HIV-1 Recombination In Natural Target Cells

描述(由申请人提供)：艾滋病毒-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，病毒多样化已导致疫苗开发受到公认关切的不同系统进化分支和亚型。虽然人们对顺序单碱基替换引起的病毒序列变化的研究投入了很大的精力，但重组对HIV-1多样性和进化的贡献还不是很清楚。以前量化逆转录病毒重组或检查影响重组的因素的研究要么是使用纯化的病毒成分进行的，要么是使用转化的成纤维细胞进行的，这些细胞不是体内感染的目标。我们已经开发出方法，可以快速和定量地研究感染期间基本上任何类型的人类细胞的重组，包括原始T细胞和巨噬细胞。初步研究显示：(1)通过检测相关目标细胞群体中的HIV-1重组，HIV-1的重组能力比先前认识的更强，(2)某些细胞类型的HIV-1重组率比其他细胞类型高得多，(3)单核细胞的分化导致重组率增加3倍，(4)HIV-1重组可受到逆转录酶内突变的影响，逆转录酶体内的突变会改变酶活性，并使临床对抗逆转录病毒药物产生耐药性。基于这些初步研究，我们假设感染期间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说：1.确定髓系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AIMS 1、2和4.4中逆转录和重组动力学之间的关系。4.确定影响RT活性和耐药性的突变在重组中的作用。

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.