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Michael F. Summers - One of the best experts on this subject based on the ideXlab platform.
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nmr detection of intermolecular interaction sites in the dimeric 5 leader of the hiv 1 genome
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Sarah C Keane, Xiao Heng, Verna Van, Heather M Frank, Carly A Sciandra, Sayo Mccowin, Justin Santos, Michael F. SummersAbstract:HIV type-1 (HIV-1) contains a pseudodiploid RNA genome that is selected for packaging and maintained in virions as a noncovalently linked dimer. Genome dimerization is mediated by conserved elements within the 5′-leader of the RNA, including a palindromic dimer initiation signal (DIS) that has been proposed to form kissing hairpin and/or extended duplex intermolecular contacts. Here, we have applied a 2H-edited NMR approach to directly probe for intermolecular interactions in the full-length, dimeric HIV-1 5′-leader (688 nucleotides; 230 kDa). The interface is extensive and includes DIS:DIS base pairing in an extended duplex state as well as intermolecular pairing between elements of the upstream Unique-5′ (U5) sequence and those near the gag start site (AUG). Other pseudopalindromic regions of the leader, including the transcription activation (TAR), polyadenylation (PolyA), and primer binding (PBS) elements, do not participate in intermolecular base pairing. Using a 2H-edited one-dimensional NMR approach, we also show that the extended interface structure forms on a time scale similar to that of overall RNA dimerization. Our studies indicate that a kissing dimer-mediated structure, if formed, exists only transiently and readily converts to the extended interface structure, even in the absence of the HIV-1 nucleocapsid protein or other RNA chaperones.
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an rna structural switch regulates diploid genome packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
Yasuyuki Miyazaki - One of the best experts on this subject based on the ideXlab platform.
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an rna structural switch regulates diploid genome packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
Eric L. Garcia - One of the best experts on this subject based on the ideXlab platform.
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NMR Detection of Structures in the HIV-1 5'-Leader RNA that Regulate Genome Packaging
Science (New York N.Y.), 2011Co-Authors: Xiao Heng, Lianko Garyu, Sarah Monti, Eric L. Garcia, Siarhei Kharytonchyk, Bilguujin Dorjsuren, Gowry Kulandaivel, Simonne Jones, Atheeth HiremathAbstract:The 5'-leader of the HIV-1 genome regulates multiple functions during viral replication via mechanisms that have yet to be established. We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residues of the unique-5' region (U5) in the dimer. U5:AUG formation promotes dimerization by displacing and exposing a dimer-promoting hairpin and enhances binding by the nucleocapsid (NC) protein, which is the cognate domain of the viral Gag polyprotein that directs packaging. Our findings support a packaging mechanism in which translation, dimerization, NC binding, and packaging are regulated by a common RNA structural switch.
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an rna structural switch regulates diploid genome packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
Steven R King - One of the best experts on this subject based on the ideXlab platform.
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an rna structural switch regulates diploid genome packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
Kelsey Loeliger - One of the best experts on this subject based on the ideXlab platform.
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an rna structural switch regulates diploid genome packaging by moloney murine leukemia virus
Journal of Molecular Biology, 2010Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes by mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukaemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5'-untranslated region (5'-UTR) that contains all residues necessary for efficient RNA packaging (Ψ WT , residues 147–623) also exhibits dimerization-dependent affinity for NC, with the native dimer ([Ψ WT ]2) binding 12 ± 2 NC molecules with high affinity (Kd = 17 ± 7 nM) and the monomer, stabilized by substitution of dimer-promoting loop residues by hairpin-stabilizing sequences (Ψ M ), binding 1–2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (~100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism, and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.
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An RNA Structural Switch Regulates Diploid Genome Packaging by Moloney Murine Leukemia Virus
Journal of molecular biology, 2009Co-Authors: Yasuyuki Miyazaki, Eric L. Garcia, Steven R King, Kilali Iyalla, Kelsey Loeliger, Patrice Starck, Sameera Syed, Alice Telesnitsky, Michael F. SummersAbstract:Retroviruses selectively package two copies of their RNA genomes via mechanisms that have yet to be fully deciphered. Recent studies with small fragments of the Moloney murine leukemia virus (MoMuLV) genome suggested that selection may be mediated by an RNA switch mechanism, in which conserved UCUG elements that are sequestered by base-pairing in the monomeric RNA become exposed upon dimerization to allow binding to the cognate nucleocapsid (NC) domains of the viral Gag proteins. Here we show that a large fragment of the MoMuLV 5' untranslated region that contains all residues necessary for efficient RNA packaging (Psi(WT); residues 147-623) also exhibits a dimerization-dependent affinity for NC, with the native dimer ([Psi(WT)](2)) binding 12+/-2 NC molecules with high affinity (K(d)=17+/-7 nM) and with the monomer, stabilized by substitution of dimer-promoting loop residues with hairpin-stabilizing sequences (Psi(M)), binding 1-2 NC molecules. Identical dimer-inhibiting mutations in MoMuLV-based vectors significantly inhibit genome packaging in vivo (approximately 100-fold decrease), whereas a large deletion of nearly 200 nucleotides just upstream of the gag start codon has minimal effects. Our findings support the proposed RNA switch mechanism and further suggest that virus assembly may be initiated by a complex comprising as few as 12 Gag molecules bound to a dimeric packaging signal.