A Hairpin Structure in the R Region of the Human Immunodeficiency Virus Type 1 RNA Genome Is Instrumental in Polyadenylation Site Selection

ABSTRACT Some retroviruses with an extended repeat (R) region encode the polyadenylation signal within the R region such that this signal is present at both the 5′ and 3′ ends of the viral transcript. This necessitates differential regulation to either repress recognition of the 5′ polyadenylation signal or enhance usage of the 3′ signal. The human immunodeficiency virus type 1 (HIV-1) genome encodes an inherently efficient polyadenylation signal within the 97-nucleotide R region. Polyadenylation at the 5′ HIV-1 polyadenylation site is inhibited by downstream splicing signals, and usage of the 3′ polyadenylation site is triggered by an upstream enhancer element. In this paper, we demonstrate that this on-off switch of the HIV-1 polyadenylation signal is controlled by a secondary RNA structure that occludes part of the AAUAAA hexamer motif, which we have termed the polyA hairpin. Opening the 5′ hairpin by mutation triggered premature polyadenylation and caused reduced synthesis of viral RNA, indicating that the RNA structure plays a pivotal role in repression of the 5′ polyadenylation site. Apparently, the same hairpin structure does not interfere with efficient usage of the 3′ polyadenylation site, which may be due to the presence of the upstream enhancer element. However, when the 3′ hairpin was further stabilized by mutation, we measured a complete loss of 3′ polyadenylation. Thus, the thermodynamic stability of the polyA hairpin is delicately balanced to allow nearly complete repression of the 5′ site yet efficient activation of the 3′ site. This is the first report of regulated polyadenylation that is mediated by RNA secondary structure. A similar hairpin motif that occludes the polyadenylation signal can be proposed for other lentiviruses and members of the spumaretroviruses, suggesting that this represents a more general gene expression strategy of complex retroviruses.