Secondary Structure of the Mature Ex Virio Moloney Murine Leukemia Virus Genomic RNA Dimerization Domain

Abstract

Retroviral genomes are dimeric, comprised of two sense-strand RNAs linked at their 5 ends by noncovalent base pairing and tertiary interactions. Viral maturation involves large-scale morphological changes in viral proteins and in genomic RNA dimer structures to yield infectious virions. Structural studies have largely focused on simplified in vitro models of genomic RNA dimers even though the relationship between these models and authentic viral RNA is unknown. We evaluate the secondary structure of the minimal dimerization domain in genomes isolated from Moloney murine leukemia virions using a quantitative and single nucleotide resolution RNA structure analysis technology (selective 2-hydroxyl acylation analyzed by primer extension, or SHAPE). Results are consistent with an architecture in which the RNA dimer is stabilized by four primary interactions involving two sets of intermolecular base pairs and two loop-loop interactions. The dimerization domain can independently direct its own folding since heating and refolding reproduce the same structure as visualized in genomic RNA isolated from virions. Authentic ex virio RNA has a SHAPE reactivity profile similar to that of a simplified transcript dimer generated in vitro, with the important exception of a region that appears to form a compact stem-loop only in the virion-isolated RNA. Finally, we analyze the conformational changes that accompany folding of monomers into dimers in vitro. These experiments support well-defined structural models for an authentic dimerization domain and also emphasize that many features of mature genomic RNA dimers can be reproduced in vitro using properly designed, simplified RNAs.Retroviruses, including both simple model viruses and complex viruses like human immunodeficiency virus (HIV), contain genomes in the form of two coding RNA strands, noncovalently linked at their 5Ј ends (11,19,20,26,38,45). This 5Ј linkage is termed the genomic RNA dimer. Packaging of RNA genomes into new virions is highly specific, even in the presence of a large background of cellular RNA (1,7,26). This packaging function is carried out by the Gag protein (14,30,43), which recognizes RNA sequences that overlap with the RNA dimerization domain (16,26,42,44). The specific Gagdimer interaction represents an elegant and direct mechanism by which exactly two RNA genomes are packaged into each nascent virion.The genomic RNA dimer is initially assembled into an immature and noninfectious viral particle (9,(18)(19)(20). After the immature particle buds from the host cell, it undergoes extensive morphological changes to form the mature and infectious virion (21, 51). Maturation is initiated through cleavage of the Gag polyprotein by the viral protease to yield smaller Gagderived proteins and also involves changes in the structure of the RNA dimer region. The RNA dimer structure appears to be more compact and, for many retroviruses, more thermostable in mature than in immature virions (18)(19)(20)26).The closely related Moloney murine leukemia and sarcoma viruse...

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

“…SHAPE uses a chemical reaction at the RNA 2′-hydroxyl position to measure local nucleotide flexibility (34,35); flexibility can be reduced either by base pairing or by bound protein. We found that the two RNA strands in the dimer are held together by intermolecular base pairs in two palindromic stretches, termed PAL1 and PAL2, and by G-C base-pairing interactions in a highly conserved double stem-loop motif (SL1-SL2) (31,33,(36)(37)(38). These elements are separated by two distinct flexible elements, between PAL1 and SL0 and between PAL2 and SL1, respectively (Fig.…”

“…1A) (33). SHAPE uses a chemical reaction at the RNA 2′-hydroxyl position to measure local nucleotide flexibility (34,35); flexibility can be reduced either by base pairing or by bound protein.…”

“…It was proposed many years ago that dimeric RNAs are selectively packaged (15,47,48), and an important initial proposal emphasized that dimerization of MuLV vRNA constitutes a switch that exposes UCUG sequences (25,26,33). In fact, Gag actually binds weakly (K d ≥ 1 μM) to short single-stranded RNAs containing only this motif (Fig.…”

“…MuLV dimers were formed from a 331-nt RNA (33). Purified recombinant MuLV Gag and NC were incubated with MuLV dimers (18 μL, in 40 mM NaCl or potassium acetate for Gag and NC, respectively, and 0.8 mM MgCl 2 ) and were treated with 1-methyl-7-nitroisatoic anhydride (1M7, 2 μL; 2 mM in anhydrous DMSO) or with neat DMSO.…”

“…Sites of 2′-O-adduct formation in the authentic MuLV genome or in simplified transcript RNAs were analyzed by capillary electrophoresis using fluorescently labeled DNA primers and reverse transcriptase-mediated primer extension (33).…”