RIBOSOMAL FRAMESHIFTING IN HIV AND OTHER RETROVIRAL INFECTED CELLS

我们在以前的研究中已经表明，在 存在低修饰的tRNAs(即缺乏高度特异的tRNAs 修饰碱基)以及这些tRNA在细菌中的利用 相应逆转录病毒中的核糖体移码。一直以来 该实验室和其他实验室的研究表明，移码突变 MMTV中的站点显著减少了移帧 相同的tRNA物种同时解码野生型和突变型信号。它 也已知不含Q碱基Asp-tRNA优先解码AAC AAU密码子。这些研究表明，需要一个低修饰的trna。 对于核糖体移帧，如果需要这样的tRNA，它 为抑制逆转录病毒的表达提供了途径。使用非洲爪哇 卵母细胞，我们之前开发了一种用于监测MMTV的体外试验 核糖体移码。从MMTV移码位点产生的RNA， 已被克隆到适当的表达载体中， 卵母细胞显微注射及逆转录病毒翻译产物的分离 通过一种特定的免疫分析。对移帧的水平进行了监测 在微量注射Q碱基或天冬酰胺tRNA后，有或没有Q 基地。这些研究给出了不确定的结果，在这一点上 另一方面，一些结果表明需要低修饰的tRNA， 但另一方面，低修饰的tRNA并不是必需的。显然，更多 需要研究来解决一个重要的问题，即 低修饰的tRNA对于移码是必不可少的。在协作环境中 与德克萨斯大学加尔维斯顿的S·威尔逊博士一起研究，我们有 制备了编码HIV引物tRNA[Lys-3]的合成基因，其中 可以产生大量的tRNA[Lys-3]。这些研究已经 提供了强有力的证据表明tRNA的核酸结合域[Lys- 3]在HIV中，逆转录酶位于273-302残基之间。

We have shown in previous studies that a correlation exists between the presence of hypomodified tRNAs (i.e., tRNAs lacking a specific highly modified base) in infected cells and the utilization of these tRNAs in ribosomal frameshifting in the corresponding retrovirus. It has been shown in this and other laboratories that a mutation at the frameshift site in MMTV results in a dramatic reduction in frameshifting even though the same tRNA species decodes both the wild type and mutant signals. It is also known that Asp-tRNA without Q base preferentially decodes AAC over AAU codons. These studies suggest that a hypomodified tRNA is required for ribosomal frameshifting and in the event such a tRNA is required, it provides an avenue for inhibition of retroviral expression. Using Xenopus oocytes, we previously developed an in vitro assay for monitoring MMTV ribosomal frameshifting. RNA generated from the MMTV frameshift site, which had been cloned into an appropriate expression vector, was microinjected into oocytes and the retroviral translation product isolated by a specific immunoassay. The level of frameshifting was monitored following microinjection of Q base or asparagine tRNA with or without Q base. These studies have given inconclusive results where, on the one hand, some of the results suggested that hypomodified tRNA is required, but on the other, hypomodified tRNA is not required. Clearly, more studies are needed to resolve the important issue of whether a hypomodified tRNA is essential for frameshifting. In a collaborative study with Dr. S. Wilson, University of Texas, Galveston, TX, we have prepared a synthetic gene encoding the HIV primer, tRNA[Lys-3], where large quantities of tRNA[Lys-3] can be generated. These studies have provided strong evidence that the nucleic acid binding domain of tRNA[Lys- 3] in HIV reverse transcriptase is located between residues 273-302.