DEVELOPMENTAL VIROLOGY RESEARCH--RESISTANCE TO ANTI-RETROVIRAL AGENTS

目前，大多数药物正在积极的临床研究中， 治疗人类免疫缺陷病毒（HIV）， 靶向病毒逆转录酶（RT）的化合物。 主 本提案的目标是：（1）详细研究 病毒耐药性和（2）确定克服病毒耐药性的新策略 耐药性 最直接和临床相关的手段， 上述目标是计划的纵向病毒分离， 选择接受抗病毒治疗的患者。 将对分离株进行筛选 就其对所用药物的敏感性以及对 其他抗病毒化合物。 鉴于大多数抗艾滋病毒化合物 靶向RT，关键是要知道临床治疗的机制， 抗性是RT相对于这些改变的结果。 化合物、其活性代谢物或宿主的 新陈代谢. 为了进一步表征酶的分子基础， 当病毒产生耐药性时，病毒RNA将通过PCR技术扩增， 进一步通过质粒pkk 322 - 2载体进行克隆和包装，以转化E. coli JM 109细胞。 将检查提取的RT的 与抗病毒化合物或其代谢物的动力学相互作用。 DNA 将对耐药重组体的RT进行测序，以评估 无论是点突变还是病毒基因组上的"热点"， 对化合物的耐药性或交叉耐药性， RT抑制。 研究可能的宿主因素参与 耐药性的发展，核苷类似物的代谢， 从患者外周血单核细胞（PBMC）和 将检查血浆。 血浆中天然存在的 能克服核苷治疗作用的核苷 还将探索类似物。 总之，这些研究应该提供一个分子的理解， 机制负责出现临床耐药艾滋病毒的任何 给予药物，并提供临床相关的选择依据， 开发替代药物来克服耐药性。

Currently, most agents under active clinical investigation for the treatment of the human immunodeficiency virus (HIV), are virustatic compounds that target the viral reverse transcriptase (RT). The primary objectives of this proposal are (1) to study in detail the mechanisms of viral drug resistance and (2) to identify new strategies for overcoming drug resistance. The most direct and clinically relevant means of pursuing the aforementioned aims is the planned longitudinal virus isolation from selected patients on antiviral therapy. The isolates will be screened serially in terms of their sensitivity to the drug employed as well as to other antiviral compounds. Given that most of the anti-HIV compounds target the RT, it is crucial to know whether the mechanism of clinical resistance is the result of an alteration in the RT with respect to these compounds, their active metabolites or an alteration in the host's metabolism. To characterize further the molecular basis of the enzyme resistance, the viral RNA will be amplified via the PCR technique and further cloned and packaged via a plasmid pkk 322-2 vector to transform E. coli JM 109 cells. The extracted RT will be examined with regard to its kinetic interaction with antiviral compounds or their metabolites. DNA sequencing of the resistant recombinants' RT will be performed to assess whether point mutations or a 'hot-spot' on the viral genome renders resistance or cross-resistance to compounds exerting their action through RT inhibition. To study the possible host factors involved in the development of resistance, the metabolism of the nucleoside analogues as harvested from both patient peripheral blood mononuclear cells (PBMC) and plasma will be examined. The plasma levels of naturally occurring nucleosides which can overcome the therapeutic action of nucleoside analogues will also be explored. In summary, these studies should provide an understanding of the molecular mechanisms responsible for the emergence of clinically resistant HIV to any given drug, and provide a clinically relevant rational for selecting and developing alternative agents to overcome that resistance.