RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System

Evans, Matthew J.; Bacharach, Eran; Goff, Stephen P.

doi:10.1128/jvi.78.14.7677-7684.2004

Cited by 20 publications

(14 citation statements)

References 44 publications

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“…1B). This model is also supported by a prior yeast three-hybrid study that concluded that MuLV positions 212-354 are necessary for high-affinity binding by Gag (32).…”

Section: Discussionsupporting

confidence: 70%

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

Gherghe

Lombo

Leonard

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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All retroviral genomic RNAs contain a cis-acting packaging signal by which dimeric genomes are selectively packaged into nascent virions. However, it is not understood how Gag (the viral structural protein) interacts with these signals to package the genome with high selectivity. We probed the structure of murine leukemia virus RNA inside virus particles using SHAPE, a high-throughput RNA structure analysis technology. These experiments showed that NC (the nucleic acid binding domain derived from Gag) binds within the virus to the sequence UCUG-UR-UCUG. Recombinant Gag and NC proteins bound to this same RNA sequence in dimeric RNA in vitro; in all cases, interactions were strongest with the first U and final G in each UCUG element. The RNA structural context is critical: High-affinity binding requires base-paired regions flanking this motif, and two UCUG-UR-UCUG motifs are specifically exposed in the viral RNA dimer. Mutating the guanosine residues in these two motifs-only four nucleotides per genomic RNA-reduced packaging 100-fold, comparable to the level of nonspecific packaging. These results thus explain the selective packaging of dimeric RNA. This paradigm has implications for RNA recognition in general, illustrating how local context and RNA structure can create information-rich recognition signals from simple single-stranded sequence elements in large RNAs.retrovirus | RNA recognition code | RNA SHAPE chemistry E xpression of a single viral protein, termed Gag, is sufficient for assembly of retrovirus-like particles in mammalian cells. If present in the cell, the viral genomic RNA (vRNA) is selectively packaged into nascent particles; this selectivity is due to a cisacting packaging signal in the RNA, termed Ψ (1, 2). Remarkably, when no Ψ-containing RNA is present, Gag still assembles efficiently, encapsidating cellular mRNAs nonselectively in place of the vRNA (3-5).There are many indications that Ψ represents a high-affinity binding site for the Gag protein both in HIV-1 and in simpler retroviruses (6-14). However, the molecular mechanisms underlying selective encapsidation of vRNAs are incompletely understood, as are the features that enable Gag to bind preferentially to vRNA rather than to other cellular RNAs. Gag proteins contain several distinct domains, always including matrix (MA), capsid, and nucleocapsid (NC). vRNA packaging is mediated by the multidomain Gag protein, but Gag is cleaved following release of the virus from the cell. The NC domain plays a principal role in interactions with nucleic acids and is largely responsible for the specific interaction between Gag and its cognate viral RNA (12,13). This domain of Gag is highly basic and contains one or more "zinc knuckles" with a conserved spacing of Zn 2þ -coordinating cysteine and histidine residues. Mutations that abolish Zn 2þ coordination impair selective encapsidation of vRNA during virus assembly (6, 15). In addition, MA domains of many retroviral Gag proteins interact with nucleic acids (16-21) and may also contribute to specific i...

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“…1B). This model is also supported by a prior yeast three-hybrid study that concluded that MuLV positions 212-354 are necessary for high-affinity binding by Gag (32).…”

Section: Discussionsupporting

confidence: 70%

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

Gherghe

Lombo

Leonard

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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“…We used high-sensitivity SHAPE, developed precisely for the structural analysis of authentic, low-abundance RNAs, to analyze the MuLV dimerization and packaging regulatory domain (24,(27)(28)(29), of approximately 170 nucleotides. RNAs were extracted from virions under mild, nondenaturing conditions designed to preserve preexisting RNA structures.…”

Section: Resultsmentioning

confidence: 99%

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

Grohman

Gorelick

Kottegoda

et al. 2014

J Virol

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Retroviral virions initially assemble in an immature form that differs from that of the mature infectious particle. The RNA genomes in both immature and infectious particles are dimers, and interactions between the RNA dimer and the viral Gag protein ensure selective packaging into nascent immature virions. We used high-sensitivity selective 2=-hydroxyl acylation analyzed by primer extension (SHAPE) to obtain nucleotide-resolution structural information from scarce, femtomole quantities of Moloney murine leukemia virus (MuLV) RNA inside authentic virions and from viral RNA extracted from immature (protease-minus) virions. Our secondary structure model of the dimerization and packaging domain indicated that a stable intermolecular duplex known as PAL2, previously shown to be present in mature infectious MuLV particles, was sequestered in an alternate stem-loop structure inside immature virions. The intermediate state corresponded closely to a late-folding intermediate that we detected in time-resolved studies of the free MuLV RNA, suggesting that the immature RNA structure reflects trapping of the intermediate folding state by interactions in the immature virion. We propose models for the RNA-protein interactions that trap the RNA in the immature state and for the conformational rearrangement that occurs during maturation of virion particles. IMPORTANCEThe structure of the RNA genome in mature retroviruses has been studied extensively, whereas very little was known about the RNA structure in immature virions. The immature RNA structure is important because it is the form initially selected for packaging in new virions and may have other roles. This lack of information was due to the difficulty of isolating sufficient viral RNA for study. In this work, we apply a high-sensitivity and nucleotide-resolution approach to examine the structure of the dimerization and packaging domain of Moloney murine leukemia virus. We find that the genomic RNA is packaged in a high-energy state, suggesting that interactions within the virion hold or capture the RNA before it reaches its most stable state. This new structural information makes it possible to propose models for the conformational changes in the RNA genome that accompany retroviral maturation.A n early step in the formation of an infectious retroviral particle involves the association of retroviral RNA with the cell membrane via interactions with the viral Gag protein. Budding from the cell yields a particle in which Gag protein domains are arrayed roughly in concentric spheres and the RNA is bound in the interior of the particle. Subsequent cleavage of the Gag structural protein results in large-scale rearrangements to yield a mature particle with a complex structure (1, 2). The genomic RNA also undergoes conformational rearrangements during particle maturation (3-5). In both immature and mature particles, two copies of the genomic RNA are present, and it is known that the structure adopted by the immature form of the viral RNA is less thermodynamically stable ...

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“…This dimerization region overlaps with sequences that were previously shown to be sufficient to allow heterologous RNAs to be packaged, albeit at reduced levels, as dimers into new virions (26). This region also forms high-affinity interactions with the viral Gag protein (16). Structural analysis of this minimal dimerization active sequence (the MiDAS RNA), performed with highly purified RNA under simplified solution conditions in vitro, supports a consensus model for the dimerization domain that involves three major structural components ( Fig.…”

mentioning

confidence: 99%

“…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.…”

mentioning

confidence: 99%

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

Gherghe

Leonard

Gorelick

et al. 2010

J Virol

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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...

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RNA Sequences in the Moloney Murine Leukemia Virus Genome Bound by the Gag Precursor Protein in the Yeast Three-Hybrid System

Cited by 20 publications

References 44 publications

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

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

An Immature Retroviral RNA Genome Resembles a Kinetically Trapped Intermediate State

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

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