在2004年於台灣人類免疫不全病毒第一型 (HIV-1)感染者有顯著的增加，主要是發現HIV-1 CRF07_BC在靜脈毒癮者間迅速傳播，至今對於HIV-1 CRF07_BC的病毒學上特性及藥物感受性並不清楚。實驗室先前研究發現台灣CRF07_BC感染者在其p6片段都有7-11胺基酸的短少，且已知p6* (即p6pol)片段的突變對於蛋白質酶水解Gag蛋白有影響，因此，本研究的目的為 (1) p6*片段短少對於其蛋白質酶效用之探討; (2) 台灣HIV-1 CRF07_BC感染者所分離出之病毒株對於抗反轉錄藥物之感受性分析。
我們首先構築了包含p6* 7個和11個胺基酸短少的gag-pol質體，在293T細胞產生缺陷病毒進行西方墨點法；也從血漿中萃取HIV-1 CRF07_BC病毒的RNA，利用聚合酶連鎖反應放大並得到pol 基因序列，再將序列上傳至史丹佛大學HIV抗藥性資料庫網站做抗藥性及序列變異性的分析；另外由HIV-1 CRF07_BC感染者的週邊血液單核球細胞培養出病毒，利用藥物感受性試驗分析HIV-1 CRF07_BC病毒對蛋白質酶抑制劑和反轉錄酶抑制劑的抗藥性表現型。除此之外，在In vitro利用蛋白質酶抑制劑Ritonavir篩選CRF07_BC病毒株，藉由基因選植及基因演化樹分析研究不同時間點的病毒序列及親緣關係。
結果發現p6*有減少7或11個胺基酸的HXB2病毒株蛋白質酶水解及病毒釋放效率相對於參考株 (HXB2)較差，釋放效率只有參考株的41％ (p6*減少7個胺基酸的HXB2)和35％ (p6*減少11個胺基酸的HXB2) 。進一步分析其病毒粒子密度及蛋白質酶水解的時效，帶有p6*短少的病毒粒子密度介於0.111-0.1117 g/cm3，經由時間點的收集發現病毒p6*包含7個和11個胺基酸短少其水解Gag蛋白需要到48-96小時才會緩慢的作用；在抗藥性基因型的實驗中，由史丹佛大學HIV抗藥性資料庫網站結果顯示CRF07_BC病毒對蛋白酶抑制劑、非核苷酸反轉錄酶抑制劑及核苷酸反轉錄酶抑制劑都不具有抗藥性的突變存在；而在抗藥性表現型的實驗中，在連續稀釋藥物濃度下顯示大部分的CRF07_BC病毒都對蛋白質酶抑制劑有抗藥性 (2 - 1,250倍)，甚至在藥物高濃度時也無法抑制病毒的產生，但對反轉錄酶抑制劑都不具有抗藥性。另外在In vitro藥物篩選實驗發現至少有四群CRF07_BC病毒存在於此檢體病毒株中，包括了野生株、反轉錄酶胺基酸變異 (K172R, P294Q)、反轉錄酶胺基酸變異 (K172R, F214L)及p6* (N22S, N39D)+蛋白質酶(I77V)+反轉錄酶 (K135R, K172R)，由基因演化樹分析亦可證明這些胺基酸序列分別為四個單系群。
本研究對於台灣CRF07_BC病毒株病毒學上特性及基因多變性和藥物易感受性提供重要的資訊，並且對於未來疫苗的研發和使用藥物的策略具有重要的參考依據。

Human immunodeficiency virus type 1 (HIV-1) infection in Taiwan was drastically increased since 2004. An outbreak of HIV-1 circulating recombinant form (CRF) 07_BC among injection drug users (IDUs) was noted in 2004. However, little is known about the information on biological properties and drug susceptibility of Taiwanese CRF07_BC. Our previously study confirmed that there were 7-11 amino acid deletions of p6* (p6pol) in Taiwanese CRF07_BC. Besides, the significant relationship between mutations of p6* and protease-mediated Gag processing were well-known. Therefore, the goal of this study is to characterize biological properties and drug susceptibility of Taiwanese CRF07_BC. The specific aim of this study is 1) to study the effects of deletions in p6pol protein on viral protease-mediated Gag processing of Taiwanese CRF07_BC 2) to analyze genotypic and phenotypic drug resistance of treatment naïve patients infected with CRF07_BC in Taiwan.
The gag-pol plasmids with 7 or 11 amino acid deletions of p6* were constructed and these two plasmids were transfected to 293T cells to produce defective viruses for western blotting. CRF07_BC viruses were isolated from patient’s peripheral blood mononuclear cells (PBMCs). Viral RNA was extracted from patients’ plasma, and the proviral nucleotide sequences of the pol region were determined using reverse transcription and polymerase chain reaction (PCR). Genotypic drug resistance mutations were identified using the Stanford University HIV Drug Resistance Database. Phenotypic assays for drug susceptibility were used to determine the susceptibility to various protease inhibitors (PIs) and reverse transcriptase (RT) inhibitors. In addition, an in vitro selection experiment was performed to select TW_D854 resistance variants from ritonavir (RTV, Protease inhibitor). Virus sequences and similarity at different time points were performed by cloning and phylogenetic analysis.
Our results showed that efficiency of virus protease-mediated Gag processing and release were lower in 7 or 11 amino acid deletions of p6* (The efficiency of virus release were 41% in 7 amino acid deletions, and 35% in 11 amino acid deletions, respectively) than in reference strain (HXB2). Furthermore, the results in density and timing of protease processing of virus particles indicated that the densities of p6 defective virus particles were between 0.111 and 0.1117 g/cm3. Moreover, viral protease-mediated Gag processing was observed after 48 hours of transfection in viruses with 7 or 11 amino acid deletions of p6*. The results in phenotypic assay suggested that most CRF07_BC isolates had resistance for PIs (2-1250 folds), and most of the virus could not be inhibited even if the PIs were at high concentrations. In vitro, TW_D854 isolate was passaged for 15 days in culture in the presence of ritonavir to select Taiwanese CRF07_BC with a drug-resistant phenotype. The results showed that at least four groups of virus were present in TW_D854 patient, including wild-type, RT double mutant (K172R, P294Q), RT double mutant (K172R, F214L) and multiple region mutant (including p6* (N22S, N39D), protease (I77V) and RT (K135R, K172R)).
This study will provide insight into the genetic diversity and drug susceptibility of Taiwanese CRF07_BC and will be helpful for future vaccine design and antiretroviral treatment strategies.

表目錄 4
圖目錄 5
中 文 摘 要 6
英 文 摘 要 8
第一章 研究背景介紹 10
人類免疫不全病毒 (HIV)與後天免疫缺乏症候群 (AIDS) 10
全球HIV-1/AIDS流行現況 10
HIV-1 CRF07_BC與台灣HIV-1/AIDS流行現況 11
HIV-1的基因與病毒構造 12
傳播方式 13
HIV-1蛋白質酶和轉架蛋白p6pol (p6*) 13
高效能抗反轉錄病毒療法 15
第二章 研究動機與目的 17
研究動機 17
研究目的 17
第三章 材料與方法 18
第一節 病毒培養 18
第二節 質體的製備 18
第三節 病毒抗藥性決定 20
第四節 HIV-1 蛋白質酶功能分析 23
第五節 In vitro HIV-1對於抗反轉錄病毒藥物產生的變異分析 (quasispecies) 25
第六節 基因系統演化樹分析 (phylogenetic tree analysis) 26
第四章 結 果 27
第一節 HIV-1 p6* 短少對蛋白質酶活性的影響 27
第二節 台灣HIV-1 CRF07_BC感染者臨床特徵描述 28
第三節 基因型抗藥性分析 29
第四節 表現型抗藥性分析 29
第五節 利用PIs藥物篩選CRF07_BC病毒株 30
第五章 討 論 33
第一節 探討p6* 短少影響蛋白質酶水解Gag蛋白的功能 33
第二節 探討HIV-1 CRF07BC 病毒株抗藥性之結果 35
第三節 探討利用藥物篩選方式看其quasispecies之變異 36
第六章 參考文獻 39


表目錄
表一、HIV-1 CRF07_BC感染者之臨床特徵 46
表二、台灣CRF07_BC病毒株蛋白質酶胺基酸序列變異分析 47
表三、台灣CRF07_BC病毒株反轉錄酶胺基酸序列變異分析 48
表四、台灣CRF07_BC病毒株對於蛋白質酶抑制劑易感受性分析 49
表五、台灣CRF07_BC病毒株對於反轉錄酶抑制劑易感受性分析 50
表六、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在不同時間點胺基酸之變異 51
表七、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在Day 0時核苷酸之變異 52
表八、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在Day 5時核苷酸之變異 53
表九、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在Day 10時核苷酸之變異 54
表十、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在Day 15時核苷酸之變異 55
表十一、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在不同時間點其相似核苷酸之變異及分佈 56
表十二、CRF07_BC病毒株 (TW_D854) 在含有Ritonavir藥物培養在不同時間點其相似胺基酸之變異及分佈 57


圖目錄
圖一、p6片段7和11胺基酸短少質體之構築(骨幹為HXB2) 58
圖二、西方墨點法分析p6*短少之蛋白質酶水解gag 之效率及釋放效率。 59
圖三、利用蔗糖梯度離心(Sucrose gradient)及西方墨點法分析不同時間點p6*短少之蛋白質酶水解Gag 能力及病毒之密度。 60
圖四、CRF07_BC感染者病毒株對於蛋白質酶抑制劑和反轉錄酶抑制劑易感受性分析。 62
圖五、病毒在高濃度蛋白質酶抑制劑時抗藥性之百分比。 63
圖六、病毒在含有Ritonavir藥物培養下在不同時間點所有病毒其pol 序列的分布情形 64
圖七、病毒在含有Ritonavir藥物培養下不同時間點時pol 序列的分布情形 68

附圖一、蛋白質酶基因對蛋白酶抑制劑相關的抗藥性突變點 69
附圖二、反轉錄酶基因對核甘酸類反轉錄鋂抑制劑及非核甘酸類反轉錄鋂抑制劑相關的抗藥性突變點 70
附圖三、HIV-1 蛋白質酶的立體結構圖 (二聚體)及其相對於蛋白酶抑制劑主要和次藥的抗藥性突變點。 71

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