RIBOSOMAL FRAMESHIFTING IN HIV AND OTHER RETROVIRAL INFECTED CELLS

以前，我们已经提供了强有力的证据表明，在人类中的宿主tRNA 免疫缺陷病毒（HIV）感染和其他逆转录病毒感染的细胞 参与这些逆转录病毒核糖体移码的是 低修饰的（即，这些转运RNA缺少一个特定的高度修饰的碱基 在反密码子环内）。 一个非常重要的悬而未决的问题是 是否在逆转录病毒核糖体中利用低修饰的tRNA 移码 如果需要修饰不足的tRNA，则改变 该过程（即，将这样的tRNA改变为相应的完全 在逆转录病毒感染的细胞中修饰的tRNA）提供了一种可能的 抑制逆转录病毒表达的手段。 为了确定是否修改了hypo 在核糖体移码中需要isoceptor，我们构建了 HIV、SRV-1和小鼠乳腺肿瘤病毒的核糖体移码信号 （MMTV），这将明确证明，如果低修饰的tRNA是 在这个事件中需要。 所有甲硫氨酸密码子（AUG）均发生突变 在HIV和MMTV gag-pol基因中， 甲硫氨酸密码子已插入到它们的-1阅读框中 就在移码位点的下游 序列已被 插入到每个构建体中对应于氨基末端， GAG和GAG-POL的羧基末端，使得所得蛋白质可以 通过免疫沉淀进行特异性分离。 此外该 低修饰的tRNA和相应的完全修饰的tRNA， 已经纯化了HIV和MMTV中的移码信号。 我们 将在明年进行体外蛋白质合成和分离 由此产生的移码肽将证明哪种tRNA是 来促进框架转换。 此外，我们已经开始种植HL 60 在化学成分确定的培养基中， 硒的存在和不存在。 这些研究可能（通过类比 硒对其他哺乳动物基因表达的影响 逆转录病毒）显示硒抑制HIV表达。 然后我们将 检查硒对核kB-2因子的影响， HIV表达所需的，其他研究的初步研究 实验室已经表明硒抑制其表达。

Previously we have provided strong evidence that the host tRNAs in human immunodeficiency virus (HIV)-infected and other retroviral-infected cells involved in ribosomal frameshifting of these retroviruses are hypomodified (i.e., these tRNAs lack a specific, highly modified base within the anticodon loop). A very significant unresolved question is whether a hypomodified tRNA is utilized in retroviral ribosomal frameshifting. If an undermodified tRNA is required, then alteration of this process (i.e., changing such a tRNA to the corresponding fully modified tRNA in cells infected with retrovirus) provides a possible means of inhibiting retroviral expression. To determine if hypo-modified isoacceptors are required in ribosomal frameshifting, we constructed ribosomal frameshift signals for HIV, SRV-1 and mouse mammary tumor virus (MMTV) that will unequivocally demonstrate if hypomodified tRNAs are required in this event. All methionine codons (AUG) have been mutated in the HIV and MMTV gag-pol genes to leucine codons (CUG) and a single methionine codon has been inserted into their -1 reading frames immediately downstream of the frameshift site. Sequences have been inserted into each construct corresponding to the amino terminus and carboxy terminus of GAG and GAG-POL so that the resulting proteins can be specifically isolated by immunoprecipitation. In addition, the hypo-modified tRNAs and the corresponding fully modified tRNAs which decode the frameshift signals in HIV and MMTV have been purified. We will, in the next year, carry out in vitro protein synthesis and isolate the resulting frameshift peptide that will demonstrate which tRNA is required to promote frameshifting. Further, we have begun growing HL60 cells chronically infected with HIV on chemically defined media in the presence and absence of selenium. These studies may likely (by analogy to the affect of selenium on the expression of other mammalian retroviruses) show that selenium inhibits HIV expression. We will then examine the affect of selenium on the nuclear kB-2 factor which is required for HIV expression and which preliminary studies from other laboratories have suggested that selenium inhibits its expression.