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標題: | 人類免疫缺乏病毒CRF07_BC重組亞型病毒株之功能性探討 Functional Characterization of HIV-1 CRF07_BC Strain |
作者: | Huei-Ling Huang 黃惠玲 |
指導教授: | 張淑媛 |
關鍵字: | CRF07_BC亞型病毒,蛋白酶,p6, CRF07_BC,protease,p6, |
出版年 : | 2012 |
學位: | 碩士 |
摘要: | 影響人類免疫缺乏病毒(HIV)疾病進程快慢的主要因素可分為病毒因子和宿主的遺傳因子。藉由了解疾病進程較為緩慢之感染者體內病毒的特徵,將有助於發展預防HIV感染及臨床治療的策略。在2004年之前,台灣HIV的傳播主要是經由性行為接觸;約70% HIV感染者經由同性間性行為感染B亞型病毒,其餘則經由異性間性行為感染CRF01_AE亞型病毒。然而,在2004年,靜脈藥癮族群爆發CRF07_BC亞型病毒感染。根據臨床資料,我們發現,相較於B亞型病毒感染者,這些CRF07_BC感染者在接受高效能抗反轉錄病毒療法(HAART)前,其體內HIV病毒量較低、CD4+血球數目較高。因此,本研究的目的為人類免疫缺乏病毒CRF07_BC重組亞型病毒株之功能性探討。
我們構築出CRF07_BC 病毒株全長基因體的molecular clone (pCRF07_BC)以進行此重組亞型病毒之功能性探討。比較B亞型與CRF07_BC重組亞型病毒株之全長基因體序列,分析CRF07_BC重組亞型之病毒基因是否帶有缺失性突變,並利用功能性試驗(functional assay)探討病毒基因序列變異對病毒表現型的影響。最後,利用CRF07_BC molecular clone製造出野生型或突變型CRF07_BC病毒,以確認上述實驗結果。 經由核酸序列分析,發現相較於B亞型HXB2病毒株之LTR序列,pCRF07_BC有2個核酸刪除性突變分別發生於NF-KB ⅰ及SP-1 I轉錄因子結合位。除此之外,相較於HXB2之LTR序列,pCRF07_BC在核酸位置340-349之間,共發生4個核酸插入性突變及2個核酸替換性突變,因而產生第三個NF-KB轉錄因子結合位。另外,比較pCRF07_BC與HIV資料庫中B、C、CRF07_BC亞型參考株之全長基因體序列,分析CRF07_BC亞型之病毒基因是否帶有缺失性突變,導致蛋白質功能缺失。發現CRF07_BC之p6基因片段發生21個核酸刪除性突變,造成CRF07_BC之p6gag發生30PIDKELY36刪除性突變;p6pol蛋白質發生38ADRQGTV44刪除性突變。也發現CRF07_BC在Vif蛋白質Cul5 motif與BC box motif之間有天門冬醯酸(Asparagine)插入性突變;在Vpr蛋白質NLS motif有A89T突變;在Nef蛋白質N端蛋白質豆蔻醯化(myristoylation)功能區段內有G3A突變。 之後分析臨床病毒株的p6基因片段,我們發現所有的32個CRF07_BC病毒株之p6gag及 p6pol蛋白質均發生如上述之7個胺基酸刪除性突變,發生刪除性突變的比例為100% (32/32)。由於p6刪除性突變發生的位點位於病毒蛋白酶切割位點的附近,可能影響蛋白酶切割polyprotein的效率,所以我們利用西方墨點法比較感染細胞內病毒蛋白質成份組成,來分析CRF07_BC病毒的蛋白質切割效率,發現相較於B亞型臨床病毒株,CRF07_BC臨床病毒株之p24gag/Pr55gag的比值較低,顯示CRF07_BC亞型病毒蛋白酶切割Gag polyprotein的效率較差。為了測試這些in vitro實驗結果,我們選殖CRF07_BC molecular clone製造出野生型或帶有p6插入性突變之突變型CRF07_BC亞型病毒,發現將p6基因片段補回pCRF07_BC質體後,蛋白酶切割Gag polyprotein的效率上升,且病毒顆粒釋放的比率增加,顯示p6刪除性突變是造成CRF07_BC重組亞型病毒蛋白酶切割Gag polyprotein效率較差的原因。 HIV disease is a culmination of a complex interplay between both viral and host factors. Understanding the virologic characteristics of long-term non-progressers will help to develop strategies to prevent HIV infection and clinical treatment. Before 2004, sexual transmission is responsible for most HIV-1 infections in Taiwan. HIV-1 subtype B and CRF01_AE are the two predominant virus strains: subtype B accounts for about 70% of HIV infections and is responsible for a relative high proportion of infections among homosexual men; CRF01_AE accounts for 25% of HIV infections and is associated with heterosexual transmission. However, CRF07_BC was responsible for the outbreak of HIV-1 among intravenous drug users between 2004 and 2005 in Taiwan. According to clinicians’ observations, we found that, before highly active antiretroviral therapy, these CRF07_BC infected individuals tend to have lower viral loads and higher CD4 counts than those patients recently infected subtype B viruses. Therefore, the specific aim of this study is to determine the functional characteristics of HIV-1 CRF07_BC. The full-length genomes of CRF07_BC infectious molecular clone was sequenced, analyzed. Compared to LTR of HXB2, two single base deletions are amongst the NF-KB ⅰand SP-1 I binding sites, respectively. Interestingly, compared to the subtype B’s (HXB2) LTR, there is a potential third NF-KB binding site in the subtype CRF07_BC’s LTR region due to the 4 base insertions and two base substitutions at nt 340–349. In addition to, we founded HIV CRF07_BC have 7-amino-acids-deletion mutation in both p6gag and p6pol proteins, one-amino-acid insertion mutation at position 140 residue in Vif protein, A89T mutation in Vpr NLS motif and G3A mutation in Nef myristoylation functional domain. Viral RNA was extracted from thirty-two patients’ plasma, and the viral nucleotide sequences of the pol region were determined using reverse transcription and nested polymerase chain reaction (PCR). Deletion of 7 amino acids in both p6gag and p6pol proteins were also noted among the Taiwanese CRF07_BC strains (32/32, 100%). The cell lysates of infected cells were used for Western blotting analysis to determine the efficiency of Gag-Pol polyprotein processing. Our results showed that CRF07_BC harbor lower Gag polyprotein processing efficiency than subtype B. CRF07_BC full-length molecular clone was constructed to confirm this finding. Site-directed mutagenesis was performed to address the effects of deletion in p6 on Gag cleavage efficiency. Our results demonstrate that 7-amino-acids-deletion in p6 can affect virus release by impairing Gag cleavage efficiency. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/64656 |
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顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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