RIBOSOMAL FRAMESHIFTING IN HIV AND OTHER RETROVIRAL INFECTED CELLS

此前，我们提供了强有力的证据表明，人类体内的宿主tRNAs 免疫缺陷病毒(HIV)感染细胞和其他逆转录病毒感染细胞 参与这些逆转录病毒的核糖体移码 低修饰(即，这些tRNA缺乏特定的、高度修饰的碱基 在反密码子循环内)。一个非常重要的悬而未决的问题是 低修饰tRNA是否用于逆转录病毒核糖体 帧移位。如果需要未充分修饰的tRNA，则改变 这一过程(即，将这样的tRNA改变为相应的完全 感染逆转录病毒的细胞中的修饰tRNA)提供了一种可能的 抑制逆转录病毒表达的手段。以确定低度修饰的 我们构建了核糖体移码所需的同种受体 HIV、SRV-1和小鼠乳腺肿瘤病毒的核糖体移码信号 (MMTV)，这将明确表明低修饰的tRNA是否 在此事件中是必需的。所有蛋氨酸密码子(AUG)都发生了突变 在HIV和MMTV的Gag-Poll基因中，亮氨酸密码子(CUG)和单个 蛋氨酸密码子已插入到它们的阅读框中 就在移框站点的下游。序列已经被 插入到对应于所述氨基末端的每个构造体中 GAG和GAG-POL的羧基末端，从而得到的蛋白质可以 用免疫沉淀法进行特异性分离。此外， 低修饰的tRNA和相应的完全修饰的tRNA，其 解码HIV和MMTV中的移码信号已被提纯。我们 将在明年进行体外蛋白质合成和分离 由此产生的移码多肽将证明哪一个tRNA是 促进移帧所必需的。此外，我们已经开始种植HL60 在化学定义的培养基上慢性感染艾滋病毒的细胞 硒的存在和缺失。这些研究可能(通过类比) 论硒对其他哺乳动物基因表达的影响 逆转录病毒)表明，硒抑制艾滋病毒的表达。到时候我们会的 检测硒对核kB-2因子的影响 HIV表达所需的，以及来自其他 实验室表明，硒可以抑制它的表达。

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.