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Dept of Molecular Pharmacology
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3. Long Terminal Repeats: The Retroviral Promoter

The long terminal repeat (LTR) is the control center for gene expression. As may be expected because of the integrated phase of their life cycle, retroviruses have somewhat typical eucaryotic promoters with transcriptional enhancers and some also have regulatory elements responsive to either viral or specialized cellular (e.g. hormonal) trans-activating factors (HIV, MMTV). Enhancer functions have also been mapped to the gag (ALSV) and gag-pol (SIV and HIV) regions of some viruses but their role in the virus life cycle has yet to be established. In any case, all of the requisite signals for gene expression are found in the LTRs: Enhancer, promoter, transcription initiation (capping), transcription terminator and polyadenylation signal. Expression directed by the viral LTR signals is carried out entirely by host cell enzymes (RNA pol II, poly A synthetase, guanyl transferase). The enhancer and other transcription regulatory signals are contained in the U3 region of the 5' LTR, and the TATA box is located roughly 25 bp from the beginning of the R sequence. The presence and position of specific sequence signals used for transcription and binding sites for known or suspected transcription sites are shown in the figure below. The poly A addition signal (AAUAAA) in HTLV-1 is an unusually long distance upstream from the polyadenylation site (250 nt, by definition the end of R). It is hypothesized that the signal is brought in proximity to the addition site by secondary structure in the RNA.

The integrated provirus has two LTRs, and the 5' LTR normally acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R, is capped, and proceeds through U5 and the rest of the provirus, usually terminating by the addition of a poly A tract just after the R sequence in the 3' LTR. Transcription is not terminated in the 5' LTR, probably due to failure of formation of secondary structure in the RNA which uncouples RNA polymerase, analogous to bacterial attenuation. Occasionally the stop signal in the 3' LTR is "read-through" and terminated in host flanking sequences, these hybrid transcripts could then be efficiently packaged and transmitted in virions. This process may play an important role in transduction of oncogenes.

The 3' LTR is not normally functional as a promoter, although it has exactly the same sequence arrangement as the 5' LTR. Instead, the 3' LTR acts in transcription termination and polyadenylation. Transcriptional interference occurs when the two LTRs are oriented as in a provirus; the 5' LTR has dominant control as a promoter. When the integrity of the 5' LTR is disrupted, the 3' LTR can act as a promoter, and this is the basis for one form of retroviral oncogenesis. Thus transcription from the 5' LTR interferes with the function of the 3' LTR as a promoter-enhancer. Experiments by Cullen et al. (Nature 307:239, 1984) demonstrated this phenomenon very clearly. By progressively removing portions of a provirus and using an indicator gene downstream of the 3' LTR, the transcriptional activity from both LTRs could be assessed. Cellular restriction of LTR function can occur. Transcriptional enhancers are tissue and cell specific in their function. For retroviruses like MuLV, embryonal tissues and cells do not host viral gene expression whereas differentiated or adult tissues and cells express viral RNA to high levels. These differences, which are due to enhancer "activators" and "repressors" have been studied in cell lines that can be differentiated in cell cultures. Changes in the structures of the binding sites in the LTRs can be correlated with tissue-specific expression patterns of mutant MLV and their induction of tumor formation in those tissues.



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