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Michael F Summers

Publications and source records attributed to Michael F Summers.

14 recordsLinked to original sources
PubMed2006

Point mutations in the HIV-1 matrix protein turn off the myristyl switch.

During the late phase of human immunodeficiency virus type-1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to lipid raft regions of specific cellular membranes, where they assemble and bud to form new virus particles. Gag binds preferentially to the plasma membrane (PM) of most hematopoietic cell types, a process mediated by interactions between the cellular PM marker phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P(2)) and Gag's N-terminally myristoylated matrix (MA) domain. We recently demonstrated that PI(4,5)P(2) binds to a conserved cleft on MA and promotes myristate exposure, suggesting a role as both a direct membrane anchor and myristyl switch trigger. Here we show that PI(4,5)P(2) is also capable of binding to MA proteins containing point mutations that inhibit membrane binding in vitro, and in vivo, including V7R, L8A and L8I. However, these mutants do not exhibit PI(4,5)P(2) or concentration-dependent myristate exposure. NMR studies of V7R and L8A MA reveal minor structural changes that appear to be responsible for stabilizing the myristate-sequestered (myr(s)) species and inhibiting exposure. Unexpectedly, the myristyl group of a revertant mutant with normal PM targeting properties (V7R,L21K) is also tightly sequestered and insensitive to PI(4,5)P(2) binding. This mutant binds PI(4,5)P(2) with twofold higher affinity compared with the native protein, suggesting a potential compensatory mechanism for membrane binding.

HIV-1↗
PubMed2006

Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly.

During the late phase of HIV type 1 (HIV-1) replication, newly synthesized retroviral Gag proteins are targeted to the plasma membrane of most hematopoietic cell types, where they colocalize at lipid rafts and assemble into immature virions. Membrane binding is mediated by the matrix (MA) domain of Gag, a 132-residue polypeptide containing an N-terminal myristyl group that can adopt sequestered and exposed conformations. Although exposure is known to promote membrane binding, the mechanism by which Gag is targeted to specific membranes has yet to be established. Recent studies have shown that phosphatidylinositol (PI) 4,5-bisphosphate [PI(4,5)P(2)], a factor that regulates localization of cellular proteins to the plasma membrane, also regulates Gag localization and assembly. Here we show that PI(4,5)P(2) binds directly to HIV-1 MA, inducing a conformational change that triggers myristate exposure. Related phosphatidylinositides PI, PI(3)P, PI(4)P, PI(5)P, and PI(3,5)P(2) do not bind MA with significant affinity or trigger myristate exposure. Structural studies reveal that PI(4,5)P(2) adopts an "extended lipid" conformation, in which the inositol head group and 2'-fatty acid chain bind to a hydrophobic cleft, and the 1'-fatty acid and exposed myristyl group bracket a conserved basic surface patch previously implicated in membrane binding. Our findings indicate that PI(4,5)P(2) acts as both a trigger of the myristyl switch and a membrane anchor and suggest a potential mechanism for targeting Gag to membrane rafts.

Allosteric Site↗
PubMed2005

High affinity nucleocapsid protein binding to the muPsi RNA packaging signal of Rous sarcoma virus.

The genomes of all retroviruses contain sequences near their 5' ends that interact with the nucleocapsid domains (NC) of assembling Gag proteins and direct their packaging into virus particles. Retroviral packaging signals often occur in non-contiguous segments spanning several hundred nucleotides of the RNA genome, confounding structural and mechanistic studies of genome packaging. Recently, a relatively short, 82 nucleotide region of the Rous sarcoma virus (RSV) genome, called muPsi, was shown to be sufficient to direct efficient packaging of heterologous RNAs into RSV-like particles. We have developed a method for the preparation and purification of large quantities of recombinant RSV NC protein, and have studied its interactions with native and mutant forms of the muPsi encapsidation element. NC does not bind with significant affinity to truncated forms of muPsi, consistent with earlier packaging and mutagenesis studies. Surprisingly, NC binds to the native muPsi RNA with affinity that is approximately 100 times greater than that observed for other previously characterized retroviral NC-RNA complexes (extrapolated dissociation constant K(d)=1.9 nM). Tight binding with 1:1 NC-muPsi stoichiometry is dependent on a conserved UGCG tetraloop in one of three predicted stem loops, and an AUG initiation codon controvertibly implicated in genome packaging and translational control. Loop nucleotides of other stem loops do not contribute to NC binding. Our findings indicate that the structural determinants of RSV genome recognition and NC-RNA binding differ considerably from those observed for other retroviruses.

Amino Acid Sequence↗
PubMed2005

Composition and sequence-dependent binding of RNA to the nucleocapsid protein of Moloney murine leukemia virus.

All retroviruses package two copies of their genomes during virus assembly, both of which are required for strand transfer-mediated recombination during reverse transcription. Genome packaging is mediated by interactions between the nucleocapsid (NC) domains of assembling Gag polyproteins and an RNA packaging signal, located near the 5' end of the genome, called Psi. We recently discovered that the NC protein of the Moloney murine leukemia virus (MLV) can bind with high affinity to conserved UCUG elements within the MLV packaging signal [D'Souza, V., and Summers, M. F. (2004) Nature 431, 586-590]. Selective binding to dimeric RNA is regulated by a conformational RNA switch, in which the UCUG elements are sequestered by base pairing in the monomeric RNA and do not bind NC, but become exposed for NC binding upon dimerization. Dimerization-dependent structural changes occur in other regions of the Psi-site, exposing guanosine-containing segments that might also bind NC. Here we demonstrate that short RNAs containing three such sequences, ACAG, UUUG, and UCCG, can bind NC with significant affinity (K(d) = 94-315 nM). Titration experiments with oligoribonucleotides of varying lengths and compositions, combined with NMR-based structural studies, reveal that binding is strictly dependent on the presence of an unpaired guanosine, and that relative binding affinities can vary by more than 1 order of magnitude depending on the nature of the three upstream nucleotides. Binding is enhanced in short RNAs containing terminal phosphates, indicating that electrostatic interactions contribute significantly to binding. Our findings extend a previously published model for genome recognition, in which the NC domains of assembling Gag molecules interact with multiple X(i-3)-X(i-2)-X(i-1)-G(i) elements (X is a variable nucleotide) that appear to be preferentially exposed in the dimeric RNA.

Base Composition↗
PubMed2005

How retroviruses select their genomes.

As retroviruses assemble in infected cells, two copies of their full-length, unspliced RNA genomes are selected for packaging from a cellular milieu that contains a substantial excess of non-viral and spliced viral RNAs. Understanding the molecular details of genome packaging is important for the development of new antiviral strategies and to enhance the efficacy of retroviral vectors used in human gene therapy. Recent studies of viral RNA structure in vitro and in vivo and high-resolution studies of RNA fragments and protein-RNA complexes are helping to unravel the mechanism of genome packaging and providing the first glimpses of the initial stages of retrovirus assembly.

Animals↗
PubMed2004

Structural basis for packaging the dimeric genome of Moloney murine leukaemia virus.

All retroviruses specifically package two copies of their genomes during virus assembly, a requirement for strand-transfer-mediated recombination during reverse transcription. Genomic RNA exists in virions as dimers, and the overlap of RNA elements that promote dimerization and encapsidation suggests that these processes may be coupled. Both processes are mediated by the nucleocapsid domain (NC) of the retroviral Gag polyprotein. Here we show that dimerization-induced register shifts in base pairing within the Psi-RNA packaging signal of Moloney murine leukaemia virus (MoMuLV) expose conserved UCUG elements that bind NC with high affinity (dissociation constant = 75 +/- 12 nM). These elements are base-paired and do not bind NC in the monomeric RNA. The structure of the NC complex with a 101-nucleotide 'core encapsidation' segment of the MoMuLV Psi site reveals a network of interactions that promote sequence- and structure-specific binding by NC's single CCHC zinc knuckle. Our findings support a structural RNA switch mechanism for genome encapsidation, in which protein binding sites are sequestered by base pairing in the monomeric RNA and become exposed upon dimerization to promote packaging of a diploid genome.

Base Pairing↗
PubMed2004

Flexibility in the P2 domain of the HIV-1 Gag polyprotein.

The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA(CTD)) through the NC domain of Gag (CA(CTD)-p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA(CTD) and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA(CTD) and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an alpha-helical conformation. The presence of a transient coil-to-helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA(CTD)-p2-NC forms dimers and self-associates with an equilibrium constant (Kd = 1.78 +/- 0.5 microM) similar to that observed for the intact capsid protein (Kd = 2.94 +/- 0.8 microM), suggesting that Gag self-association is not significantly influence by the P2 domain.

Capsid Proteins↗
PubMed2004

NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus.

The full length, positive-strand genome of the Moloney Murine Leukemia Virus contains a "core encapsidation signal" that is essential for efficient genome packaging during virus assembly. We have determined the structure of a 101-nucleotide RNA that contains this signal (called mPsi) using a novel isotope-edited NMR approach. The method is robust and should be generally applicable to larger RNAs. mPsi folds into three stem loops, two of which (SL-C and SL-D) co-stack to form an extended helix. The third stem loop (SL-B) is connected to SL-C by a flexible, four-nucleotide linker. The structure contains five mismatched base-pairs, an unusual C.CG base-triple platform, and a novel "A-minor K-turn," in which unpaired adenosine bases A340 and A341 of a GGAA bulge pack in the minor groove of a proximal stem, and a bulged distal uridine (U319) forms a hydrogen bond with the phosphodiester of A341. Phylogenetic analyses indicate that these essential structural elements are conserved among the murine C-type retroviruses.

Animals↗
PubMed2003

Entropic switch regulates myristate exposure in the HIV-1 matrix protein.

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (K(assoc) = 2.5 +/- 0.6 x 10(8) M(-2)). The equilibrium constant is shifted approximately 20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.

Gene Products, gag↗
PubMed2003

Antiviral inhibition of the HIV-1 capsid protein.

During the assembly stage of the human immunodeficiency virus (HIV) replication cycle, several thousand copies of the viral Gag polyprotein associate at the cell membrane and bud to form an immature, non-infectious virion. Gag is subsequently cleaved by the protease, which liberates the capsid proteins for assembly into the polyprotein shell of the central core particle (or capsid) of the mature virus. Viral infectivity is critically dependent on capsid formation and stability, making the capsid protein a potentially attractive antiviral target. We have identified compounds that bind to an apical site on the N-terminal domain of the HIV-1 capsid protein and inhibit capsid assembly in vitro. One compound, N-(3-chloro-4-methylphenyl)-N'-[2-[([5-[(dimethylamino)-methyl]-2-furyl]-methyl)-sulfanyl]ethyl]urea) (CAP-1), is well tolerated in cell cultures, enabling in vivo antiviral and mechanistic studies. CAP-1 inhibits HIV-1 infectivity in a dose-dependent manner, but does not interfere with viral entry, reverse transcription, integration, proteolytic processing, or virus production, indicating a novel antiviral mechanism. Significantly, virus particles generated in the presence of CAP-1 exhibit heterogeneous sizes and abnormal core morphologies, consistent with inhibited CA-CA interactions during virus assembly and maturation. These findings lay the groundwork for the development of assembly inhibitors as a new class of therapeutic agents for the treatment of AIDS.

Anti-HIV Agents↗
PubMed2003

Structure of the intact stem and bulge of HIV-1 Psi-RNA stem-loop SL1.

The Psi-RNA packaging signal of the human immunodeficiency virus type-1 (HIV-1) genome contains a 35 nucleotide stem-loop, termed SL1, which is important for efficient genome packaging during virus assembly and for reverse transcription during infectivity. The predicted secondary structure of SL1 consists of an upper stem with a GC-rich loop that facilitates dimerization, a lower stem, and an intervening bulge (G5, A24-G25-G26) that is both strictly conserved and essential for efficient packaging of the viral genome. The structure of the upper stem in both the kissing and duplex dimer forms have been determined recently. Here, we report the structure of an engineered form of SL1 (SL1(m)) that contains a GAGA tetraloop substituted for the GC-rich loop. This construct does not aggregate and remains monomeric at concentrations up to 1mM, enabling structural studies of the intact stems and bulge. The structure was refined using 1H-13C residual dipolar couplings. The upper stem (C6-G12, C17-G23) is in close agreement with X-ray structures of kissing and duplex dimer forms of related oligoribonucleotides, and nucleotides C1-G4 and C27-G30 form the expected A-helical lower stem. Residues G5 and A24 of the predicted bulge form a G-A mismatch that stacks with the upper stem, and residues G25 and G26 stack between the G-A mismatch and the lower stem in a manner that produces a hole in the center of the bulge and a 25(+/-4) degrees bend between the upper and lower stems. SL1(m) exhibits relatively poor affinity for the HIV-1 nucleocapsid protein, suggesting that the bulge plays other roles in genome packaging.

Base Sequence↗
PubMed2002

Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein.

The capsid protein (CA) of the mature human immunodeficiency virus (HIV) contains an N-terminal beta-hairpin that is essential for formation of the capsid core particle. CA is generated by proteolytic cleavage of the Gag precursor polyprotein during viral maturation. We have determined the NMR structure of a 283-residue N-terminal fragment of immature HIV-1 Gag (Gag(283)), which includes the intact matrix (MA) and N-terminal capsid (CA(N)) domains. The beta-hairpin is unfolded in Gag(283), consistent with the proposal that hairpin formation occurs subsequent to proteolytic cleavage of Gag, triggering capsid assembly. Comparison of the immature and mature CA(N) structures reveals that beta-hairpin formation induces a approximately 2 A displacement of helix 6 and a concomitant displacement of the cyclophylin-A (CypA)-binding loop, suggesting a possible allosteric mechanism for CypA-mediated destabilization of the capsid particle during infectivity.

Amino Acid Sequence↗
PubMed2001

Amino acid substitutions in Gag protein at non-cleavage sites are indispensable for the development of a high multitude of HIV-1 resistance against protease inhibitors.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

Amino Acid Sequence↗