The human immunodeficiency virus type 1 (HIV-1) is well-known for its genetic variability, which leads for instance to the development of drug-resistant virus variants. The molecular basis for the genetic flexibility of this virus is the error-prone Reverse Transcriptase enzyme. We employ the genetic flexibility of HIV-1 to study the function of regulatory viral RNA motifs in tissue culture infections by a method termed forced evolution. Mutant viruses with specific alterations in RNA motifs that cause a severe replication defect were cultured for a prolonged period to select for revertant viruses with improved replication characteristics. This method turned out to be very productive for the molecular analysis of structured RNA motifs that control different steps of the viral replication cycle. We will review the results obtained in the analysis of two RNA stem-loop structures that are encoded within the repeat region (R element) of the HIV-1 genome: the TAR and polyA hairpins. The different repair strategies observed for mutant forms of these hairpin structures will be described in detail, followed by a discussion of the functional insight gained by these studies. Because the R region forms both the extreme 5' and 3' end of the retroviral RNA genome, the two structured RNA motifs could in theory have distinct replicative functions at either end of the genome. The 5' copy of the TAR hairpin forms the binding site for the viral Tat trans-activator protein and is important for transcriptional up-regulation of viral gene expression. The polyA hairpin motif occludes part of the AAUAAA polyadenylation signal and appears to be critical for down-regulation of 5' polyadenylation. The same sequence is used efficiently as a polyadenylation signal at the 3' end of HIV-1 RNA, which is due to the presence of upstream enhancer elements. Putative additional functions of these RNA hairpins, e.g. in packaging of HIV-1 RNA into virions and the process of reverse transcription, will be discussed.
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