RIBOSOMAL FRAMESHIFTING IN HIV AND OTHER RETROVIRAL INFECTED CELLS

我们的实验室此前已经证明，参与其中的tRNA HIV和其他逆转录病毒感染细胞中的核糖体移码 低修饰(即，这些tRNA缺少特定的、高度修饰的 基地)。因此，在受感染的细胞中， 低修饰tRNAs的出现及其在核糖体中的应用 在相应的逆转录病毒中移码。一个非常重要的 那么，未解决的问题是不是真的需要一个低修饰的tRNA？ 核糖体移码？在需要这种tRNA的情况下，那么 逆转这一过程(即，将未充分修饰的tRNA转化为 逆转录病毒感染细胞中相应的完全修饰的tRNA)提供了 抑制逆转录病毒表达的途径。我们的关注点是 因此，去年的重点是开发一种能够 明确证明是否需要低修饰的tRNA 在移帧方面。我们正在改变MMTV和HIV基因 对各个移码位点进行编码以移除所有蛋氨酸密码子 并立即在阅读框中插入一个蛋氨酸密码子 在移码站点的下游。此外，我们正在净化 低修饰的tRNAs和相应的完全修饰的tRNAs 解码HIV和MMTV中的移帧信号。三重标记实验 将使用35S-蛋氨酸和14C-(亚硫代)-和 ~H-(完全修饰)-氨基酰基-tRNA(移码所需 位点)在兔网织红细胞裂解物中编程为相应的 突变的HIV或MMTV克隆产生的信使核糖核酸。隔离 所产生的移码多肽被35S-蛋氨酸和 对这种多肽的序列分析应该明确地证明 无论是低修饰还是完全修饰的tRNA负责 将其氨基酸捐献给移码部位。

Our laboratory has previously demonstrated that the tRNAs involved in ribosomal frameshifting in HIV and other retroviral infected cells are hypomodified (i.e., these tRNAs are missing a specific, highly modified base). Thus, a correlation exists in infected cells between the occurrence of hypomodified tRNAs and their utilization in ribosomal frameshifting in the corresponding retrovirus. A very important unresolved question then is a hypomodified tRNA actually required in ribosomal frameshifting? In the event such a tRNA is required, then reversal of this process (i.e., converting the undermodified tRNA to the corresponding fully modified tRNA in retroviral infected cells) provides an avenue for inhibiting retroviral expression. Our attention in the last year has therefore focused on developing an assay that will unequivocally demonstrate whether or not hypomodified tRNAs are required in frameshifting. We are in the process of changing MMTV and HIV genes encoding the respective frameshift sites to remove all methionine codons and inserting one methionine codon in the -1 reading frame immediately downstream of the frameshift site. Further, we are purifying the hypomodified tRNAs and the corresponding fully modified tRNAs which decode the frameshift signals in HIV and MMTV. Triple label experiments will be carried out employing 35S-methionine and 14C-(hypomodified)- and 3H-(fully modified)-aminoacyl-tRNA (which is required at the frameshift site) in rabbit reticulocyte lysates programmed with the corresponding mRNA generated from mutant HIV or MMTV clones. Isolation of the resulting frameshift peptide which is labeled with 35S-methionine and sequence analysis of this peptide should unequivocally demonstrate whether the hypomodified or fully modified tRNA is responsible for donating its amino acid to the frameshift site.