粒,包装获得14株重组病毒,感染性滴度为10^(4)~10^(6)半数组织细胞感染剂量(TCID50)/ml,在MT2细胞高效复制,上清液中HIV-1 P24抗原浓度可达830~2700 ng/ml。5个突变或突变组合(T66K、F121Y、Q148K、N155H、Q148K/N155H)均可导致CRF01_AE和B亚型毒株对RAL和EVG高度耐药,与野生病毒相比IC_(50)分别提高200倍和2000倍以上,相同突变导致RAL和EVG对CRF01_AE的IC_(50)提高的倍数均显著低于B亚型(P<0.01)。Q148K/N155H突变导致B亚型和CRF01_AE对DTG和BIC高度耐药,IC_(50)提高50倍以上,其他突变对DTG和BIC的药物敏感性几乎无影响。结论构建了基于CRF01_AE和B亚型的14株携带不同INSTI耐药突变的HIV-1毒株,5个突变可导致对RAL和EVG的高水平交叉耐药,同一突变导致B亚型毒株耐药程度显著高于CRF01_AE毒株。Q148K和N155H突变组合可导致DTG和BIC的高度耐药,表明DTG和BIC耐药的遗传屏障高,可有效抑制携带INSTI耐药突变的毒株,且无明显的亚型耐药差异。
Objective To analyze the effects of the main drug resistance mutations in the integrase(IN)region on the resistance of HIV-1 CRF01_AE strains,and compare the differences with subtype B strains.Methods Seven IN region mutations or combined mutations(T66K,F121Y,Q148K,N155H,G118R,R263K,Q148K/N155H)were selected from the HIV drug resistance database of Stanford University in the United States,and introduced to the IN region of HIV-1 B subtype infectious clone pNL4-3 and CRF01_AE infectious clone pGX002 by seamless cloning,homologous recombination and point mutation.The mutant plasmids were transfected into 293T cells for virus packaging.The culture was expanded in MT2 cells and infectious titers were detected.Half maximal inhibitory concentrations(IC_(50))of four integrase inhibitors(INSTIs),raltegravir(RAL),elvitegravir(EVG),dolutegravir(DTG)and bictegravir(BIC),against 14 mutant viruses were detected and compared with the IC_(50) against the wild-type viruses.Results B subtype and CRF01_AE plasmids carrying seven IN region mutations or combined mutations were successfully constructed,and 14 recombinant viruses were packaged with an infectious titer of 10^(4)-10^(6) median tissue culture infective dose(TCID50)/ml.The recombinant viruses replicated efficiently in MT2 cells.The concentrations of HIV-1 p24 antigen contained in the supernatants of cell culture reached 830-2700 ng/ml.Five mutations or combined mutations(T66K,F121Y,Q148K,N155H,Q148K/N155H)caused CRF01_AE and B subtype strains to be highly resistant to RAL and EVG,resulting in an increase in the IC_(50) by 200 times and 2000 times or more as compared with the IC_(50) against the wild-type viruses.The same mutation-caused fold changes of IC_(50) of RAL and EVG against CRF01_AE were significantly lower than that of subtype B(P<0.01).Q148K/N155H mutation caused B subtype and CRF01_AE to be highly resistant to DTG and BIC,with IC_(50) increased by more than 50 times.Other mutations had little effects on the sensitivity to DTG and BIC.Conclusions Fourteen HIV-1 strains carrying seven INSTI resistance mutations based on B subtype and CRF01_AE were constructed.Five mutations resulted in high resistance to RAL and EVG,and there was a high level of cross-resistance.Resistance to RAL and EVG caused by the same mutation was higher in B subtype than in CRF01_AE.The combined mutation of Q148K and N155H was associated with greater resistance to DTG and BIC,indicating that the genetic barrier of DTG and BIC resistance was high.DTG and BIC could effectively inhibit the strains carrying INSTI resistance mutations without obvious subtype difference.
作者
姚腾冲
韩婧婉
李韩平
贾磊
王晓林
李林
李敬云
Yao Tengchong;Han Jingwan;Li Hanping;Jia Lei;Wang Xiaolin;Li Lin;Li Jingyun(HIV/AIDS Department,Beijing Institute of Microbiology and Epidemiology,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100071,China)
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2022年第2期81-87,共7页
Chinese Journal of Microbiology and Immunology
