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- EMDB-48719: Cryo-EM Structure of HIV-1 Reverse Transcriptase p66 Homodimer in... -

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Entry
Database: EMDB / ID: EMD-48719
TitleCryo-EM Structure of HIV-1 Reverse Transcriptase p66 Homodimer in Complex with 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy]phenoxy}naphthalene-2-carbonitrile (JLJ648), a Non-nucleoside Inhibitor
Map data
Sample
  • Virus: Human immunodeficiency virus 1
    • Protein or peptide: Reverse transcriptase/ribonuclease H
  • Ligand: 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy]phenoxy}naphthalene-2-carbonitrile
KeywordsREVERSE TRANSCRIPTASE / ANTIVIRAL / DRUG DESIGN / HIV-1 / VIRAL PROTEIN
Function / homology
Function and homology information


HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency ...HIV-1 retropepsin / symbiont-mediated activation of host apoptosis / retroviral ribonuclease H / exoribonuclease H / exoribonuclease H activity / host multivesicular body / DNA integration / viral genome integration into host DNA / RNA-directed DNA polymerase / establishment of integrated proviral latency / viral penetration into host nucleus / RNA stem-loop binding / RNA-directed DNA polymerase activity / RNA-DNA hybrid ribonuclease activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / host cell / viral nucleocapsid / DNA recombination / DNA-directed DNA polymerase / aspartic-type endopeptidase activity / Hydrolases; Acting on ester bonds / DNA-directed DNA polymerase activity / symbiont-mediated suppression of host gene expression / viral translational frameshifting / lipid binding / symbiont entry into host cell / host cell nucleus / host cell plasma membrane / virion membrane / structural molecule activity / proteolysis / DNA binding / zinc ion binding / membrane
Similarity search - Function
Reverse transcriptase connection / Reverse transcriptase connection domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain ...Reverse transcriptase connection / Reverse transcriptase connection domain / Reverse transcriptase thumb / Reverse transcriptase thumb domain / Integrase Zinc binding domain / Zinc finger integrase-type profile. / Integrase-like, N-terminal / Integrase DNA binding domain / Integrase, C-terminal domain superfamily, retroviral / Integrase, N-terminal zinc-binding domain / Integrase, C-terminal, retroviral / Integrase DNA binding domain profile. / Immunodeficiency lentiviral matrix, N-terminal / gag gene protein p17 (matrix protein) / RNase H / Integrase core domain / Integrase, catalytic core / Integrase catalytic domain profile. / Retropepsin-like catalytic domain / Matrix protein, lentiviral and alpha-retroviral, N-terminal / Retroviral nucleocapsid Gag protein p24, C-terminal domain / Gag protein p24 C-terminal domain / RNase H type-1 domain profile. / Ribonuclease H domain / Retropepsins / Retroviral aspartyl protease / Aspartyl protease, retroviral-type family profile. / Peptidase A2A, retrovirus, catalytic / Reverse transcriptase domain / Retrovirus capsid, C-terminal / Reverse transcriptase (RNA-dependent DNA polymerase) / Reverse transcriptase (RT) catalytic domain profile. / 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. / Aspartic peptidase, active site / Eukaryotic and viral aspartyl proteases active site. / Aspartic peptidase domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Reverse transcriptase/Diguanylate cyclase domain / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesHuman immunodeficiency virus 1
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsHollander K / Devarkar SC / Tang S / Ma S / Xiong Y / Jorgensen WL / Anderson KS
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH/NAD AI155072 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI186613 United States
CitationJournal: NPJ Drug Discov / Year: 2025
Title: Mechanistic basis for a novel dual-function Gag-Pol dimerizer potentiating CARD8 inflammasome activation and clearance of HIV-infected cells.
Authors: Klarissa Hollander / Swapnil C Devarkar / Su Tang / Ritudhwaj Tiwari / Shumeng Ma / Won Gil Lee / Elizabeth Denn / Qiankun Wang / Krasimir A Spasov / Jake A Robbins / Kathleen M Frey / ...Authors: Klarissa Hollander / Swapnil C Devarkar / Su Tang / Ritudhwaj Tiwari / Shumeng Ma / Won Gil Lee / Elizabeth Denn / Qiankun Wang / Krasimir A Spasov / Jake A Robbins / Kathleen M Frey / William L Jorgensen / Yong Xiong / Liang Shan / Karen S Anderson /
Abstract: A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside reverse transcriptase inhibitors (NNRTIs) that promote pyroptosis of HIV-1 infected cells. These ...A strategy to functionally cure AIDS by eliminating latent HIV-1 reservoirs involves non-nucleoside reverse transcriptase inhibitors (NNRTIs) that promote pyroptosis of HIV-1 infected cells. These NNRTIs stimulate dimerization of the Gag-Pol polyprotein, resulting in premature HIV-1 protease (PR) dimerization and cleavage of intracellular CARD8. A unique cell-based high-throughput screen was developed to identify potent compounds activating the CARD8 inflammasome through Gag-Pol dimerization. Our in-house library of NNRTIs was evaluated, including a series of catechol diethers, which are potent, nontoxic antivirals. JLJ648 was identified as a promising dual-function antiviral and Gag-Pol dimerizer. Cryo-EM studies of HIV reverse transcriptase p66 bound to JLJ648 revealed populations of homodimers and, surprisingly, a homotetramer. This novel homotetramer structure resembling an 'infinity knot' revealed two JLJ648-bound homodimers forming an extensive interface and nucleated around a dimer of JLJ648 molecules. Structure-guided mutagenesis studies indicate that Gag-Pol homotetramerization may play a critical role in facilitating PR self-cleavage and triggering pyroptosis.
History
DepositionJan 20, 2025-
Header (metadata) releaseSep 10, 2025-
Map releaseSep 10, 2025-
UpdateSep 17, 2025-
Current statusSep 17, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48719.png

Downloads & links

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Map

FileDownload / File: emd_48719.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.22869089 - 0.40908664
Average (Standard dev.)0.00022309422 (±0.006974378)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_48719_msk_1.map
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Additional map: #1

Fileemd_48719_additional_1.map
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Half map: #2

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Half map: #1

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Sample components

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Entire : Human immunodeficiency virus 1

EntireName: Human immunodeficiency virus 1
Components
  • Virus: Human immunodeficiency virus 1
    • Protein or peptide: Reverse transcriptase/ribonuclease H
  • Ligand: 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy]phenoxy}naphthalene-2-carbonitrile

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Supramolecule #1: Human immunodeficiency virus 1

SupramoleculeName: Human immunodeficiency virus 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1 / NCBI-ID: 11676 / Sci species name: Human immunodeficiency virus 1 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: Yes / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: Reverse transcriptase/ribonuclease H

MacromoleculeName: Reverse transcriptase/ribonuclease H / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RNA-directed DNA polymerase
Source (natural)Organism: Human immunodeficiency virus 1
Molecular weightTheoretical: 64.916348 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ...String:
MVPISPIETV PVKLKPGMDG PKVKQWPLTE EKIKALVEIC TEMEKEGKIS KIGPENPYNT PVFAIKKKDS TKWRKLVDFR ELNKRTQDF WEVQLGIPHP AGLKKKKSVT VLDVGDAYFS VPLDEDFRKY TAFTIPSINN ETPGIRYQYN VLPQGWKGSP A IFQSSMTK ILEPFKKQNP DIVIYQYMDD LYVGSDLEIG QHRTKIEELR QHLLRWGLTT PDKKHQKEPP FLWMGYELHP DK WTVQPIV LPEKDSWTVN DIQKLVGKLN WASQIYPGIK VRQLCKLLRG TKALTEVIPL TEEAELELAE NREILKEPVH GVY YDPSKD LIAEIQKQGQ GQWTYQIYQE PFKNLKTGKY ARMRGAHTND VKQLTEAVQK ITTESIVIWG KTPKFKLPIQ KETW ETWWT EYWQATWIPE WEFVNTPPLV KLWYQLEKEP IVGAETFYVD GAANRETKLG KAGYVTNKGR QKVVPLTNTT NQKTE LQAI YLALQDSGLE VNIVTDSQYA LGIIQAQPDK SESELVNQII EQLIKKEKVY LAWVPAHKGI GGNEQVDKLV SAGENL YFQ

UniProtKB: Gag-Pol polyprotein

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Macromolecule #2: 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)etho...

MacromoleculeName: 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy]phenoxy}naphthalene-2-carbonitrile
type: ligand / ID: 2 / Number of copies: 1 / Formula: A1BTU
Molecular weightTheoretical: 415.464 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.65 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
25.0 mMNaClSodium Chloride
1.0 mMC9H15O6PTCEP
5.0 PercentC3H8O3Glycerol

Details: 50mM Tris pH 7.0, 25mM NaCl, 5% Glycerol, 1mM TCEP
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 20 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec.
VitrificationCryogen name: ETHANE / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4we1

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 316118
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4we1


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9mxr:
Cryo-EM Structure of HIV-1 Reverse Transcriptase p66 Homodimer in Complex with 5-{2-[2-(2-oxo-4-sulfanylidene-3,4-dihydropyrimidin-1(2H)-yl)ethoxy] phenoxy}naphthalene-2-carbonitrile (JLJ648), a Non-nucleoside Inhibitor

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