HIV-1 Recombination In Natural Target Cells

描述（由申请人提供）：HIV-1的多样化和进化使病毒能够持续存在，尽管有强有力的免疫反应和有效的抗逆转录病毒治疗。在全球范围内，病毒多样化导致了不同的系统发育分支和亚型，这是疫苗开发公认的问题。虽然人们对序列单碱基替换导致的病毒序列变化的研究投入了大量的注意力，但对重组对HIV-1多样化和进化的贡献却知之甚少。以前的研究量化逆转录病毒重组或检查影响重组的因素，要么在体外使用纯化的病毒成分，要么在体内使用非感染目标的转化成纤维细胞。我们已经开发了一种方法，可以快速和定量地研究任何人类细胞类型感染期间的重组，包括原代T细胞和巨噬细胞。初步研究表明：(1)通过检测相关靶细胞群中的HIV-1重组，HIV-1的重组性比之前认为的要高；(2)某些细胞类型的HIV-1重组率明显高于其他细胞类型；(3)单核细胞的分化导致重组率增加3倍。(4) HIV-1重组可能受到逆转录酶内突变的影响，这种突变会改变酶的活性，从而导致临床对抗逆转录病毒药物产生耐药性。基于这些初步研究，我们假设感染过程中的重组率与感染细胞的分化、激活和/或增殖有关。我们还假设重组率与逆转录动力学直接相关，这是由感染细胞和影响酶功能的RT突变决定的。我们将通过以下具体目标来检验这些假设：1。确定骨髓分化对重组率的影响。2. 确定T细胞活化和增殖对重组的影响。3. 确定目标1、2和4中逆转录动力学与重组之间的关系。4. 确定在影响RT活性和耐药性的突变重组中的作用。

DESCRIPTION (provided by applicant): Diversification and evolution of HIV-1 enables the virus to persist despite a vigorous immune response and the administration of potent antiretroviral therapy. Globally, viral diversification has led to distinct phylogenetic clades and subtypes of recognized concern to vaccine development. While much attention has been devoted to the study of viral sequence changes resulting from sequential single base substitutions, the contribution of recombination to HIV-1 diversification and evolution is less well understood. Previous studies quantifying retroviral recombination or examining factors which affect recombination have been performed either in vitro using purified viral components, or with transformed fibroblastic cells that are not targets of infection in vivo. We have developed methods which permit rapid and quantitative study of recombination during infection of essentially any human cell type, including primary T cells and macrophages. Preliminary studies reveal (1) that by examining HIV-1 recombination in the relevant target cell population, HIV-1 is revealed to be more recombinogenic than previously appreciated, (2) that HIV-1 recombination rates are substantially higher in some cell types than in others, (3) that differentiation of monocytic cells leads to a 3 fold increase in recombination rates, and (4) that HIV-1 recombination can be influenced by mutations within the reverse transcriptase enzyme which alter enzyme activity and confer clinical resistance to antiretroviral drugs. Based on these preliminary studies, we hypothesize that the rate of recombination during infection is linked to the differentiation, activation and/or proliferation of the infected cell. We also hypothesize that recombination rates are directly linked to the kinetics of reverse transcription as determined by both the infected cell and by mutations in RT, which affect enzyme function. We will test these hypotheses through the following specific aims: 1. Determine the effects of myeloid differentiation on recombination rates. 2. Determine the effects of T cell activation and proliferation on recombination. 3. Determine the relationship between the kinetics of reverse transcription and recombination within aims 1, 2 and 4. 4. Determine the role in recombination of mutations which influence RT activity and drug resistance.