The ability of the HIV-1 AAUAAA signal to bind polyadenylation factors is controlled by local RNA structure

The 5' and 3' ends of HIV-1 transcripts are identical in sequence. This repeat region (R) folds a stem-loop structure that is termed the poly(A) hairpin because it contains polyadenylation or poly(A) signals: the AAUAAA hexamer motif, the cleavage site and part of the GU-rich downstream element. Obviously, HIV-1 gene expression necessitates differential regulation of the two poly(A) sites. Previous transfection experiments indicated that the wild-type poly(A) hairpin is slightly inhibitory to the process of polyadenylation, and further stabilization of the hairpin inhibited polyadenylation completely. In this study, we tested wild-type and mutant transcripts with poly(A) hairpin structures of differing thermodynamic stabilities for the in vitro binding of polyadenylation factors. Mutant transcripts with a destabilized hairpin efficiently bound the polyadenylation factors, which were provided either as purified proteins or as nuclear extract. The RNA mutant with a stabilized hairpin did not form this 'poly(A) complex'. Additional mutations that repair the stability of this hairpin restored the binding capacity. Thus, an inverse correlation was measured between the stability of the poly(A) hairpin and its ability to interact with polyadenylation factors. The wild-type HIV-1 transcript bound the polyadenylation factors suboptimally, but full activity was obtained in the presence of the USE enhancer element that is uniquely present upstream of the 3' poly(A) site. We also found that sequences of the HIV-1 leader, which are uniquely present downstream of the 5' poly(A) site, inhibit formation of the poly(A) complex. This inhibition could not be ascribed to a specific leader sequence, as we measured a gradual loss of complex formation with increasing leader length. We will discuss the regulatory role of RNA structure and the repressive effect of leader sequences in the context of differential HIV-1 polyadenylation