An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Miyazaki, Yasuaki; Garcia, Eric L.; King, Steven R.; Iyalla, Kilali; Loeliger, Kelsey; Starck, Patrice; Syed, Sameera; Telesnitsky, Alice; Summers, Michael F.

doi:10.1016/j.jmb.2009.11.033

Cited by 48 publications

(52 citation statements)

References 79 publications

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“…6) that when all four UCUG elements within the two UCUG-UR-UCUG motifs were changed to UCUA, the efficiency with which genomic RNA was packaged was reduced by ∼200-fold. A comparable effect is obtained by deleting large regions of the packaging signal (150-350 nts) or by introducing mutations that compromise dimerization (29,46). These results have important implications for the mechanism of vRNA packaging and for RNA recognition, in general.…”

Section: Discussionmentioning

confidence: 89%

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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Section: Discussionmentioning

confidence: 89%

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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“…When RNA mutations that destabilize the dimer-competent fold of the HIV-1 5′-L are reconstituted into viral genomes, and these are coexpressed with wild-type genomes, the mutant RNAs fail to compete with the wild-type RNA for packaging (26). Similarly, mutations that prevent dimerization of the recombinant 5′-L RNA of the Moloney murine leukemia virus (MLV) block packaging (40), suggesting that dimerization-dependent control mechanisms are conserved among evolutionarily distant retroviruses (40,41). MLV-derived vectors harboring monomer-stabilizing mutations remain unpackaged in the absence of competing wild-type leader RNAs (40), whereas mutations that shift the equilibrium of the HIV-1 5′-L in favor of the monomer but do not prevent dimerization do not prevent the HIV-1 RNA from functioning as both genome and mRNA in the absence of wild-type competition (26,39,42).…”

Section: Discussionmentioning

confidence: 99%

“…Similarly, mutations that prevent dimerization of the recombinant 5′-L RNA of the Moloney murine leukemia virus (MLV) block packaging (40), suggesting that dimerization-dependent control mechanisms are conserved among evolutionarily distant retroviruses (40,41). MLV-derived vectors harboring monomer-stabilizing mutations remain unpackaged in the absence of competing wild-type leader RNAs (40), whereas mutations that shift the equilibrium of the HIV-1 5′-L in favor of the monomer but do not prevent dimerization do not prevent the HIV-1 RNA from functioning as both genome and mRNA in the absence of wild-type competition (26,39,42). These and other observations support a mechanism in which a single viral transcript equilibrates between structures associated with alternate replication functions (9,12,16,26,29).…”

Section: Discussionmentioning

confidence: 99%

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Kharytonchyk

Monti

Smaldino

et al. 2016

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

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106

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The promoter in HIV type 1 (HIV-1) proviral DNA contains three sequential guanosines at the U3-R boundary that have been proposed to function as sites for transcription initiation. Here we show that all three sites are used in cells infected with HIV-1 and that viral RNAs containing a single 5′ capped guanosine ( Cap 1G) are specifically selected for packaging in virions, consistent with a recent report [Masuda et al. (2015) Sci Rep 5:17680]. In addition, we now show that transcripts that begin with two or three capped guanosines ( Cap 2G or Cap 3G) are enriched on polysomes, indicating that RNAs synthesized from different transcription start sites have different functions in viral replication. Because genomes are selected for packaging as dimers, we examined the in vitro monomer-dimer equilibrium properties of Cap 1G, Cap 2G, and Cap 3G 5′-leader RNAs in the NL4-3 strain of HIV-1. Strikingly, under physiological-like ionic conditions in which the Cap 1G 5′-leader RNA adopts a dimeric structure, the Cap 2G and Cap 3G 5′-leader RNAs exist predominantly as monomers. Mutagenesis studies designed to probe for base-pairing interactions suggest that the additional guanosines of the 2G and 3G RNAs remodel the base of the PolyA hairpin, resulting in enhanced sequestration of dimerpromoting residues and stabilization of the monomer. Our studies suggest a mechanism through which the structure, function, and fate of the viral genome can be modulated by the transcriptionally controlled presence or absence of a single 5′ guanosine.HIV-1 | 5′-leader | transcription | RNA | structure

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“…A model for the switch between translation and packaging has been proposed based on NMR spectroscopy data showing that the DIS loop of SL1 folds back and interacts with an upstream region in the viral RNA, thereby preventing dimerization of SL1 and promoting translation (Lu et al 2011a). In MLV, dimerization of the gRNA exposes high-affinity binding sites for the NC domain of Gag (D'Souza and Summers 2004;Gherghe et al 2010;Miyazaki et al 2010), suggesting that packaging and Gag binding to ψ are coordinated with dimerization. In HIV-1, a 159-nt "core encapsidation signal" was recently described that binds NC with similar affinity as the full-length 5 ′ leader sequence (Heng et al 2012).…”

Section: Thementioning

confidence: 99%

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

Webb

Jones

Parent

et al. 2013

RNA

131

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Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Z eff ) and nonelectrostatic (i.e., specific) component of binding, K d(1M) . Gag binds to Psi RNA with a dramatically reduced K d(1M) and lower Z eff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Z eff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Z eff . These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus

Cited by 48 publications

References 79 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

Transcriptional start site heterogeneity modulates the structure and function of the HIV-1 genome

Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: Implications for viral genomic RNA packaging

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