HIV-1耐药相关特征性突变位点及复制适应性研究

发布时间：2018-02-28 09:01

本文关键词： HIV 耐药相关突变位点多态性位点适应性　出处：《中国疾病预防控制中心》2015年博士论文　论文类型：学位论文

【摘要】：HIV目前在世界各地广泛传播,抗病毒治疗的开展可以有效的提高患者的生命质量,延长生存寿命,并能达到减少艾滋病传播的目的。但是HIV耐药的出现却严重影响了抗病毒治疗的效果。HIV耐药的产生是一个极其复杂的动态过程,这一过程除了包含多种环境因素外,还与病毒基因相关,是多个位点共同作用的结果。参与耐药发生的病毒基因位点包括耐药相关突变位点和多态性位点,它们通过改变病毒的复制能力,进而达到调整HIV在患者体内的准种分布,实现病毒最优适应性的目的。大多数耐药相关突变位点可以降低病毒的适应性,病毒在进化过程中可能会产生协同突变的多态性位点,补偿耐药位点所造成的病毒适应性下降,但是这一说法还缺少实验证据。本研究通过对我国CRF01_AE亚型、CRF07_BC亚型和B’亚型的HIV序列分析,从生物信息学的角度证明了多态性位点与耐药相关突变位点的协同突变现象。进一步通过体外实验验证这些耐药相关特征性突变位点对于病毒适应性的影响,并对其影响适应性的机制进行初步的探索。该研究对于理解耐药发生的分子基础有重要意义,而且对于改善抗病毒治疗方案,实现对HIV的有效防控提供了重要的基础数据。一、北京市朝阳区2011-2013年男男性行为人群HIV感染情况及耐药基因特征分析本部分通过对北京市朝阳区MSM中未治疗HIV感染者的病毒pol基因分析,获得了该人群的基因亚型分布情况及耐药基因特征,进一步通过比较携带耐药相关突变位点的HIV基因序列与不携带耐药相关突变位点的HIV基因序列,获得了未治疗CRF01_AE亚型中耐药相关突变位点与多态性位点的协同突变现象。研究结果显示,北京市朝阳区MSM处于HIV高流行水平,流行的主要基因亚型依次为CRF01_AE亚型、CRF07_BC亚型和B亚型。该部分的研究人群为未治疗的HIV感染者,其中有25.56%的HIV感染者出现了至少一种相关耐药突变位点。该人群中CRF01_AE亚型中,耐药相关突变位点V179D/E与7个多态性位点(R238K、A272P、T11K、I173K、K174Q、S207Q、S211K)之间具有协同突变关系。二、CRF07_BC亚型和B,亚型耐药相关突变位点与多态性位点的协同突变分析本部分利用R语言技术及相关的软件包CorMut,对CRF07_BC亚型和B’亚型的序列进行分析,获得这两种基因亚型中耐药相关突变位点与多态性位点的协同突变现象。本部分通过比较新疆、四川等地的CRF07 BC亚型中600例未治疗的HIV-1感染病人和344例接受治疗的HIV-1感染病人序列,分析获得了该亚型治疗组中RT区耐药相关突变位点(K103N、M184V、Q197K、G190A、Y181C、L228R、M230L)和多态性位点(A36E、R135I、R277K、L283I、D291E)具有协同突变关系,PR区耐药相关突变位点(L10I)和多态性位点(I132L)之间具有协同突变关系；对600例未治疗的HIV-1感染病人进行分析,获得了未治疗组中RT区耐药相关突变位点(Y188C、K103N、E138G、V108I、L74F)和多态性位点(M16K、K166R、E248V、L283I、E297A、T200M)具有协同突变关系,PR区耐药相关突变位点(L10I)和多态性位点(I64V、A71V)具有协同突变关系。本部分通过比较河南、安徽两地的B’亚型中178例未治疗的HIV-1感染病人和327例接受治疗的HIV-1感染病人序列,分析获得了该亚型治疗组中RT区耐药相关突变位点(Y181C、L210W、M41L、E44A、E203D、H208Y、T215Y、L228R、K65R、Q151M、V75T)和多态性位点(S162C、Q207E、R211K、L214F、I293V)之间具有协同突变关系；对178例未治疗的HIV-1感染病人进行分析,获得了未治疗组中RT区耐药相关突变位点(V106I、M41L、L210W、T215Y、H221Y)与多态性位点(I135V、S68G、I178L、R277K)具有协同突变关系。三、HIV-1耐药相关特征性突变位点对病毒复制适应性的影响本部分研究中,我们首次建立了HIV-1 CRF07_BC亚型的体外生长竞争实验体系,结合HIV-1 B亚型体外生长竞争实验体系,通过体外实验,研究分析第二部分所发现的CRF07_BC亚型和B’亚型中耐药相关特征性突变位点对于病毒适应性的影响,并通过检测P51蛋白相对含量、逆转录酶活性和蛋白质三级结构预测的方法,对其改变病毒适应性的机制进行初步的探讨。对于HIV-1 B'亚型未治疗组中所发现的协同突变位点进行研究发现,HIV-1RT区T215Y+I178L协同突变的重组病毒其适应性明显高于T215Y单点突变的重组病毒适应性,而且与之相比P51蛋白相对含量和逆转录酶活性也明显上升。这提示1178L可能通过影响P51蛋白相对含量及逆转录酶活性的方式补偿耐药相关突变位点T215Y所造成的病毒适应性下降。对于HIV-1 B'亚型治疗组中所发现的协同突变位点进行研究发现,在无药环境中,HIV-1 RT区T215Y+R211K协同突变的重组病毒其适应性明显高于T215Y单点突变的重组病毒适应性；在AZT存在的情况下,T215Y+R211K协同突变的重组病毒的适应性要高于野毒株；这提示在治疗患者体内,R211K的出现,可以使病毒适应性上升,这样的现象可能会加速耐药发展的进程。
[Abstract]:HIV is currently widely spread all over the world, the development of antiviral therapy can effectively improve the quality of life of patients, prolong the survival life, and can achieve the purpose of reducing the spread of AIDS. But HIV resistance has seriously affected the effect of antiviral therapy resistant.HIV production is a complicated dynamic process, this process in addition to contain a variety of environmental factors, but also related with viral gene, multiple sites is the result of joint action. Viral genes involved in resistance loci including resistance related mutations and polymorphisms, their ability to change through the replication of the virus, and then adjust the HIV quasispecies distribution in the patient's body, to achieve the optimal adaptation of the virus purpose. Most resistance related mutations can reduce the virus virus adaptability, may produce polymorphic loci mutation in the evolutionary process of collaborative, Drop compensation resistance loci caused by virus adaptation, but this argument is the lack of experimental evidence. Through the study on China's CRF01_AE subtype, sequence analysis of HIV CRF07_BC subtype and B subtype ", proved that the polymorphic loci associated with resistance mutation sites collaborative phenomenon from the perspective of bioinformatics further. In vitro experiments verify the characteristics of resistance related mutations in virus affecting the adaptability, and its influence on the adaptability of the mechanism was preliminarily explored. The research has an important significance for understanding the molecular basis of drug resistance, but also for improving the anti viral treatment programs provide important basic data to achieve effective prevention and control of HIV. 1. The analysis of Beijing city Chaoyang District 2011-2013 years of MSM HIV infection and drug resistance characteristics through this part of the untreated HIV Beijing city in Chaoyang District MSM Analysis of virus pol gene infection, the distribution of the population genetic subtype and drug resistance gene characteristics, further by comparing the carrying drug-resistant mutations in HIV gene sequence loci associated with drug-resistant mutations do not carry the HIV gene sequences obtained in untreated collaborative resistant CRF01_AE subtype mutation related mutation and polymorphism the site of Beijing city. The results showed that the Chaoyang District MSM HIV in high prevalence, major gene prevalent subtypes were CRF01_AE subtype, CRF07_BC subtype and B subtype. The study population is not part of the treatment of HIV infection, including 25.56% HIV infected patients had at least one related drug resistance mutations in this population. CRF01_AE subtypes, resistance related mutations V179D/E and 7 polymorphic loci (R238K, A272P, T11K, I173K, K174Q, S207Q, S211K) have a synergistic relationship with mutation. Two, CRF07_BC subtype and B subtype resistance related mutations and polymorphisms of the cooperative mutation analysis of this part of the use of R language technology and related software CorMut sequences of CRF07_BC subtype and B subtype analysis, cooperative mutation phenomenon received the two kinds of genotypes of resistance related mutations loci and polymorphic loci. This part compares Xinjiang, Sichuan and other places of CRF07 BC subtype in patients with sequence and 344 patients received the treatment of HIV-1 infection in 600 cases of infected untreated HIV-1 was obtained RT subtype in the treatment group, the drug resistance related mutations (K103N, M184V, Q197K, G190A Y181C, L228R, M230L), and polymorphic loci (A36E, R135I, R277K, L283I, D291E) has a synergistic relationship between the mutation and PR resistance associated mutation region (L10I) and polymorphic loci (I132L) has a synergistic relationship between mutations; 600 cases of untreated HIV-1 infection Carries on the analysis, obtained from untreated RT resistant mutations were in the group (Y188C, K103N, E138G, V108I, L74F) and polymorphic loci (M16K, K166R, E248V, L283I, E297A, T200M) has a synergistic relationship between the mutation and PR resistance associated mutation region (L10I) and polymorphic loci (I64V A71V) has a synergistic mutation relationship. This part compares Henan, Anhui two B subtypes in patients' sequence and 327 patients received the treatment of HIV-1 infection in 178 cases of infected untreated HIV-1 was obtained RT subtype in the treatment group, the drug resistance related mutations (Y181C, L210W, M41L, E44A. E203D, H208Y, T215Y, L228R, K65R, Q151M, V75T) and polymorphic loci (S162C, Q207E, R211K, L214F, I293V) has a synergistic relationship between mutations; 178 cases of untreated HIV-1 infected patients were analyzed, obtained in untreated RT resistant mutations were in the treatment group (V106I, M41L L210W, T215Y, H221Y). With polymorphic loci (I135V, S68G, I178L, R277K) has a synergistic relationship between the mutation of HIV-1. Three, resistance characteristic effects of mutation site on the viral replication adaptability of this part of the study, we first established the HIV-1 subtype CRF07_BC in vitro competition experiment system, combined with the growth of HIV-1 B subtype in vitro competition experiment system in vitro experiments, research, analysis of drug resistance of CRF07_BC subtype and B subtype found the second part of the relevant features of the virus effects of mutation site adaptability, and by detecting the relative content of P51 protein, prediction method of reverse transcriptase activity and protein three level structure, carry on the preliminary discussion to the mechanism of virus adaptability change for the HIV-1 B'subtype is not found in the treatment group collaborative mutations were found that the recombinant virus HIV-1RT T215Y+I178L cooperative mutation its adaptability is obviously higher than that of T215Y The single point mutation of the recombinant virus and adaptability, and the relative content of the reverse transcriptase activity of P51 protein also increased significantly compared with it. The results suggest that 1178L and reverse transcriptase activity by affecting P51 protein relative content of the compensation of drug resistance related mutations caused by T215Y virus adaptability decreased. For the detection of HIV-1 subtype B'in the treatment group the synergistic mutations were found in the drug free environment, the recombinant virus HIV-1 RT T215Y+R211K mutation was significantly higher than that of the adaptive collaborative T215Y single point mutant recombinant virus adaptability; in the presence of AZT, the recombinant virus T215Y+R211K cooperative mutation adaptability is higher than that of wild strain; this suggests that in the treatment of patients. The emergence of R211K, can make the virus adaptation of this phenomenon may rise, will accelerate the resistance development.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R512.91

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