Molecular cloning and characterization of deoxyuridine triphosphatase from feline immunodeficiency virus (FIV).
Molecular cloning and characterization of deoxyuridine triphosphatase from feline immunodeficiency virus (FIV).
复制标题
猫免疫缺陷病毒（FIV）脱氧尿苷三磷酸酶的分子克隆和表征。
DOI：
10.1006/viro.1993.1501
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发表时间：
1993
期刊：
影响因子：
3.7
通讯作者：
Elder,JH
中科院分区：
文献类型：
--
作者：
Wagaman,PC;Hasselkus-Light,CS;Henson,M;Lerner,DL;Phillips,TR;Elder,JH
猫慢病毒FIV含有dUTR（DU）作为由viralpolgene编码的酶盒的一部分（Elderet al.，66，1791-1794）。该酶由Pol多聚蛋白加工而成，并包装成感染性病毒粒子。我们在这里报告的病毒酶的基本特性，包括底物特异性，离子的要求，和最佳pH值。我们还报道了DU在大肠杆菌中的过表达以及在完整前病毒或单独DU的情况下该酶的插入突变。该酶需要Mg 2+的全部活动和竞争研究，采用未标记的dNTPs表明，DU有一个绝对的偏好dUTP。FIV DU的最佳pH为pH 7.0。蛋白质的限制决定了一种Mr 14，350，这与从病毒体分离的DU的离子喷雾质谱法的测定精确一致。DU、RT和IN之间连接处的切割位点（如通过每种蛋白质的N-末端氨基酸测序所定义）与天冬氨酸蛋白酶切割位点的预测一致。在DU的Tyr 75处的框内插入突变消除了活性。用含有该突变的前病毒转染的细胞表达病毒体相关的逆转录酶活性，但缺乏DU活性。因此，产生的病毒体复制慢于那些拥有野生型DU。测试目前正在进行中，以评估DU诱变onvivophenotype的后果。
The feline lentivirus, FIV, contains dUTPase (DU) as part of the enzyme cassette encoded by the viralpolgene (Elderet al., 1992,J. Virol.66, 1791-1794). The enzyme is processed from the Pol polyprotein and is packaged into infectious virions. We report here the basic characteristics of the viral enzyme, including substrate specificity, ion requirements, and pH optimum. We also report the overexpression of DU inEscherichia coliand insertional mutagenesis of the enzyme in the context of the complete provirus or DU alone. The enzyme requires Mg2+for full activity and competition studies employing unlabeled dNTPs indicated that DU has an absolute preference for dUTP. The pH optimum for FIV DU is pH 7.0. The limits of the protein dictate a species ofMr14,350, which agrees precisely with the determination by ion spray mass spectroscopy of DU isolated from virions. Cleavage sites at the junctions between DU, RT, and IN, as defined by N-terminal amino acid sequencing of each protein species, are consistent with predictions for sites of cleavage by aspartate protease. In-frame insertional mutagenesis at Tyr 75 of DU abolishes activity. Cells transfected with proviruses containing this mutation express virion-associated reverse transcriptase activity but lack DU activity. The resultant virions replicate slower than those possessing wild-type DU. Tests are currently underway to evaluate the consequences of DU mutagenesis onin vivophenotype.