TAM类突变对B'亚型HIV病毒耐药和复制能力的影响  

The impact of thymidine analogue resistance mutations on the level of drug resistance and replicative capacity of HIV-1 subtype B'viruses

作　　者：周茜[1,2,3] 邢辉[1] 方志明[1] 陈彬[1] 王铮[1] 邵一鸣 廖玲洁[1] 黄汉菊[2] 

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206 [2]华中科技大学同济医学院,湖北武汉430030 [3]宜昌市疾病预防控制中心,湖北宜昌443000

出　　处：《中国热带医学》2013年第6期655-658,共4页China Tropical Medicine

基　　金：十二五科技重大专项基金项目(No.2012ZX10001002);国家自然科学基金项目(No.81101281)

摘　　要：目的研究T215Y和L210W等TAM类突变在B'亚型pol区遗传背景下对HIV病毒的耐药程度和复制适应性的影响。方法血浆样本来自我国中部地区1名已接受抗病毒治疗且耐药的患者,利用SGA方法获得其3个病毒准种的含TAM突变的pol基因片段,采用PCR引入点突变分别将T215Y和L210W回复突变为野生型密码子,将突变前后的pol区片段替换pNL4-3-BstII感染性克隆的相应片段,构建重组感染性克隆,利用荧光素酶和P24抗原检测分别评价重组病毒的耐药程度和复制能力。结果成功构建了3个重组感染性克隆pNL-PAT-1、pNL-PAT-2、pNL-PAT-3,TAM类突变分别为:T215Y、M41L/L210W/T215Y和M41L/L210W/T215Y,回复突变后的重组感染性克隆分别为pNL-PAT-1-T215Y(-)、pNL-PAT-2-L210W(-)和pNL-PAT-3-L210W(-);与原始重组病毒比较,T215Y和L210W回复突变后的重组病毒对AZT的IC50分别下降4.9、8.1和2.7倍,感染pNL-PAT-1-T215Y(-)的培养上清中P24含量较原始毒株pNL-PAT-1高,而两株含M41L/T215Y的病毒与含M41L/L210W/T215Y突变病毒的复制能力比较结果并不一致,其中pNL-PAT-2-L210W(-)感染上清的P24含量较原始毒株略有降低,而pNL-PAT1-3-L210W(-)的P24含量则较原始毒株明显升高。结论在B'亚型pol区遗传背景下,T215Y和L210W均可引起HIV病毒对AZT的耐药程度升高,T215Y还可造成病毒复制能力的下降,但是L210W对复制能力的影响不一,基因多态性可能对病毒的耐药和复制能力具有一定的影响。Objective To study the influence of thymidine analogue resistance mutations（TAMs） such as T215Y and L210W on antiretroviral drug resistance and replicative capacity of HIV-1 viruses under the genetic background of pol fragment of subtype B＇strains.Methods The plasma sample was collected from a patient infected with HIV-1 subtype B＇ strains who was under antiretroviral therapy and with TAM mutations.Partial pol gene was amplified from three different viral quasispecies with single genome amplification（SGA）,T215Y and L210W was reverse mutated to wild type codons by PCR based point mutation.Recombinant infectious clones were constructed by replacing pol fragment of pNL4-3-BstII with counterparts from the patient＇s viruses.The level of drug resistance and replicative dynamics were evaluated via luciferase assay and P24 measurement.Results Three recombinant infectious clones were successfully constructed,named as pNLPAT-1,pNL-PAT-2 and pNL-PAT-3 with mutations T215Y,M41L/L210W/T215Y and M41L/L210W/T215Y,respectively.Three corresponding infectious clones with reverse mutations at 215 and 201 positions were pNL-PAT-1-T215Y（-）,pNLPAT-2-L210W（-） and pNL-PAT-3-L210W（-）.Compared with the IC50 values to AZT in the original viruses,those in the mutated viruses had been lowered for 4.9,8.1 and 2.7 times.pNL-PAT-1-T215Y（-） had a higher level of P24 in supernatant than pNL-PAT-1 did.The viruses with reverse mutation of L210W had different levels of replication as compared with the original ones： pNL-PAT-2-L210W（-） had a slightly lower level of P24 than pNL-PAT-2 did,but pNL-PAT-3-L210W（-） had a much higher level of P24 as compared with pNL-PAT-3.Conclusions TAM mutations such as T215Y and L210W can raise the resistance to AZT of subtype B＇strains to certain levels.T215Y can impair viral replicative fitness.However,L210W might have different impacts on replicative capacity in different HIV-1 strains.Genetic diversity may have a role in drug resistance and viral replicative fitness.

关 键 词：人类免疫缺陷病毒 感染性克隆 耐药突变 复制能力 

分 类 号：R512.91[医药卫生—内科学]