Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

Deshmukh, Lalit; Ghirlando, Rodolfo; Clore, G. Marius

doi:10.1073/pnas.1501985112

Cited by 45 publications

(72 citation statements)

References 48 publications

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“…1A). ΔGag exhibits a monomer-dimer equilibrium at high ionic strength (K dimer ∼35 μM at ≥300 mM NaCl), but at low salt (≤100 mM NaCl) forms immature virus-like particles (3). We also made use of the construct, ΔGag W316A M317A (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…These encounter complexes are likely to have a significant impact on the (26) suggested that the six-helix bundle formed by SP1 (27) in immature Gag assemblies may block access to the CAjSP1 junction, thereby slowing down the proteolysis at this site. However, SP1 and neighboring residues are intrinsically disordered and fully accessible in solution in the context of ΔGag, as evidenced by NMR chemical shifts, relaxation parameters, and backbone amide RDCs (3,24), and yet hydrolysis at the CAjSP1 junction is remarkably slow in vitro (compare Fig. 1B), even when mutated to the sequence of the SP1jNC junction (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…1A). MA, CA, and NC are globular domains, whereas SP1, SP2, and p6 are intrinsically disordered in solution (3). At neutral pH in vitro, the five cleavage sites in Gag are hydrolyzed by protease in the following order: SP1jNC > SP2jp6 ∼ MAjCA > CAjSP1 ∼ NCjSP2 (4).…”

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confidence: 99%

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Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

Deshmukh

Louis

Ghirlando

et al. 2016

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

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Cleavage of the group-specific antigen (Gag) polyprotein by HIV-1 protease represents the critical first step in the conversion of immature noninfectious viral particles to mature infectious virions. Selective pressure exerted by HIV-1 protease inhibitors, a mainstay of current anti–HIV-1 therapies, results in the accumulation of drug resistance mutations in both protease and Gag. Surprisingly, a large number of these mutations (known as secondary or compensatory mutations) occur outside the active site of protease or the cleavage sites of Gag (located within intrinsically disordered linkers connecting the globular domains of Gag to one another), suggesting that transient encounter complexes involving the globular domains of Gag may play a role in guiding and facilitating access of the protease to the Gag cleavage sites. Here, using large fragments of Gag, as well as catalytically inactive and active variants of protease, we probe the nature of such rare encounter complexes using intermolecular paramagnetic relaxation enhancement, a highly sensitive technique for detecting sparsely populated states. We show that Gag-protease encounter complexes are primarily mediated by interactions between protease and the globular domains of Gag and that the sites of transient interactions are correlated with surface exposed regions that exhibit a high propensity to mutate in the presence of HIV-1 protease inhibitors.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

Deshmukh

Louis

Ghirlando

et al. 2016

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

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“…RSV protease was purified as previously described (26), and stored at 1 mg/ml in buffer (20 mM MES [pH 6.5], 100 mM NaCl, 2 M ZnCl 2 ) at Ϫ80°C until use. For digestions of protein with GT25 oligonucleotide, protein was mixed with oligonucleotide as described for VLP assembly, and the mixture was incubated for 1 h prior to the addition of protease (a method similar to one used to analyze HIV Gag cleavage [27]). PR digestions were performed in buffer (20 mM MES [pH 6.5], 100 mM NaCl) at a 1:1 mass ratio of PR to Gag⌬MBD protein for 10 min.…”

Section: Methodsmentioning

confidence: 99%

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

Schur

Dick

Hagen

et al. 2015

J Virol

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The polyprotein Gag is the primary structural component of retroviruses. Gag consists of independently folded domains connected by flexible linkers. Interactions between the conserved capsid (CA) domains of Gag mediate formation of hexameric protein lattices that drive assembly of immature virus particles. Proteolytic cleavage of Gag by the viral protease (PR) is required for maturation of retroviruses from an immature form into an infectious form. Within the assembled Gag lattices of HIV-1 and Mason-Pfizer monkey virus (M-PMV), the C-terminal domain of CA adopts similar quaternary arrangements, while the N-terminal domain of CA is packed in very different manners. Here, we have used cryo-electron tomography and subtomogram averaging to study in vitro-assembled, immature virus-like Rous sarcoma virus (RSV) Gag particles and have determined the structure of CA and the surrounding regions to a resolution of ϳ8 Å. We found that the C-terminal domain of RSV CA is arranged similarly to HIV-1 and M-PMV, whereas the N-terminal domain of CA adopts a novel arrangement in which the upstream p10 domain folds back into the CA lattice. In this position the cleavage site between CA and p10 appears to be inaccessible to PR. Below CA, an extended density is consistent with the presence of a six-helix bundle formed by the spacer-peptide region. We have also assessed the affect of lattice assembly on proteolytic processing by exogenous PR. The cleavage between p10 and CA is indeed inhibited in the assembled lattice, a finding consistent with structural regulation of proteolytic maturation. IMPORTANCERetroviruses first assemble into immature virus particles, requiring interactions between Gag proteins that form a protein layer under the viral membrane. Subsequently, Gag is cleaved by the viral protease enzyme into separate domains, leading to rearrangement of the virus into its infectious form. It is important to understand how Gag is arranged within immature retroviruses, in order to understand how virus assembly occurs, and how maturation takes place. We used the techniques cryo-electron tomography and subtomogram averaging to obtain a detailed structural picture of the CA domains in immature assembled Rous sarcoma virus Gag particles. We found that part of Gag next to CA, called p10, folds back and interacts with CA when Gag assembles. This arrangement is different from that seen in HIV-1 and Mason-Pfizer monkey virus, illustrating further structural diversity of retroviral structures. The structure provides new information on how the virus assembles and undergoes maturation.T he major structural component of retroviruses is the polyprotein Gag. The expression of Gag alone is sufficient to mediate the assembly and release of virus-like particles (VLPs). Gag proteins from all retroviruses contain an N-terminal membranebinding matrix (MA) domain, two capsid (CA) domains, and a nucleocapsid (NC) domain (1). These domains are structurally similar across retroviral genera but differ greatly in sequence. Outside these core do...

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“…Essential information about CA molecular structure and lattice structure has been obtained from a large number of investigations by electron microscopy (2-4, 7), x-ray crystallography (5,6,8,(17)(18)(19)(20), and multidimensional nuclear magnetic resonance (NMR) spectroscopy of soluble monomeric and dimeric constructs (21)(22)(23)(24)(25)(26)(27)(28)(29). Results from these investigations show that CA is primarily ␣-helical, with independently folding N-terminal (helices 1-7) and C-terminal (helices 8 -11) domains (NTD and CTD), connected by a short linker that allows the relative orientations of NTD and CTD to vary widely in the unassembled, soluble state.…”

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confidence: 99%

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

Lü¹,

Bayro

Tycko

2016

Journal of Biological Chemistry

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We present the results of solid state nuclear magnetic resonance (NMR) experiments on HIV-1 capsid protein (CA) assemblies with three different morphologies, namely wild-type CA (WT-CA) tubes with 35-60 nm diameters, planar sheets formed by the Arg 18 -Leu mutant (R18L-CA), and R18L-CA spheres with 20 -100 nm diameters. The experiments are intended to elucidate molecular structural variations that underlie these variations in CA assembly morphology. We find that multidimensional solid state NMR spectra of 15 N, 13 C-labeled CA assemblies are remarkably similar for the three morphologies, with only small differences in 15 N and 13 C chemical shifts, no significant differences in NMR line widths, and few differences in the number of detectable NMR cross-peaks. Thus, the pronounced differences in morphology do not involve major differences in the conformations and identities of structurally ordered protein segments. Instead, morphological variations are attributable to variations in conformational distributions within disordered segments, which do not contribute to the solid state NMR spectra. Variations in solid state NMR signals from certain amino acid side chains are also observed, suggesting differences in the intermolecular dimerization interface between curved and planar CA lattices, as well as possible differences in intramolecular helix-helix packing.

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Conformation and dynamics of the Gag polyprotein of the human immunodeficiency virus 1 studied by NMR spectroscopy

Cited by 45 publications

References 48 publications

Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

Transient HIV-1 Gag–protease interactions revealed by paramagnetic NMR suggest origins of compensatory drug resistance mutations

The Structure of Immature Virus-Like Rous Sarcoma Virus Gag Particles Reveals a Structural Role for the p10 Domain in Assembly

Major Variations in HIV-1 Capsid Assembly Morphologies Involve Minor Variations in Molecular Structures of Structurally Ordered Protein Segments

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