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標題: | 第一型人類免疫缺乏病毒於p6蛋白PTAP具插入胺基酸之功能探討 Functional characterization of HIV-1 with insertions near the p6 PTAP motif |
作者: | Yi-Ting Chen 陳怡婷 |
指導教授: | 張淑媛(Sui-Yuan Chang) |
關鍵字: | p6蛋白,PTAP motif 重複,Vpr, p6 protein,PTAP motif duplication,Vpr, |
出版年 : | 2019 |
學位: | 碩士 |
摘要: | 第一型人類免疫缺乏病毒(HIV-1)的組裝與釋出由其結構蛋白Gag所主導。Gag之組成由N端依序為基質蛋白(Matrix, p17)、衣殼蛋白(Capsid, p24)、核蛋白殼(Nucleocapsid, p7)以及C端的p6蛋白。病毒之出芽需藉由p6蛋白的late domain召集細胞內之內吞體分選轉運複合體(Endosomal-sorting complex required for transport, ESCRT)來進行。p6蛋白亦於病毒組裝時結合Vpr蛋白,並將其帶入病毒顆粒內,以利進一步的病毒複製。p6蛋白基因的變異性相對高,在實驗室過去所收集的HIV-1臨床病毒樣本中,可於p6區域發現許多胺基酸的插入(Insertion)變異。這些變異是否會如先前本實驗室發現在p6有七個胺基酸刪除(30-36)的重組型CRF07_BC,會造成病毒釋出效率及生長較差的現象目前仍不清楚。
本研究針對2013年至2018年共2086個來自臨床之HIV-1 p6蛋白基因樣本之胺基酸序列進行分析,並統計最常見之插入胺基酸模式。統計結果發現最常見的插入位置為鄰近late domain的P11,其中佔最多數的插入胺基酸序列為APP (304/586, 51.9%)與EPSAPP(63/586, 10.8%)。首先以演化樹分析確認感染此兩種病毒病人並非由單一來源傳播,接著進一步分析發現感染此兩種病毒之病人族群基礎點的血漿病毒量較高。為了進一步了解這些胺基酸插入對於病毒複製的影響,我們建構重組病毒株,並分析其表型。帶有APP與EPSAPP插入的病毒分別命名為Ins3與Ins6。實驗結果顯示Ins6病毒生長能力較原生型(WT)佳;而病毒蛋白表現部分,透過分析結構蛋白Gag的切割,所得之切割效率(processing efficiency)與相對釋放係數(relative releasing factor)皆與WT沒有差異,也確認病毒蛋白酶表現量亦無差異。最後發現Ins6病毒顆粒的Vpr含量較高,可能藉此促進病毒複製功能。最後也進行藥物感受性分析,結果顯示帶有插入胺基酸之病毒對LPV、EFV、FTC和DTG等藥物的感受性較差。 The assembly of human immunodeficiency virus type 1(HIV-1) virus is driven by the Gag precursor Pr55Gag, which is composed of matrix (MA), capsid (CA), nucleocapsid (NC), and p6Gag protein. Virus budding is facilitated by p6Gag protein through recruiting endosomal sorting complex required for transport (ESCRT) machinery of host cells. The p6Gag protein can also help to recruit the accessory protein Vpr into virus particles. The genetic sequence of p6Gag is quite polymorphic, and amino acid-insertions in p6Gag have been observed in our clinical samples. However, whether these polymorphisms could contribute to phenotypic changes similar to our previously reported 7-amino acid deletion in p6Gag of CRF07_BC remains unclear. The study aimed to analyze the amino acid insertion patterns of p6Gag among 2086 clinical HIV-1 samples collected from 2013 to 2018, and further investigate whether there are phenotypic changes due to the genetic variations. We found the most common insertion site are located at P11, which is located beside one of the late domains, and the most frequent observed insertion patterns are 3-amino acid insertion APP (304/586, 51.9%) and 6-amino acid insertion EPSAPP (63/586, 10.8%). Phylogenetic analysis was conducted to exclude the potential spreading of a single source virus for the observed insertion patterns. Those patients who infected with APP-insertion or EPSAPP-insertion virus tend to have higher baseline plasma viral load. To further characterize the phenotypes of viruses with APP-insertion or EPSAPP-insertion, recombinant virus clones were constructed and the respective clones were named as Ins3 and Ins6, respectively. The Ins3 and Ins6 viruses demonstrated greater growth capacity than wild type. However, Gag processing analysis showed no significant difference of relative releasing factor as well as processing efficiency between these two mutant viruses and WT; expression levels of protease was also similar. Interestingly, we found Ins6 virus incorporated more Vpr than WT, which might contribute to the increased replication capacity. Finally, the drug susceptibility of these mutatnt viruses was determined and viruses with insertion showed reduced susceptibility to LPV, EFV, FTC and DTG. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21271 |
DOI: | 10.6342/NTU201903664 |
全文授權: | 未授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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