Nam S H, Copeland T D, Hatanaka M, Oroszlan S
Laboratory of Molecular Virology and Carcinogenesis, National Cancer Institute-Frederick Cancer Research and Development Center, Maryland 21702-1201.
J Virol. 1993 Jan;67(1):196-203. doi: 10.1128/JVI.67.1.196-203.1993.
For study of the pol gene expression of human T-cell leukemia virus type I (HTLV-I), RNA was transcribed in vitro from proviral DNA and translated in rabbit reticulocyte lysates. This cell-free translation resulted in two major translation products representing the Gag and Gag-Pro polyproteins. By contrast, the Gag-Pro-Pol polyprotein could be readily observed only when translation was performed with mutant mRNA in which the protease (pro) reading frame was aligned to gag to eliminate the frameshifting event in the gag-pro overlap. The results indicated that two independent ribosomal frameshifting events are required for expression of the HTLV-I pol gene product. Studies with mutant DNAs facilitated the characterization of the primary structure of the HTLV-I mRNA responsible for the ribosomal frameshift in the pro-pol overlap and demonstrated that the frameshift occurs at the signal sequence UUUAAAC. Direct amino acid sequencing of the transframe protein localized the site of the frameshift to the asparagine codon AAC.
为了研究I型人类T细胞白血病病毒（HTLV-I）的pol基因表达，从原病毒DNA体外转录RNA，并在兔网织红细胞裂解物中进行翻译。这种无细胞翻译产生了两种主要的翻译产物，分别代表Gag和Gag-Pro多蛋白。相比之下，只有在用突变mRNA进行翻译时，才能很容易地观察到Gag-Pro-Pol多蛋白，其中蛋白酶（pro）阅读框与gag对齐，以消除gag-pro重叠处的移码事件。结果表明，HTLV-I pol基因产物的表达需要两个独立的核糖体移码事件。对突变DNA的研究有助于表征负责pro-pol重叠处核糖体移码的HTLV-I mRNA的一级结构，并证明移码发生在信号序列UUUAAAC处。对转框蛋白进行直接氨基酸测序，将移码位点定位到天冬酰胺密码子AAC。
