【作者】 张磊；

【导师】 邵一鸣；

【作者基本信息】 中国疾病预防控制中心 ， 免疫学， 2014， 博士

【摘要】 研究目的和意义：HIV-1病毒利用宿主细胞表面的受体CD4和辅助受体触发对细胞的感染,辅助受体主要为CCR5和CXCR4。可利用CCR5的病毒为R5病毒,可利用CXCR4的为X4病毒,两种辅助受体都可以利用的为R5X4双嗜病毒。R5病毒是HIV-1传播、感染早期及慢性感染阶段的主要毒株。X4毒株的出现与随后CD4+T细胞的加速衰减及疾病快速进展相关。在B亚型病毒感染中,约50%疾病进展晚期的患者会出现辅助受体转变毒株R5X4和/或X4。嗜性转变在其它亚型中也有报道,如subtype A、C、D、F、G、CRF01_AE和CRF02_AG。但是至今还未发现CRF07_BC病毒的CXCR4嗜性毒株,对其特征一无所知。根据这个现象我们提出了以下三个科学问题。第一,为什么目前CRF07_BC无CXCR4嗜性毒株?第二,CRF07_BC毒株以后是否会出现CXCR4嗜性毒株?第三,如果CRF07_BC也能发生嗜性转变,那么阻碍其发生嗜性转变的原因是什么?我们认为CRF07_BC毒株目前仍未出现嗜性转变可能是由于其发生嗜性转变的遗传屏障高,嗜性转变受结构和功能的限制,以及与毒株的流行时间及进化相关。对嗜性转变的研究主要是基于B亚型毒株,最近也有C亚型毒株嗜性转变研究报道。因此,基于B亚型和C亚型建立的辅助受体预测方法是否也适于CRF07_BC毒株?CRF07BC的X4毒株与其它亚型X4毒株在V3区是否具有相似的特征?依据B亚型和C亚型X4毒株V3区的特征,我们能否通过分子生物学手段在实验室构建出CRF07_BC X4突变株?以及HIV-1病毒嗜性转变与其膜蛋白在进化中存在什么关系,能否通过HIV-1病毒进化特征预测出CRF07_BC出现CXCR4嗜性毒株的时间?了解HIV-1嗜性转变的特征有助于我们深入了解其发病机制并能够有效地指导HRRTA治疗。获得功能性CRF07_BC CXCR4嗜性毒株能够加快对该亚型病毒特性的研究,为疫苗研究和药物开发提供有利工具。本研究在CRF07_BC感染性克隆pXJDC13的基础上系统地研究了B亚型和C亚型X4毒株V3特征位点对CRF07_BC感染性克隆嗜性的影响。在自然界还未发现CRF07_BC CXCR4毒株的情况下,我们通过合理设计成功地构建CXCR4嗜性转变毒株,使我们从进化的角度更进一步了解嗜性转变的遗传屏障。深入分析HIV-1env基因的进化特征并预测在自然界中出现CRF07_BC嗜性毒株的CXCR4时间,对Env基因的进化分析能够使我们更深入地了解HIV-1膜蛋白进化特征与病毒的流行传播、适应性及嗜性转变等功能的关系。本研究对致病机CRF07_BC理的研究,实施有效的抗病毒治疗方案及进行疫苗和药物研发具有重要的意义。方法和结果：和C亚型X4毒株V3特征对1. HIV-1B嗜性的影响CRF07_BC从序列数据库中下载B亚型、C亚型及Los Alamos HIV的V3CRF07_BC序列,用分析B亚型和C亚型中VESPA嗜性毒株在V3区的特征。并CXCR4分析与B亚型或C亚型CRF07_BC嗜性毒株之间的特征性差异位点。CXCR4根据分析结果及前期报道的与嗜性转变相关的V3区特征,我们在CCR5嗜性感染性克隆CRF07_BC上进行定向突变改造,共设计构建pXJDC13V3突变株35株。用辅助受体预测软件CRF07_BC V3及C-PSSM、geno2pheno对突变株的辅助受体利用进行预测。综合这三种预测方法的预测结CoRSeqV3-C果,3/35为R5毒株,17/35为R5X4双嗜毒株,15/35为X4毒株。参照B亚型X4毒株V3特征,在上共构建了23个V3突变株。突变位点包括pXJDC13和S11R, Q18R、A22T、G24R等。其中21个突变株具有感染性,但是全部D25Q/E/H仍为CCR5嗜性,不能诱发MT-2细胞形成合胞体。参照C亚型X4毒株V3特征构建了12个V3突变株。突变位点包括及S11R、I12V、P16R、Q18R、D25R第13位和14位之间两个氨基酸IG或MG插入。其中仅有一个突变株M55有感染性,其它的12个突变株均由于突变而失去了感染性。而且突变株M55的感染性只有原始克隆pXJDC13的0.14%左右,且仍为CCR5嗜性。2. CRF07_BC CXCR4嗜性毒株的构建及功能鉴定首先,为了确定能够使CRF07_BC V3发生嗜性转变的特征性位点,将pXJDC13及其V3突变株的V3区用融合PCR方法克隆至B亚型CXCR4嗜性感染性克隆pNIA-3骨架上构建V3嵌合病毒。用基于pNIA-3的V3嵌合病毒系统确定能够引发CRF07_BC V3嗜性转变的关键突变位点。共构建了21个嵌合病毒,且所有嵌合病毒都具有感染性。10个嵌合病毒的V3区含有B亚型X4相关特征,仅有NL4-3/M44V3为双嗜毒株,剩余的9个仍为CCR5嗜性。NL4-3/M44V3的V3区带有5个突变位点。带有C亚型X4相关特征的10个嵌合病毒中,3个嵌合病毒仍为CCR5嗜性,剩余7个为双嗜毒株。在这7个双嗜毒株中,有6个毒株具有一个共同的特征,即第13和14位间有两个氨基酸IG插入及P16R突变。而另一个双嗜毒株在13和14位间带有MG插入及S11R突变。这两个与嗜性转变相关的V3特征分别为设计的M45和M61,但是这两类突变在CRF07_BC骨架上无感染性。第13位和14位间插入突变IG或MG及S11R不仅对pXJDC13Env的感染性具有毒害作用,而且对其它C亚型C3(Du156.12), C4(Du172.17), C12(ZM55F.PB28a), C13(ZM109F.PB4)和CRF07_BC CH70, CH91, CH110, CH119和CH120Env克隆的感染性具有同样的毒害作用。为了进一步确定V3区外限制嗜性转变的区域,我们用融合PCR方法将pNL4-3的V1-V5、V1-V3、V3-V5或V3等区域克隆至pXJDC13骨架上,共构建了4个嵌合病毒。但是这4个嵌合病毒均无法有效包装,不能产生有感染性的病毒颗粒。将C亚型CXCR4嗜性Env克隆1109-F-30的V1或V1V2区克隆至CRF07_BC V3突变株M45和M61上构建了4个嵌合病毒。其中3个克隆恢复了感染性,M61/CX1109F-V1仍为CCR5嗜性。1109-F-30的V1V2区可以拯救活M45和M61,且M45/CX1109F-V1V2和M61/CX1109F-V1V2发生了嗜性转变,为CCR5和CXCR4双嗜病毒,可以诱发MT-2细胞形成合胞体。CCR5拮抗剂Maraviroc和CXCR4拮抗剂AMD3100均能有效阻断嗜性转变株对细胞的感染。3.HIV-1嗜性转变与env进化定量分析HIV-1gp120上V1V2和V3区的突变与病毒的嗜性转变相关。为了确定HIV-1嗜性转变是否与env的进化特征相关,本研究对比分析了CRF07_BC毒株和CRF01_AE, CRF02_AG、A1、B、C、D及G亚型env的进化特征。用BEAST软件MCMC方法分析HIV-1各亚型的tMRCA及env全长及其亚区的进化速率。HIV-1env的进化速率约为1.97×10-3～7.54×10-3substitutions/site/year。HIV-1env基因上各亚区的进化速率从高到低依次为V4、V5、V1V2、C3、V3、C2、C4、 C1、C5。主要流行亚型和CRFs的tMRCA分别为：CRF01_AE1971/1959, CRF02AG1965/1968, CRF07BC1990/1987, A11959/1939, B1956/1965, C1932/1939, D1887/1912, F11960/1952, G1966/1969=根据HIV-1各亚型出现X4病毒所需时间△T乘以V1V2V3的进化速率可计算出X4病毒在V1V2V3上所需的累积突变量。再用累积突变量除以CRF07_BC V1V2V3的进化速率估算CRF07_BC出现X4的时间。用V1V2V3累积突变量预测X4病毒出现的时间为2032(2018-2045)。为了进一步验证预测结果的准确性,将成功构建的嗜性转变株v1v2v3和CRF07_BC毒株的v1v2v3序列构建进化树。分析所构建的嗜性转变株在自然界中大概出现的时间。分析发现,在自然界中CRF07_BC病毒达到嗜性转变克隆V1V2V3突变量的时间约在2026年。这与CRF07_BC CXCR4嗜性转变株预测出现的时间基本一致。结论：在自然界中还未发现CRF07_BC CXCR4嗜性毒株情况下,本研究在CRF07_BC CCR5嗜性感染性克隆pXJDC13基础上通过合理设计,成功构建了2株CXCR4嗜性毒株。研究发现B亚型X4毒株的特征无法使CRF07_BC发生嗜性转变,而C亚型X4的特征则可以,即CRF07_BC的CXCR4嗜性转变特征与C亚型一致。V3区嗜性转变相关突变会导致毒株感染性降低或缺失,需要V1V2区域的突变来补偿V3区对感染性的有害突变。在结构分析上发现,V1V2和V3空间结构紧密,其相互作用程度最大。HIV-1某亚型X4毒株的出现及流行程度与该亚型毒株出现的时间及Env上V1V2V3的进化速率相关。该研究对提高艾滋病治疗效果具有重要的指导意义,为CRF07_BC致病机制研究及体外药物筛选等提供了有利的工具。对HIV-1env基因进化的深入分析使我们更清楚地了解HIV-1膜蛋白进化的特征,能够更好地指导下一步对Env及各亚区的研究。本研究首次定量分析了HIV-1嗜性转变与病毒起源时间及进化速率的关系,预测并验证了自然界中出现CRF07_BC CXCR4嗜性转变株的时间。综上所述,CRF07_BC至今没有出现CXCR4嗜性毒株是因为其遗传屏障高,未达到嗜性转变所需的累积突变量。本研究阐明了CRF07_BC CXCR4嗜性毒株的V3特征,通过合理设计,成功构建了2株CXCR4嗜性毒株。首次将HIV-1env基因进化特征与嗜性转变联系起来,预测并验证了CRF07_BC CXCR4嗜性毒株在自然界出现的时间。更多还原

【Abstract】 Background and objectiveThe envelope glycoproteins of human immunodeficiency virus type1(HIV-1) serve to mediate viral entry into cells. This process requires the sequential interaction of gp120with the receptor CD4and coreceptors, such as CCR5or CXCR4. Viruses that only use either CCR5or CXCR4coreceptors are termed R5-or X4-tropic, respectively, while R5X4dual-tropic viruses can use both. HIV-1infection is almost always established with R5viruses, which predominate during the acute and asymptomatic phases of infection. The appearance of X4and/or R5X4viruses is associated with a more rapid decline of CD4+cells in peripheral blood and faster progression to AIDS. Although R5viruses typically persist into late stages of disease, CXCR4-or dual-tropic viruses emerge in approximately50%of individuals infected with subtype B viruses, and coreceptor switching is also reported in subtype A, C, D, CRF01_AE, and CRF02_AG infections. So far, CRF07_BC CXCR4-using viruses have not been found in nature and its characteristics were unknown. So, we have three key questions about CRF07_BC coreceptor switch. First, why there is no CXCR4-tropic virus in CRF07_BC up to now? Second, will CRF07_BC X4virus emerge in future? Third, if yes, what slow CRF07_BC coreceptor switching? We have a hypothesis about CRF07_BC coreceptor switching. We think the genetic barrier is much higher for CRF07_BC than other subtype virus to switch coreceptor-usage. The gp120structure and function also restrict coreceptor switching. Besides, circulating time and evolutionary characteristics may also correlate with HIV-1coreceptor switching.Almost all the studies about coreceptor switching were based on subtype B virus, and recently there were some reports also in subtype C. Dose the coreceptor prediction method developed on other subtype also work in CRF07_BC? Do CRF07_BC CXCR4-tropic viruses share the same characteristics in the V3region with X4variants in other subtypes? Can we construct CRF07_BC CXCR4-tropic variants by mutagenesis according to the V3characteristics of subtype B and C X4viruses? Is there any correlation between HIV-1coreceptor switching and envelope evolution? If yes, what kind of relationship is it and can we use it to predict the CRF07_BC X4virus emerging time in nature? Knowing coreceptor switching in HIV-1will help us to better understand the pathogenesis and improve efficiency of HRRTA treatment. A functional CRF07_BC CXCR4-tropic molecular clone will definitely accelerate the study in this subtype and will be a powerful tool for therapeutics and vaccine research. In this study, firstly we tested the impact of X4-related V3features found in subtype B and C on CRF07_BC. Secondly, according to rational design, we constructed two CRF07_BC X4viruses before its emerging in nature. This makes us better understand the genetic barrier for HIV-1coreceptor switching. Thirdly, we determined the relationship between HIV-1coreceptor switching and envelope evolutionary characteristics and then predicted the CRF07_BC X4virus emerging time in nature. Systematic analysis of HIV-1envelope will show more detailed relationship between viral evolution and HIV-1transmission, fitness, coreceptor switching and so on. This study has special meaning for CRF07_BC pathogenesis study, HRRTA treatment efficiency, therapeutics and vaccine research. Methods and Results 1. Impact of X4-related V3features found in subtype B and C on CRF07_BC coreceptor switchingAll the V3sequences of subtype B, C and CRF07_BC were retrieved from Los Alamos HIV database and coreceptor-specific features were analyzed by VESPA tool. According to VESPA results and X4-related V3characteristics reported previously, we have designed and constructed a panel of V3mutants based on CRF07_BC CCR5-tropic molecular clone pXJDC13. The coreceptor usage of35mutants were predicted by C-PSSM, geno2pheno and CoRSeqV3-C. In combination of these three prediction results, there were3/35R5,17/35R5X4and15/35X4. Twenty-three CRF07BC V3mutants harbor subtype B X4-related features, including S11R, Q18R, A22T, G24R and D25Q/E/H. Twenty one out of23were infectious, but all of them were still CCR5-tropic. Twelve mutants harbor subtype C X4-related V3features, including S11R,112V, P16R, Q18R, D25Q and two amino acids IG or MG insertion between position13and14. Only mutants M55containing IG insertion was poor in infectivity and still CCR5-tropic, while the rest mutants were non-infectious.2. Construction and identification of HIV-1CRF07_BC CXCR4-tropic virusIn this part of work, according to rational design, we constructed two CRF07_BC CXCR4-tropic variants. There were two steps to construct functional X4virus. In the first step, we determined the CRF07_BC X4-related V3features in pNL4-3based chimeric virus system. The CRF07_BC mutated V3region constructed in first part were transferred to pNL4-3backbone by fusion PCR and then the viral coreceptor usage were tested. Twenty-one chimeric viruses were constructed and all of them were infectious. One of ten chimeras containing subtype B X4-related features was dual-tropic and the rest were still CCR5-tropic. Among ten chimeras containing subtype C X4-related features, three were CCR5-tropic and the other seven were dual-tropic. Two kinds of mutations within CRF07_BC V3were correlated with coreceptor switching, IG insertion and P16R or MG insertion and S11R. But these two kinds of mutations deprived infectivity of mutants M45and M61in the CRF07_BC molecular backbone. Similarly, these mutations also have deleterious effect on infectivity of other CRF07_BC (CH70, CH91, CH110, CH119, CH120) and subtype C (Dul56.12, Du172.17, ZM55F.PB28a, ZM109F.PB4) envelope clones. In the second step, based on non-infectious CRF07_BC molecular clone M45and M61, we have successfully constructed two factional CXCR4-tropic variants. The V1V2region comes from subtype C CXCR4-tropic Env clone1109-F-30rescued M45and M61, and conferred them CXCR4-using ability. These two CXCR4-tropic variants grow well on PBMCs and could induce syncitium formation on MT-2cells, and they were also sensitive to CCR5and CXCR4antagonists.3. Quantitive analysis of HIV-1coreceptor switching and envelope evolutionIn this part, we characterized the relationship between HIV-1coreceptor switching and viral envelope evolution. The full-length envelope sequences of nine subtypes were retrieved from Los Alamos HTV databaes, including subtype A, B, C, D, F, G, CRF01_AE, CRF02_AG and CRF07_BC. The tMRCA and envelope evolutionary rate were calculated using BEAST software. The envelope evolutionary rate were between1.97X10-3～7.54×10-3substitutions/site/year. Also, the envelope subregions evolutionary rate were also determined and from faster to slower were V4, V5, V1V2, C3, V3, C2, C4, C1and C5respectively. The tMRCA of each subtype were CRF01_AE1971/1959, CRF02_AG1965/1968, CRF07_BC1990/1987, subtype Al1959/1939, subtype B1956/1965, subtype C1932/1939, subtype D1887/1912, subtype F11960/1952and1966/1969. According to V1V2V3evolutionary rate, tMRCA and emerging time of CXCR4-tropic virus in each subtype, we could determine the mean accumulated mutations needed for coreceptor switching and then predict CRF07_BC CXCR4-tropic virus emerging time in nature. The CXCR4-tropic variants emerging time in CRF07_BC were2032(2018-2045) predicted by V1V2V3accumulated mutations. We also tested this prediction using the two CXCR4-tropic variants constructed in part two. The vlv2v3sequences including two CXCR4-tropic variants created were used to construct ML phylogenetic tree to analyze these variants emerging time in nature. We found that these two CRF07_BC CXCR4-tropic variants would emerge in approximately2026and this is consistent with the predicted time.ConclusionsAccording to rational design, we have successfully created two CRF07_BC CXCR4-tropic variants before its emerging in nature. Subtype C X4-related features, but not subtype B, could confer CRF07_BC CXCR4-using ability. So the coreceptor-using prediction methods developed on subtype C may work well for CRF07_BC. But subtype C X4-related features within V3deprived viral infectivity in CRF07_BC backbone and this deletetious mutations need compensate changes within V1V2to maintain a functional CXCR4-using variant. In consistent with its fuction, the envelope trimer structure revealed that V1V2region just covered on V3loop and they had a close interaction. Besides, we firstly showed that HIV-1coreceptor switching correlated with its prevalance time and evolutionary rate, especially V1V2V3rate. The accumulated mutations within V1V2V3was used to predict CRF07_BC CXCR4-tropic variants emerging time in nature and tested by two CXCR4-tropic variants constructed in this study. In conclusion, at present the genetic barrier is very high for coreceptor switching in CRF07_BC. According to rational design, we have successfully created two CRF07_BC CXCR4-tropic variants. The divergence within V1V2and V3were firstly used to predict and test CRF07_BC CXCR4-tropic variants emergence in nature. This study will help us to improve HRRTA treatment efficiency and provide a powerful tool for therapeutics and vaccine research in HIV-1, especially for CRF07_BC.更多还原