INHIBITION OF POLYADENYLATION BY STABLE RNA SECONDARY STRUCTURE

The presence of a polyadenylation signal in the repeat (R) region of t he HIV-1 genome, which is located at both the 5' and 3' ends of the vi ral transcripts, requires differential regulation of polyadenylation. The HIV-1 poly(A) site can fold in a stable stem-loop structure that i s well-conserved among different human and simian immunodeficiency vir uses. In this study, we tested the effect of this hairpin on polyadeny lation by introducing mutations that either stabilize or destabilize t he RNA structure. The HIV-1 sequences were inserted into the pSV(2)CAT reporter plasmid upstream of the SV40 early poly(A) site. These const ructs were transfected into COS cells and transcripts were analyzed fo r the usage of the HIV-1 versus SV40 poly(A) site. The wild-type HIV-1 poly(A) site was used efficiently in this context and destabilization of the poly(A) hairpin did not affect the polyadenylation efficiency. In contrast, further stabilization of the hairpin severely inhibited HIV-1 polyadenylation. Additional mutations that repair the thermodyna mic stability of this mutant hairpin restored the polyadenylation acti vity. These results indicate that the mechanism of polyadenylation can be repressed by stable RNA structure encompassing the poly(A) signal. Experiments performed at reduced temperatures also suggest an inverse correlation between the stability of the RNA structure and the effici ency of polyadenylation.