• 作者： 彭文君; 潘俊廷; 賴銘志; 邱昌芳; 林志侯
• 作者服務機構： 清華大學生命科學系; 台北榮民總醫院內部血液科
• 中文摘要： 　　為研究反轉錄病毒的核酸銜接過程，我們將愛滋病毒(HIV-1)的核酸銜接酵素(HIV-1 IN)單獨的表現在細胞中，並使用攜帶著完整鼠類的白血病毒(Mo-MuLV)基因組載體做為其受質，以設計了一個活體內的核酸銜接分析方法。取自載體pINSD的HIV-1 IN基因先被選殖入載體pXT1，以產生能表現HIV-1 IN的載體pXTl-IN。載體pMLV-K攜有完整的Mo-MuLV基因組，而且具感染性。在此載體原Mo-MuLV IN能作用上去的circle junction上的U3及U5 attachment (att)序列，也被以定點突變的方法改變成HIV-1 IN，所能作用者以產生載體pMLV*(U3U5)。然後把在載體pMLV-K及pMLV*(U3U5)上的Mo-MuLV IN的序列做部份切除，以產生載體pMLV△IN及pMLV*(U3U5)△IN。在用這些載體分別轉化感染NIH/3T3細胞之後，為偵測Mo-MuLV的基因組是否有被 HIV-1 IN所銜接，我們使用一種非放射性的反轉錄酵素(RT)活性分析法，以察看是否有病毒粒子自細胞中被釋出。我們發現被載體pMLV△IN所單獨感染的細胞並無病毒粒子的釋出。不過分別被載體pMLV△IN或pMLV*(U3U5)△IN所轉化感染，且能表現HIV-1 IN的細胞卻有病毒粒子的釋出，因為有明顯的RT活性被測到。此結果顯示單獨被表現在細胞中的HIV-1 IN，可將完整的Mo-MuLV基因組銜接上細胞的染色體。此外，我們將含有被銜接上的Mo-MuLV基因組的細胞基因組抽出，並以被部份切除的Mo-MuLVIN序列做為目標序列，以使用多鏈聚合反應，進一步證明HIV- I IN可單獨將Mo-MuLV的基因組銜接上細胞的染色體。
• 英文摘要： An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1)integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV)provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned intovector pXTl to give pXTl-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5).The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectorspMLV △ IN and pMLV*(U3U5) △ IN. Integration of these wild type and MuLV IN partially deleted or attmutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by meansof a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity wasdetected for the NIH/3T3 cell singly transfected with vector pMLV △ IN. However, some RT activities wereobserved for the HIV-1 IN expressing cell transfected either with vectors pMLV △ IN or pMLV*(U3U5) △ IN.This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was inte-grated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was furtherconfirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cellsusing the MuLV IN sequence remained from partial deletion as a target.
• 中文關鍵字： retrovirus; integration; HIV-1 integrase.
• 英文關鍵字： --