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- PDB-9h1p: Mature HIV-1 matrix from MA-SP1 cleavage mutant -

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Basic information

Entry
Database: PDB / ID: 9h1p
TitleMature HIV-1 matrix from MA-SP1 cleavage mutant
Components(Gag polyprotein) x 2
KeywordsVIRAL PROTEIN / HIV-1 / HIV / matrix / mature / spacer peptide 2 / SP2
Function / homology
Function and homology information


viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding ...viral budding via host ESCRT complex / host multivesicular body / ISG15 antiviral mechanism / viral nucleocapsid / viral translational frameshifting / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / RNA binding / zinc ion binding / membrane
Similarity search - Function
Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal ...Gag protein p6 / Gag protein p6 / : / gag protein p24 N-terminal domain / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / Retrovirus capsid, C-terminal / Retroviral matrix protein / Retrovirus capsid, N-terminal / zinc finger / Zinc knuckle / Zinc finger, CCHC-type superfamily / Zinc finger, CCHC-type / Zinc finger CCHC-type profile.
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus type 1 group M subtype B
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsStacey, J.C.V. / Hrebik, D. / Briggs, J.A.G.
Funding support Germany, United States, 4items
OrganizationGrant numberCountry
German Research Foundation (DFG)240245660 - SFB 1129 (project 5 HGK, project 6 BM, project 931 21 JAGB) Germany
German Research Foundation (DFG)DFG KR 906/7-1 (HGK) Germany
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32 AI055403 United States
Max Planck Society Germany
Citation
Journal: Nature / Year: 2025
Title: The conserved HIV-1 spacer peptide 2 triggers matrix lattice maturation.
Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Pradeep D Uchil / Robert A Dick / Walther Mothes / Hans-Georg ...Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Pradeep D Uchil / Robert A Dick / Walther Mothes / Hans-Georg Kräusslich / Barbara Müller / John A G Briggs /
Abstract: The virus particles of human immunodeficiency virus type 1 (HIV-1) are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional ...The virus particles of human immunodeficiency virus type 1 (HIV-1) are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane-binding domain, MA, forms a hexameric protein lattice that undergoes structural transition, following cleavage, into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated about 300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilize the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid monolayers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation and assign function to the SP2 peptide.
#1: Journal: bioRxiv / Year: 2024
Title: The conserved HIV-1 spacer peptide 2 triggers matrix lattice maturation.
Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Robert A Dick / Walther Mothes / Hans-Georg Kräusslich / ...Authors: James C V Stacey / Dominik Hrebík / Elizabeth Nand / Snehith Dyavari Shetty / Kun Qu / Marius Boicu / Maria Anders-Össwein / Robert A Dick / Walther Mothes / Hans-Georg Kräusslich / Barbara Müller / John A G Briggs /
Abstract: HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious ...HIV-1 particles are released in an immature, non-infectious form. Proteolytic cleavage of the main structural polyprotein Gag into functional domains induces rearrangement into mature, infectious virions. In immature virus particles, the Gag membrane binding domain, MA, forms a hexameric protein lattice that undergoes structural transition upon cleavage into a distinct, mature MA lattice. The mechanism of MA lattice maturation is unknown. Here we show that released spacer peptide 2 (SP2), a conserved peptide of unknown function situated ~300 residues downstream of MA, binds MA to induce structural maturation. By high-resolution in-virus structure determination of MA, we show that MA does not bind lipid into a side pocket as previously thought, but instead binds SP2 as an integral part of the protein-protein interfaces that stabilise the mature lattice. Analysis of Gag cleavage site mutants showed that SP2 release is required for MA maturation, and we demonstrate that SP2 is sufficient to induce maturation of purified MA on lipid layers in vitro. SP2-triggered MA maturation correlated with faster fusion of virus with target cells. Our results reveal a new, unexpected interaction between two HIV-1 components, provide a high-resolution structure of mature MA, establish the trigger of MA structural maturation, and assign function to the SP2 peptide.
History
DepositionOct 10, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 4, 2024Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Gag polyprotein
B: Gag polyprotein
C: Gag polyprotein
D: Gag polyprotein
E: Gag polyprotein
F: Gag polyprotein
G: Gag polyprotein
H: Gag polyprotein
I: Gag polyprotein
J: Gag polyprotein
K: Gag polyprotein
L: Gag polyprotein
M: Gag polyprotein
N: Gag polyprotein
O: Gag polyprotein
P: Gag polyprotein
Q: Gag polyprotein
R: Gag polyprotein
S: Gag polyprotein
T: Gag polyprotein
U: Gag polyprotein
V: Gag polyprotein
W: Gag polyprotein
X: Gag polyprotein


Theoretical massNumber of molelcules
Total (without water)198,95724
Polymers198,95724
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Gag polyprotein / Pr55Gag


Mass: 14734.616 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate NY5)
Gene: gag / Plasmid: pcHIV / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: P12493
#2: Protein/peptide
Gag polyprotein / Pr55Gag


Mass: 1845.154 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Details: spacer peptide 2
Source: (gene. exp.) Human immunodeficiency virus type 1 group M subtype B (isolate NY5)
Gene: gag / Plasmid: pcHIV / Cell line (production host): HEK293T / Organ (production host): Kidney / Production host: Homo sapiens (human) / References: UniProt: P12493
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1HIV-1 vector pNL4-3VIRUSHEK293T cells were transfected with pcHIV which was expressed and purified.all0RECOMBINANT
2HIV-1 mature matrixCOMPLEXHIV-1 mature matrix as a part of the MA-SP1 polypeptide, in a complex with SP2all1RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11110 MDaNO
2182 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21HIV-1 vector pNL4-3 (others)151458
32HIV-1 vector pNL4-3 (others)151458
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainCellPlasmid
21Homo sapiens (human)9606HEK293TKidneypcHIV
32Homo sapiens (human)9606HEK293TKidneypcHIV
Details of virusEmpty: NO / Enveloped: YES / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural host
IDEntity assembly-IDOrganismNcbi tax-ID
11Homo sapiens9606
22Homo sapiens9606
Virus shell
IDEntity assembly-IDNameDiameter (nm)
11Gag1200
22
Buffer solutionpH: 7.4 / Details: PBS
Buffer component
IDConc.NameFormulaBuffer-ID
18.1 mmol/Ldisodium hydrogen phosphateNa2HPO41
21.5 mmol/Lpotassium dihydrogen phosphateKH2PO41
3137 mmol/Lsodium chlorideNaCl1
42.7 mmol/Lpotassium chlorideKCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: purified HIV-1 MA-SP1 particles in PBS
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 600 nm / Calibrated defocus min: 450 nm / Calibrated defocus max: 4500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 88 K
Image recordingAverage exposure time: 4 sec. / Electron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 14222 / Details: EER mode
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 15 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1crYOLO1.9.2particle selection
2EPU3image acquisition
4cryoSPARC4.3CTF correction
7ISOLDE1.6model fitting
9cryoSPARC4.3initial Euler assignment
10cryoSPARC4.3final Euler assignment
11cryoSPARC4.3classification
12cryoSPARC4.33D reconstruction
13PHENIX1.21model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5704512
Details: A new model was trained in crYOLO using a training dataset annotated in a randomly selected set of 50-100 micrographs. Annotation was performed in the crYOLO boxmanager GUI placing positions ...Details: A new model was trained in crYOLO using a training dataset annotated in a randomly selected set of 50-100 micrographs. Annotation was performed in the crYOLO boxmanager GUI placing positions all over the visible surface of an HIV virus particle. The picks did not distinguish individual proteins or membranes.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 5486693 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 52.49 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: cross-correlation
Atomic model buildingPDB-ID: 2H3I

2h3i


Pdb chain-ID: A / Accession code: 2H3I / Source name: PDB / Type: experimental model

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