Dynamics of HIV-1 recombination in its natural target cells
Dynamics of HIV-1 recombination in its natural target cells
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DOI：
10.1073/pnas.0306764101
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发表时间：
2004-03-23
影响因子：
11.1
通讯作者：
Shaw, GM
中科院分区：
文献类型：
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作者：
Levy, DN;Aldrovandi, GM;Shaw, GM
据信遗传重组有助于HIV-1多样化并摆脱宿主的免疫和抗病毒药性，但是在自然目标细胞种群中，这种过程在很大程度上仍未受到检查。我们开发了一种直接测量HIV-1重组的方法，该方法采用了具有功能性增强黄色荧光蛋白（YFP）和增强的Cyan荧光蛋白（CFP）基因的报告病毒，其中重组会在感染细胞中产生改良的GFP基因和GFP。这些记者病毒允许通过流量仪分析同时量化细胞培养和动物模型中HIV-1感染，共同感染和重组的动力学。多轮感染测定法表明，生产性细胞共感染几乎没有功能抑制作用。结果，在T淋巴细胞中HIV-1复制过程中的感染率和人类胸腺移植物中的感染率平方以及严重的联合免疫缺陷型（SCID）-HU（THY/LIV）小鼠中进行了重组。这些结果表明，病毒载量的增加可能会通过重组多样化赋予病毒逃生的复合风险。培养物中T淋巴细胞中的一轮复制平均每病毒重组事件，巨噬细胞的感染导致了大约30次交叉事件，这使HIV-1的重组级比以前认识到的重组级高，并表明表明这一点表明这一点表明。感染的细胞对重组频率产生了深远的影响。
Genetic recombination is believed to assist HIV-1 diversification and escape from host immunity and antiviral therapies, yet this process remains largely unexamined within the natural target-cell populations. We developed a method for measuring HIV-1 recombination directly that employs reporter viruses bearing functional enhanced yellow fluorescent protein (YFP) and enhanced cyan fluorescent protein (CFP) genes in which recombination produces a modified GFP gene and GFP fluorescence in the infected cells. These reporter viruses allow simultaneous quantification of the dynamics of HIV-1 infection, coinfection, and recombination in cell culture and in animal models by flow-cytometric analysis. Multi-round infection assays revealed that productive cellular coinfection was subject to little functional inhibition. As a result, generation of recombinants proceeded according to the square of the infection rate during HIV-1 replication in T lymphocytes and within human thymic grafts in severe combined immunodeficient (SCID)-hu (Thy/Liv) mice. These results suggest that increases in viral load may confer a compounding risk of virus escape by means of recombinational diversification. A single round of replication in T lymphocytes in culture generated an average of nine recombination events per virus, and infection of macrophages led to approximate to30 crossover events, making HIV-1 up to an order of magnitude more recombinogenic than recognized previously and demonstrating that the infected cell exerts a profound influence on the frequency of recombination.