Roles of retroviral R regions in long terminal repeat-driven gene expression

Abstract

The R region of the retroviral long terminal repeat (LTR) marks the site of transcription initiation of provirus RNAs and has been well characterized for its role in viral replication. In the human retroviruses, the R region also functions in the expression of viral genomes. A sequence within the R region of the human immunodeficiency virus type 1, HIV-1, termed TAR, is critical for transactivation by the virally encoded transactivator protein, Tat. Although transactivation occurs at high levels in human cells, it does not occur in mouse cells. We have used mouse-human somatic cell hybrid lines to show that a factor present on human chromosome 12, or the long arm of chromosome 2, facilitates this transactivation when present in a mouse chromosome background.;We have also demonstrated that a sequence within the R regions of the murine leukemia viruses, Akv and SL3, is necessary for maximum expression. Deletion of nucleotides +4 to +32 of the MuLV R region in LTR-CAT constructs demonstrated that this sequence contributes to transcription initiation and to some post-transcriptional process.;A computer-generated secondary structure prediction revealed a possible stem-loop structure formed by this sequence at the RNA level. Comparison of retroviral R regions showed that there is a 90-100% conservation of the 5{dollar}\sp\prime{dollar} 32 nucleotides of this region among MuLVs and some related retroviruses, and that this strong conservation maintains the predicted secondary structure.;Specific mutations within the R region element were therefore designed to test the effects of altered secondary structure. These mutations indicated that both primary sequence and secondary structure seem to be important for this element's activity. Initiation of transcription seems to be dependent upon primary sequence and secondary structure, while the post-transcriptional component of the R element's activity seems to be more dependent upon secondary structure. In addition, the element seems to work best when positioned just 3{dollar}\sp\prime{dollar} to the mRNA start site.;Possibilities for the post-transcriptional component of activity include RNA stability, processing, and transport. Precedents for the element's transcriptional activity are initiator elements, which represent binding sites for transcription factors on the DNA, and the TAR element, which responds to the Tat protein in a manner that is dependent upon RNA secondary structure.