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- PDB-9be9: HIV-1 Env 16055 dGly4 NFL -

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Basic information

Entry
Database: PDB / ID: 9be9
TitleHIV-1 Env 16055 dGly4 NFL
ComponentsEnvelope glycoprotein gp140
KeywordsVIRAL PROTEIN / HIV-1 / NFL / Env / vaccine design
Function / homology
Function and homology information


positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane ...positive regulation of plasma membrane raft polarization / positive regulation of receptor clustering / host cell endosome membrane / clathrin-dependent endocytosis of virus by host cell / viral protein processing / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / virion attachment to host cell / host cell plasma membrane / virion membrane / structural molecule activity / membrane
Similarity search - Function
Envelope glycoprotein Gp160 / Retroviral envelope protein / Retroviral envelope protein GP41-like / Gp120 core superfamily / Envelope glycoprotein GP120 / Human immunodeficiency virus 1, envelope glycoprotein Gp120
Similarity search - Domain/homology
Envelope glycoprotein gp160
Similarity search - Component
Biological speciesHuman immunodeficiency virus 1
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOzorowski, G. / Lee, W.-H. / Ward, A.B.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)UM1 AI144462 United States
CitationJournal: NPJ Vaccines / Year: 2025
Title: Accelerated cGMP production of near-native HIV-1 Env trimers following electroporation transfection and immunogenicity analysis.
Authors: Shridhar Bale / Elena Gustchina / Javier Guenaga / Victor Ayala / Wen-Hsin Lee / Gabriel Ozorowski / Stephen Whitney / Richard Wilson / Sabyasachi Baboo / Jolene K Diedrich / Esmeralda D ...Authors: Shridhar Bale / Elena Gustchina / Javier Guenaga / Victor Ayala / Wen-Hsin Lee / Gabriel Ozorowski / Stephen Whitney / Richard Wilson / Sabyasachi Baboo / Jolene K Diedrich / Esmeralda D Doyle / Lauren Hudacik / Elana Ben-Akiva / Kristen A Rodrigues / Darrell J Irvine / John R Yates / James C Paulson / Andrew B Ward / Timothy Fouts / Richard T Wyatt /
Abstract: Evaluation of recombinant HIV-1 surface glycoproteins (Env) as vaccine candidates for Phase I human experimental trials often requires production of cGMP-grade well-ordered Env trimers. Here, we ...Evaluation of recombinant HIV-1 surface glycoproteins (Env) as vaccine candidates for Phase I human experimental trials often requires production of cGMP-grade well-ordered Env trimers. Here, we report an accelerated cGMP compatible approach for expression and purification of a stabilized HIV clade C-derived trimer '16055 DG4 NFL' (for native flexibly linked). This recombinant trimer was expressed from CHO-S™ cells using a MaxCyte® VLX™ electroporation-based transient transfection process. The 16055 DG4 NFL was designed with multiple internal stabilizing mutations and, as well, deletion of four N-linked glycans (DG4) proximal to the CD4 binding site (CD4bs) engineered to improve B cell recognition of this conserved neutralizing determinant. The transient process circumvents the need to develop stable cell lines expressing the Env trimers that is often the most time-consuming step impacting vaccine development timelines. The 16055 DG4 NFL trimer was purified by immunoaffinity chromatography using the broadly neutralizing antibody (bNAb), PGT145. Following additional downstream processing steps, purified trimer was vialed, frozen and stored at -80 °C. Upon thaw and analysis, the trimer displayed homogeneity and a near-native conformation as determined by size-exclusion chromatography (SEC), negative stain and cryo-electron microscopy (EM), differential scanning calorimetry (DSC) and biolayer interferometry (BLI). The immunogenicity of the trimer was tested in rabbits with bolus, escalating dose and divided dose immunization regimens. Rabbits from all three regimens elicited tier 2 autologous neutralizing antibodies that targeted the exposed protein region at the CD4bs. The trimer is currently under investigation in a human clinical trial (NCT06332339) for safety, tolerability and as a priming candidate followed by heterologous boosting to potentially elicit cross-neutralizing antibodies.
History
DepositionApr 15, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Envelope glycoprotein gp140
B: Envelope glycoprotein gp140
C: Envelope glycoprotein gp140
hetero molecules


Theoretical massNumber of molelcules
Total (without water)229,85951
Polymers213,6383
Non-polymers16,22148
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Envelope glycoprotein gp140 / Env polyprotein


Mass: 71212.586 Da / Num. of mol.: 3
Fragment: GGGGSGGGGS is an engineered insertion (linker to replace cleavage site of reference sequence
Source method: isolated from a genetically manipulated source
Details: HIV-1 Env 16055 dGly4 NFL gp140 / Source: (gene. exp.) Human immunodeficiency virus 1 / Gene: env / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: A1EAI1
#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 15
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#3: Polysaccharide alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D- ...alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 1072.964 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-2DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,6,5/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3-3/a4-b1_b4-c1_c3-d1_c6-f1_d2-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 30 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestN
Has protein modificationY

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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

ComponentName: HIV-1 Env 16055 dGly4 NFL / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Human immunodeficiency virus 1
Source (recombinant)Organism: Cricetulus griseus (Chinese hamster)
Buffer solutionpH: 7.4 / Details: Added LMNG to 0.005 mM shortly before freezing
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN 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: 2000 nm / Nominal defocus min: 700 nm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 48 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 91115 / Symmetry type: POINT

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