Structure of an RNA switch that enforces stringent retroviral genomic RNA dimerization

Abstract

Retroviruses selectively package two copies of their RNA genomes in the context of a large excess of nongenomic RNA. Specific packaging of genomic RNA is achieved, in part, by recognizing RNAs that form a poorly understood dimeric structure at their 5 ends. We identify, quantify the stability of, and use extensive experimental constraints to calculate a 3D model for a tertiary structure domain that mediates specific interactions between RNA genomes in a gamma retrovirus. In an initial interaction, two stem-loop structures from one RNA form highly stringent crossstrand loop-loop base pairs with the same structures on a second genomic RNA. Upon subsequent folding to the final dimer state, these intergenomic RNA interactions convert to a high affinity and compact tertiary structure, stabilized by interdigitated interactions between U-shaped RNA units. This retroviral conformational switch model illustrates how two-step formation of an RNA tertiary structure yields a stringent molecular recognition event at early assembly steps that can be converted to the stable RNA architecture likely packaged into nascent virions. retroviral RNA dimer ͉ RNA folding ͉ Selective 2Ј-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) chemistry ͉ site-directed cleavage R etroviral genomes usually consist of two sense-strand RNAs that are noncovalently linked near their 5Ј ends to form a dimeric structure (1-3). Recognition of this dimeric state ensures that exactly two RNA genomes are packaged into each nascent virion (1, 2). Mature retroviral virions contain almost exclusively retroviral genomic RNA plus a few select cellular RNAs (4, 5). Many other cellular RNAs, including mRNAs (6-8), are accessible to the retroviral packaging process. Specific recognition of retroviral genomic RNA against a large background of cellular RNA thus represents a striking example of molecular recognition in biology.We have recently identified a minimal dimerization active sequence (MiDAS) (9) for a representative gamma retrovirus, the Moloney murine sarcoma virus (MuSV; Fig. 1A). The MiDAS domain correlates closely with retroviral genomic sequences sufficient to package heterologous RNAs into virions (6,8,11,12), as dimers (8). The MiDAS domain also includes conserved sequence elements previously proposed to specify the noncovalent interactions that mediate RNA dimerization.Conserved sequence elements include self-complementary (palindromic) sequences (PAL1 and PAL2) and stem-loop structures 1 and 2 (SL1 and SL2) (10, 13-16). SL1 and SL2 contain GACG tetraloops that form stable loop-loop interactions with a second RNA molecule. Loop-loop interactions are mediated by canonical intermolecular C-G base pairing and additional stacking and intraand intermolecular hydrogen bonds (17) (see Fig. 6, which is published as supporting information on the PNAS web site). In addition, the self-complementary PAL1 and PAL2 sequences form extended heteroduplexes involving both strands in the dimer (refs. 13-16; C.S.B. and K.M.W., unpublished data). However, the kine...

Definition of a high-affinity Gag recognition structure mediating packaging of a retroviral RNA genome

“…Prior work has shown that the MiDAS RNA dimer contains true long-range tertiary interactions involving PAL1, PAL2, and SL1-SL2 (38,45). Thus, the simplest element in the packaging signal involves both the primary sequence and the tertiary architecture of the RNA.…”

On the significance of an RNA tertiary structure prediction

“…Our laboratories are developing accurate and efficient methods for modeling complex RNA structures (Badorrek et al 2006;Ding et al 2008a;Sharma et al 2008;Deigan et al 2009;Gherghe et al 2009;Lavender et al 2010). Many other laboratories are also making innovative contributions to the RNA modeling field (Massire and Westhof 1998;Tan et al 2006;Das and Baker 2007;Das et al 2008;Parisien and Major 2008;Yu et al 2008;Jonikas et al 2009).…”

Cell biology of retroviral RNA packaging

“…Such RNA conformational switches have been elucidated upon dimerization of HIV-1 and MLV RNAs. 11,12,19,[93][94][95][96][97][98][99] However, direct insights into RNA structure-function relationship are needed to validate this hypothesis. Another possibility is that the interaction between the gRNA and Gag, or between the gRNA and a-yet-to-be-identified cellular factor, could modulate the dual targeting of the genome.…”