Borg K T, Favaro J P, Arrigo S J, Schmidt M
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, S.C., USA.
J Biomed Sci. 1999 Jan;6(1):45-52. doi: 10.1007/BF02256423.
The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5' splice donor and the tat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.
人类免疫缺陷病毒1型调节蛋白Rev是病毒结构蛋白产生所绝对必需的。剪接位点已被视为顺式作用阻遏序列（CRS），并抑制Rev依赖性RNA的表达。为了分析剪接供体在Rev依赖性中的作用，HIV-1的野生型5'剪接供体在其他gag序列的背景下发生了突变。瞬时转染后，通过RT-PCR分析RNA表达。突变构建体产生的未剪接RNA在RNA的细胞质积累方面仍需要Rev。尽管野生型5'剪接供体被删除，但使用了tat剪接受体。通过PCR和随后对剪接RNA的克隆鉴定出一个隐蔽剪接供体。该隐蔽位点与共有序列的相似度为5/9，位于Gag起始密码子（ATG）的紧邻下游。对含有隐蔽剪接供体的RNA产物的分析表明，Rev是未剪接RNA细胞质积累所必需的，而剪接RNA则不依赖Rev。野生型构建体的转染也证明了隐蔽剪接供体的使用。这些结果表明，当野生型剪接供体失活时，隐蔽剪接供体可以被激活，并且隐蔽剪接供体可能保留Rev调节作用。这些发现还表明，隐蔽剪接位点在确定病毒RNA的Rev依赖性方面有作为CRS的潜力。
