1. Microbiology and Infectious Disease
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HIV-1 integrase tetramers are the antiviral target of pyridine-based allosteric integrase inhibitors

  1. Pratibha C Koneru
  2. Ashwanth C Francis
  3. Nanjie Deng
  4. Stephanie V Rebensburg
  5. Ashley C Hoyte
  6. Jared Lindenberger
  7. Daniel Adu-Ampratwum
  8. Ross C Larue
  9. Michael F Wempe
  10. Alan N Engelman
  11. Dmitry Lyumkis
  12. James R Fuchs
  13. Ronald M Levy
  14. Gregory B Melikyan
  15. Mamuka Kvaratskhelia  Is a corresponding author
  1. University of Colorado, United States
  2. Emory University, United States
  3. Pace University, United States
  4. The Ohio State University, United States
  5. University of Colorado Denver, United States
  6. Dana-Farber Cancer Institute, United States
  7. Harvard Medical School, United States
  8. The Salk Institute for Biological Studies, United States
  9. Temple University, United States
Research Article
Cite this article as: eLife 2019;8:e46344 doi: 10.7554/eLife.46344
8 figures, 1 table, 1 data set and 2 additional files

Figures

Figure 1 with 1 supplement
IN tetramers are preferentially targeted by pyridine and quinoline based ALLINIs.

(A) Chemical structures of pyridine-based KF116 and quinoline-based BI224436. (B) SEC based separation of IN tetramer(T), dimer(D) and monomer(M) fractions. C and D, DLS analysis of 200 nM IN fractions in the presence of 1 µM KF116 (blue lines, (C) or BI224436 (red lines, (D). DMSO controls for each DLS experiment (C and D) are shown in gray. Representative results of three independent experiments at 30 mins time point are shown.

Figure 1—figure supplement 1
Pyridine and quinoline based ALLINIs preferentially target IN tetramers.

Kinetic analysis of KF116 (blue lines in A and B) or BI224436 (red lines in C and D) induced higher-order oligomerization of tetramer (A and C) and dimer (B and D) fractions. 1 µM inhibitor was added to 200 nM IN and higher-order oligomerization was monitored by DLS. DMSO controls for each experiment are shown in gray. Representative results of three independent experiments are shown.

Figure 2 with 1 supplement
Stoichiometry of KF116 and BI224436 induced aggregation of IN.

Quantitative analysis of ALLINI induced IN aggregation. The error bars indicate the standard deviation of three independent experiments. The stoichiometry for KF116:IN and BI224436:IN were determined using piecewise linear regression.

Figure 2—figure supplement 1
KF116 and BI224436 induced aggregation of IN.

Increasing concentrations of the ALLINIs were added to the full length WT IN and the supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE gels with coomassie staining. Representative images of three independent experiments are shown.

Figure 3 with 1 supplement
Roles of individual IN domains for ALLINI induced aggregation.

(A and B) DLS analysis of 10 µM full length IN, NTD-CCD, CCD and CCD-CTD in the presence of 1 µM KF116 or BI224436. Representative results of two independent experiments at 30 mins time point are shown. DMSO control results for each respective experiment are shown in gray. (C and D) Quantitative analysis of KF116 and BI224436 induced aggregation of 5 µM full length IN, NTD-CCD, CCD and CCD-CTD by centrifugation-based aggregation assay. The error bars indicate the standard error of two independent experiments (see Figure 3—figure supplement 1 for representative primary data).

Figure 3—figure supplement 1
Contributions of individual IN domains for ALLINI induced aggregation.

Increasing concentrations of KF116 and BI224436 were added to 5 µM of full length IN, NTD-CCD, CCD and CCD-CTD. Following centrifugation, supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE with coomassie staining. The representative images of two independent experiments are shown.

Figure 4 with 4 supplements
Probing the importance of the NTD and CCD-CTD linker for ALLINI induced higher-order multimerization of IN.

(A) Schematic diagram of IN with indicated mutations in NTD and CCD-CTD linker regions. (B) Summary table of all IN mutants indicating their predominant multimeric form as analyzed by SEC and effects of ALLINI induced higher-order multimerization monitored by DLS. ‘+' and '- ' indicates susceptibility and resistance of the mutant proteins to ALLINI induced higher-order multimerization, respectively.

Figure 4—figure supplement 1
Multimeric forms of IN mutants.

SEC profiles of WT and mutant INs were analyzed by superdex 200 10/300 GL column. X-axis indicates elution volume (mL) and y-axis indicates the intensity of absorbance (mAU). Tetramers (T), Dimers (D) and Monomers (M) are indicated.

Figure 4—figure supplement 2
Higher-order IN multimerization induced by KF116 and BI224436.

DLS analysis of 200 nM IN mutants in the presence of 1 µM KF116 or BI224436 at 30 min time points. DMSO control runs are shown in gray. Representative results of three independent experiments are shown.

Figure 4—figure supplement 3
Biochemical characterization of IN mutants.

(A) Catalytic activities of mutant INs in the presence of LEDGF/p75 monitored by HTRF based assay. The bars represent the percent activity of the mutants INs relative to the WT IN. The error bars indicate the standard deviation of triplicate experiment. (B) Quantification of LEDGF/p75 binding to the mutant INs monitored by affinity pull-down of His tagged INs and tag-less LEDGF/p75 using Ni-sepharose beads. The error bars indicate the standard deviation of two independent experiments.

Figure 4—figure supplement 4
Effect of IN substitutions on the infectivity of HIV-1NL4-3.

Relative infectivity of WT and mutant viruses were tested by luciferase reporter assay in TZM-bl cells infected with 100 ng viruses. The standard deviations from three independent experiments are indicated.

Figure 5 with 1 supplement
Trans-complementation of IN tetramer interface mutants to elucidate ALLINI preferences.

(A) The salt bridge between E11 and K186 residues is highlighted in the context of IN tetramer interface. (B) DLS analysis of 200 nM E11K, K186E and E11K + K186E INs in the presence of 1 µM KF116 or BI224436 after 30 mins. DMSO controls with respective INs are shown in gray. Representative results of two independent experiments are shown.

Figure 5—figure supplement 1
Trans-complementation of inactive mutants partially restores IN functions.

(A) Catalytic activities of the mutant INs in the presence of LEDGF/p75 were monitored by HTRF based assay. The bars represent the percent activity of the mutants INs relative to the WT IN. The error bars indicate the standard deviation of triplicate experiment. (B) Quantification of LEDGF/p75 binding to the mutant INs assayed by the affinity pull-down of His tagged INs and tag-less LEDGF/p75 using Ni-sepharose beads. The error bars indicate the standard deviation of two independent experiments.

Figure 6 with 1 supplement
Structural analysis of KF116 and BI224436 interactions with IN.

(A) The top ranked model for symmetric tetramer-KF116-tetramer interactions. Each protomer is distinctly colored (green, yellow, violet, orange). Each domain is assigned to its respective protomer, as previously proposed for Maedi Visna IN (PDB: 3HPH)(Hare et al., 2009). (B) Buried surface areas (BSA) of KF116 and BI224436 induced higher-order IN multimers; (C) overlay of the crystal structures of KF116 (PDB: 4O55) and BI224436 bound to IN CCDF185H. KF116 and BI224436 are shown in magenta and cyan respectively. The arrow points to the benzimidazole group in KF116.

Figure 6—figure supplement 1
Molecular modeling of BI224436 induced higher-order IN multimerization.

The top ranked IN tetramers (A) and dimers (B) constructed with BI224436 (dark spheres).

Figure 7 with 1 supplement
Antiviral activities of ALLINIs.

(A) Antiviral activities of (-) and (+)- KF116 against WT virus. (B) Antiviral activities of KF116 and BI224436 against DTG resistant quadruple and double mutant viruses. The error is the S.D. of three independent experiments. (C) SEC analysis of mutant INs.

Figure 7—figure supplement 1
Comparative analysis of (+) and (-) enantiomers of KF116.

(A) Chemical structures and antiviral activity profiles of (+) and (-) enantiomers of KF116. (B) In vitro metabolic stabilities of KF116 and BI224436.

Figure 8 with 2 supplements
VSV-G pseudotyped fluorescent HIV-1 labeled with Vpr-IN-mNeonGreen (INmNG,a marker of viral complex) and CypA-DsRed (CA marker).

Viruses produced in the presence of 5 µM ALLINIs or raltegravir (RAL) or left untreated (DMSO) control were used to infect TZM-bl PPIA-/- cells (MOI 0.08) for 90 min. Cells were imaged on the Zeiss LSM880 using Fast-Optimal AiryScan settings, which enables sensitive detection and tracking of viral complexes with high temporal resolution and minimal photobleaching. (A) Images of CypA-DsRed labeled cores retaining INmNG signal for DMSO treated samples and loss of INmNG signal from the viral CA cores, leaving CypA-DsRed puncta for KF116 treated samples at 90 min post infection. Arrows point to single labeled CypA-DsRed complexes. (B) The sum fluorescence of INmNG within CypA-DsRed/CA puncta is plotted. (C–D) Time-lapse imaging of INmNG/CypA-DsRed labeled virus. Single particles losing the CypA-DsRed or INmNG signal were manually annotated and tracked. (C) Images and fluorescent intensity traces showing uncoating (loss of CypA-DsRed prior to INmNG loss) for single viral particle produced in the presence of DMSO. (D) Images and fluorescent intensity traces showing a gradual INmNG signal loss from a CypA-DsRed/CA puncta for viruses produced in the presence of KF116 (5 μM). Scale bar in (A, C and D) =2 µm. Statistical significance in (B) was determined by comparing respective samples with the DMSO control using a pair-wise student t-test, ***=p < 0.0001. p values > 0.5 were considered not significant (ns).

Figure 8—figure supplement 1
VSV-G pseudotyped HIV-1 viral particles fluorescently labeled with INmNG and CypA-DsRed in the presence of indicated treatments.

(A) The fraction of CypA-DsRed colocalized with INmNG signal for viruses bound on poly-lysine treated glass, or inside cells at 5 or 90 min post infection are shown. Object based colocalization of INmNG and CypA-DsRed puncta was determined. Data are representative of 3 independent experiments. Statistical significance was determined by comparing respective samples with the DMSO control using pair-wise student t-test, ***=p < 0.0001. p values > 0.5 were considered not significant (ns). (B) Representative images of single cells infected with viruses treated with the different ALLINIs, DMSO or RAL at time 0 min and 90 min post-infection. Images showing nuclei labeled with DAPI (blue), INmNG (green) and CypA-DsRed (red). Bottom most panel of images show the distribution of INmNG puncta of the same overlay images in middle panels at 90 min post infection. Scale bar = 5 µm.

Figure 8—figure supplement 2
Schematic summary of the experimental results.

(A) Schematics showing the observed productive and non-productive pathways of co-trafficking of INmNG and CypA-DsRed labeled CA cores during infection of target cells with infectious (in the absence of the inhibitor) and ALLINI-treated, non-infectious virions. (B) Schematic depictions of infectious and ALLINI-treated, non-infectious virions. In infectious virions, IN binding to viral RNA ensures that RNPs are packaged within the cone-shaped CA core. In contrast, ALLINI treatment promotes higher-order, aberrant IN multimerization which in turn impairs IN binding to viral RNA. As a result, both ALLINI induced IN aggregates and RNPs lacking IN are mislocalized outside of the protective CA core.

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain
background
(Retroviridae)
HIV-1NL4-3OtherReplication competent
HIV-1 virus particles
produced from pNL4-3
plasmid in HEK293T
producing cells;
laboratory adapted
HIV-1 strain
Strain, strain
background
(Retroviridae)
HIV-1NL-lucE-R+VSVgOtherReplication incompetent
HIV-1 virus particles
produced from pNL-luc.
E-R+and pMD.G plasmids
in HEK293T producing
cells; laboratory adapted
HIV-1 strain pseudotyped
with VSV glycoprotein
Genetic
reagent
(Retroviridae)
HIV-1NL4-3 IN (K14A)OtherReplication competent
HIV-1 virus particles
containing the mutation
K14A in the Integrase ORF
Genetic
reagent
(Retroviridae)
HIV-1NL4-3 IN
(Y15A)
OtherReplication competent
HIV-1 virus particles
containing the mutation
Y15A in the Integrase ORF
Genetic
reagent
(Retroviridae)
HIV-1NL4-3 IN
(T210 + Pro)
OtherReplication competent
HIV-1 virus particles
containing the mutation
T210 + Pro in the
Integrase ORF
Genetic
reagent
(Retroviridae)
HIV-1NL4-3 IN
(N222K)
OtherReplication competent
HIV-1 virus particles
containing the mutation
N222K in the Integrase
ORF
Genetic
reagent
(Retroviridae)
HIV-1NL-lucE-R+
N155H/K156NVSVg
OtherReplication incompetent,
VSVg pseudotyped HIV-1
virus particles containing
the following mutations
in the IN ORF: N155H/K156N
Genetic
reagent
(Retroviridae)
HIV-1NL-lucE-R+
N155H/K156N/K211R/E212TVSVg
OtherReplication incompetent,
VSVg pseudotyped HIV-1
virus particles containing
the following mutations in
the IN ORF:
N155H/K156N/K211R/
E212T
Cell line
(H.sapiens)
HeLaATCCATCC CCL-2
Cell line
(H.sapiens)
Hek293TATCCATCC CRL-3216
Cell line (H.sapiens)TZM-blNIH AIDS Reagent
Program
NIH AIDS Reagent
Program: 8129
The reagent was obtained
through the NIH AIDS
Reagent Program, Division
of AIDS, NIAID, NIH: TZM-bl
cells (Cat# 8129) from
Dr. John C Kappes,
and Dr. Xiaoyun Wu
Cell line
(H.sapiens)
TZM-bl PPIA(-/-)Francis and Melikyan, 2018
Cell line
(H.sapiens)
MT-4NIH AIDS Reagent
Program
NIH AIDS Reagent
Program: 120
The reagent was obtained
through the NIH AIDS
Reagent Program, Division
of AIDS, NIAID, NIH: MT-4
from Dr. Douglas Richman
Biological
sample (Rat)
Sprague-Dawley
rat liver microsomes
Xenotech LLCXenotech LLC: 1510115Pool of 500, male; 20 mg/mL
Biological
sample (H.sapiens)
Human Liver
Microsomes
Xenotech LLCXenotech LLC: 1610016Pool of 50, mixed
gender; 20 mg/mL
Recombinant
DNA reagent
pNL4-3 (plasmid)Adachi et al., 1986
Recombinant
DNA reagent
pNL4-3 IN
(K14A) (plasmid)
This paperSite-directed mutagenesis
in pNL4-3 to generate
IN K14A mutant
Recombinant
DNA reagent
pNL4-3 IN
(Y15A) (plasmid)
This paperSite-directed mutagenesis
in pNL4-3 to generate IN
Y15A mutant
Recombinant
DNA reagent
pNL4-3 IN
(T210 + Pro) (plasmid)
This paperSite-directed mutagenesis
in pNL4-3 to generate
IN T210 + Pro mutant
Recombinant
DNA reagent
pNL4-3 IN
(T222K) (plasmid)
This paperSite-directed mutagenesis
in pNL4-3 to generate IN
T222K mutant
Recombinant
DNA reagent
pNL-luc.E-R+
(plasmid)
Connor et al., 1995
Recombinant
DNA reagent
pNL-luc.E-R+N
155 H/K156N (plasmid)
This paperSite-directed mutagenesis
in pNL-luc.E-R+to generate
IN N155H/K156N mutant
Recombinant
DNA reagent
pNL-luc.E-R+N155
H/K156N/K211R/E212T
(plasmid)
This paperSite-directed mutagenesis
in pNL-luc.E-R+to generate
IN N155H/K156N/K211R/
E212T mutant
Recombinant
DNA reagent
pMD.G (plasmid)Naldini et al., 1996
Recombinant
DNA reagent
Vpr-INmNeon
Green (INmNG)
Francis and Melikyan, 2018
Recombinant
DNA reagent
CypA-DsRedFrancis et al., 2016;
Francis and Melikyan, 2018
Recombinant
DNA reagent
pHIVeGFP-deltaEnvFrancis and Melikyan, 2018
Recombinant
DNA reagent
Recombinant
IN (pET-15b)
Larue et al., 2012All the mutations were
carried out by site
directed mutagenesis in IN
coding region of pET-15b
Recombinant
DNA reagent
Recombinant IN
domains: NTD-CCD,
CCD and CCD-CTD
(pET-15b)
This paperWild type NL4-3 IN domains
were constructed by site
directed mutagenesis from
pET-15b truncated IN
domains containing
solubilizing mutants
(Larue et al., 2012)
Recombinant
DNA reagent
Recombinant
LEDGF (pLEDGF)
Tsiang et al., 2009
Commercial
assay or kit
p24 ELISAZeptometrixZeptometrix: 0801111
Commercial
assay or kit
MycoscopeMycoplasm
PCR detection kit
GenlantisGenlantis: MY01100
Commercial
assay or kit
CellTiter-GloPromega
Biosciences Inc
Promega
Biosciences Inc: G7571
Commercial
assay or kit
Luciferase Assay
System
Promega
Biosciences Inc
Promega
Biosciences Inc: E1500
Commercial
assay or kit
QuikChange XL site
directed mutagenesis
kit
AgilentAgilent: 200516
Commercial
assay or kit
QuikChange XL II
site directed
mutagenesis kit
AgilentAgilent: 200522
Commercial
assay or kit
LANCE Europium-
streptavidin for HTRF
assay
PerkinElmerPerkinElmer: AD0062
Commercial
assay or kit
Reporter Lysis
buffer
Promega
Biosciences Inc
Promega
Biosciences Inc: E3971
Commercial
assay or kit
X-treme Gene
HP
RocheRoche: 6366244001
Chemical
compound, drug
KF116Sharma et al., 2014
Chemical
compound, drug
BI224436MedChemExpressMedChem
Express: HY-18595
Chemical
compound, drug
nicotinamide
adenine dinucleotide
phosphate (NADPH)
Sigma-Aldrich
Chemical Company
Sigma-Aldrich:
10107824001
Chemical
compound, drug
Verapamil
Hydrochloride
Sigma-Aldrich
Chemical Company
Sigma-Aldrich:
V4629
Chemical
compound, drug
DomperidoneSigma-Aldrich
Chemical Company
Sigma-Aldrich:
D122
Chemical
compound, drug
Chlorpromazine
hydrochloride
Sigma-Aldrich
Chemical Company
Sigma-Aldrich:
C8138
Software,
algorithm
HKL 3000HKL 3000
(http://hkl-xray.com)
RRID:SCR_015023
Software,
algorithm
PhenixPhenix
(https://www.phenix-online.org/)
RRID:SCR_014224
Software,
algorithm
CootCoot
(http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot)
RRID:SCR_014222
Software,
algorithm
HADDOCKDominguez et al., 2003,
van Zundert et al., 2016
Software,
algorithm
ImageJImageJ
(https://imagej.net)
RRID:SCR_003070
Software,
algorithm
OriginLabOriginLab
software (https://www.originlab.com)
Software,
algorithm
ICY image analysis
software
ICY image analysis
software (https://icy.bioimageanalysis.org)
RRID:SCR_010587

Data availability

Diffraction data have been deposited in PDB under the accession code 6NUJ.

The following data sets were generated
  1. 1
    RCSB Protein Data Bank
    1. JJ Lindenberger
    2. M Kvaratskhelia
    (2019)
    ID 6NUJ. HIV-1 Integrase Catalytic Core Domain Complexed with Allosteric Inhibitor BI-224436.

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