Proteolytic processing of the human T-cell lymphotropic virus 1 reverse transcriptase: identification of the N-terminal cleavage site by mass spectrometry.
Proteolytic processing of the human T-cell lymphotropic virus 1 reverse transcriptase: identification of the N-terminal cleavage site by mass spectrometry.
复制标题
人 T 细胞嗜淋巴细胞病毒 1 逆转录酶的蛋白水解加工：通过质谱法鉴定 N 末端切割位点。
DOI：
10.1006/abbi.2001.2432
复制
发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Moen,LK
中科院分区：
文献类型：
--
作者：
Agbuya,PG;Sherman,NE;Moen,LK
人类 T 细胞嗜淋巴细胞病毒 1 (HTLV-1) 是一种 C 型人类逆转录病毒，是成人 T 细胞白血病和其他疾病的病原体。 HTLV-1 (E.C. 2.7.7.49) 的逆转录酶是作为 Gag-Pro-Pol 前体蛋白的一部分合成的，成熟的 Gag、Pro 和 Pol 蛋白（包括逆转录酶）是通过病毒蛋白酶催化的蛋白水解过程产生的。产生成熟 HTLV-1 逆转录酶 N 末端的蛋白水解切割位点的位置此前尚未确定。通过使用几种逆转录病毒聚合酶的序列比较以及有关核糖体移码位置的信息，我们初步鉴定了这个 N 末端加工位点。 PCR 扩增用于构建一个克隆，该克隆跨越 HTLV-1 的 pro-pol 连接区域，以产生重组 Pro-Pol 蛋白，该蛋白跨越其他人提出的切割位点以及我们的序列比对识别的切割位点的位置。 HTLV-1 蛋白酶裂解重组 Pro-Pol 蛋白，生成 5.5 kDa 片段。通过毛细管 LC-MS 和 MS/MS 分析该片段显示 N 末端切割位点位于原 ORF 的 Leu147–Pro148 之间。这是首次对HTLV-1逆转录酶的真实氨基酸序列进行物理鉴定。这里报告的数据为进一步研究蛋白质-核相互作用的功能和结构方面提供了基础。
Human T-cell lymphotropic virus 1 (HTLV-1) is a type C human retrovirus, which is the causative agent of Adult T-cell Leukemia and other diseases. The reverse transcriptase of HTLV-1 (E.C. 2.7.7.49) is synthesized as part of a Gag–Pro–Pol precursor protein, and the mature Gag, Pro, and Pol proteins, including the reverse transcriptase, are created by proteolytic processing catalyzed by the viral protease. The location of the proteolytic cleavage site, which creates the N-terminus of mature HTLV-1 reverse transcriptase, has not been previously identified. By using sequence comparisons of several retroviral polymerases, as well as information about the location of the ribosomal frameshift, we tentatively identified this N-terminal processing site. PCR amplification was used to construct a clone, which spans a region of the pro–pol junction of HTLV-1, to produce a recombinant Pro–Pol protein spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombinant Pro–Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysis of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cleavage site to be between Leu147–Pro148of the pro ORF. This is the first physical identification of the authentic amino acid sequence of the reverse transcriptase of HTLV-1. The data reported here provides a basis for further investigation of the function and structural aspects of protein-nucleic interaction.