Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest
- Department of Medicine, University of Cambridge, United Kingdom
- Cambridge Institute of Therapeutic Immunology and Infectious Disease (CITIID), University of Cambridge, United Kingdom
- Department of Clinical Neuroscience, University of Cambridge, United Kingdom
- Cambridge Institute for Medical Research (CIMR), University of Cambridge, United Kingdom
Figures
Figure 1 with 4 supplements
Flow cytometric screen of HIV-1 Vif point mutants.
(A) Depletion of endogenous PPP2R5D by HIV-1 Vif. CEM-T4s were transduced with lentiviruses encoding either EGFP-SBP-ΔLNGFR (Ctrl) or EGFP-P2A-Vif (Vif) at an MOI of 3, then lysed in 2% SDS and …
Figure 1—figure supplement 1
Further details for site-directed mutagenesis and flow cytometric screen.
(A) Overview of PCR and Gibson assembly-based approach to site-directed mutagenesis. PCR products are digested with DpnI (1 hr, 37°C) to degrade template. Vector is digested with XhoI and KpnI, gel …
Figure 1—figure supplement 2
Complete results of flow cytometric screen.
(A–B) Depletion of PPP2R5B (A) or APOBEC3G (B) by all Vif point mutants targeted in our library. 293Ts stably expressing HA-tagged PPP2R5B or APOBEC3G were transfected with constructs encoding …
Figure 1—figure supplement 3
Stability of selected Vif point mutants in 293Ts.
293Ts were transfected with constructs encoding EGFP-P2A-Vif, then lysed in 2% SDS and analysed by immunoblot with anti-Vif, anti-GFP (transfection control) and anti-β-actin (loading control) …
Figure 1—figure supplement 4
Depletion of APOBEC3F by selected Vif point mutants.
(A) 293Ts stably expressing HA-tagged APOBEC3F were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody and analysed by flow …
Figure 2 with 2 supplements
Depletion of endogenous APOBEC3 and PPP2R5 family proteins by HIV-1 Vif point mutants.
(A) Depletion of endogenous PPP2R5D by selected Vif point mutants. CEM-T4s were transduced with lentiviruses encoding EGFP-P2A-Vif at an MOI of 3, then lysed in 2% SDS and analysed by immunoblot …
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Figure 2—source data 1
Complete data from proteomic experiment 1 (selected Vif point mutants).
Complete dataset (unfiltered) from TMT-based quantitative proteomic experiment illustrated in Figure 2B. For each protein, normalised, unscaled protein abundances, the number of unique peptides used for protein quantitation, and the protein FDR confidence are shown.
- https://cdn.elifesciences.org/articles/53036/elife-53036-fig2-data1-v2.xlsx
Figure 2—figure supplement 1
Stability of selected Vif point mutants in CEM-T4s.
Expression of selected Vif point mutants in cells from proteomic experiment 1 (Figure 2B). For each Vif point mutant, abundance is shown as a ratio to Vif WT.
Figure 2—figure supplement 2
Calculation of t-scores and p-values.
Illustrative t-score/p-value calculations for PPP2R5A in cells transduced with EGFP-SBP-ΔLNGFR (control lentivector) or WT Vif. Graphs show protein abundance (x axis) vs probability density (y …
Figure 3 with 2 supplements
Regulation of cell cycle by HIV-1 Vif point mutants.
(A) Phosphorylation of aurora kinases in the presence of selected Vif point mutants. CEM-T4s were transduced with lentiviruses encoding EGFP-P2A-Vif at an MOI of 3, then lysed in 2% SDS and analysed …
Figure 3—figure supplement 1
Additional controls for cell cycle analysis (Vif point mutants).
(A–B) Regulation of cell cycle by selected Vif point mutants. CEM-T4s were transduced with lentiviruses encoding EGFP-P2A-Vif at an MOI of 3, then fixed in 90% methanol, stained with 7-AAD and …
Figure 3—figure supplement 2
Depletion of endogenous DPH7 and FMR1 by selected Vif point mutants.
(A) Depletion of DPH7 (left panel) and FMR1 (right panel) by selected Vif point mutants in cells from proteomic experiment 1 (Figure 2B). For each Vif point mutant, abundances of DPH7 and FMR1 are …
Figure 4 with 3 supplements
Regulation of cell cycle by depletion of PPP2R5 family subunits.
(A–B) Regulation of cell cycle by individual vs pooled PPP2R5A-E siRNA. HeLas were transfected with the indicated siRNA, then fixed in 90% methanol, stained with 7-AAD and analysed by flow cytometry …
Figure 4—figure supplement 1
Additional controls for cell cycle analysis (okadaic acid and Vif).
(A) Regulation of cell cycle by PP2A inhibition. CEM-T4s were treated with either 100 nM okadaic acid or DMSO (vehicle) for 16 hr, then fixed in 90% methanol, stained with 7-AAD and analysed by flow …
Figure 4—figure supplement 2
Additional controls for cell cycle analysis (PPP2R5A-E siRNA).
(A) Abundances of PPP2R5A-E transcripts in HeLas and CEM-T4s. Total RNA was analysed by quantitative reverse transcription PCR (qRT-PCR), and mRNA copy numbers expressed relative to Tata Binding …
Figure 4—figure supplement 3
Additional controls for cell cycle analysis (DPH7 and FMR1 siRNA).
(A–B) Regulation of cell cycle by DPH7 and FMR1 siRNA. HeLas were transfected with the indicated siRNA, then fixed in 90% methanol, stained with 7-AAD and analysed by flow cytometry after 48 hr. …
Figure 5 with 2 supplements
Analysis of naturally occurring HIV-1 Vif variants.
(A) Amino acid polymorphism amongst 2171 naturally occurring HIV-1 M group Vif variants (clade B). Sequence logos (left panel) and bar chart (right panel) highlight frequencies of amino acids …
Figure 5—figure supplement 1
Amino acid polymorphism amongst 3412 naturally occurring non-recombinant HIV-1 M group Vif variants (all clades).
Bar chart highlights frequencies of amino acids corresponding to residues 31, 33 and 128 of NL4-3 Vif.
Figure 5—figure supplement 2
Depletion of APOBEC3G by selected Vif variants.
Depletion of APOBEC3G by selected Vif variants. 293Ts stably expressing HA-tagged APOBEC3G were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with …
Figure 6 with 3 supplements
Depletion of endogenous APOBEC3 and PPP2R5 family proteins by naturally occurring HIV-1 Vif variants.
(A) Overview of proteomic experiment 2 (naturally occurring Vif variants and corresponding point mutants). CEM-T4s were transduced with lentiviruses encoding EGFP-P2A-Vif at an MOI of 3, then …
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Figure 6—source data 1
Complete data from proteomic experiment 2 (naturally occurring Vif variants and corresponding point mutants).
Complete dataset (unfiltered) from TMT-based quantitative proteomic experiment illustrated in Figure 6A. For each protein, normalised, unscaled protein abundances, the number of unique peptides used for protein quantitation, and the protein FDR confidence are shown.
- https://cdn.elifesciences.org/articles/53036/elife-53036-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Sequence of Exonic Splicing Silencer of Vpr (ESSV) in NL4-3 and YU2 Vif variants.
The ESSV (highlighted in bold) is a short nucleotide element within the HIV-1 Vif open reading frame (exon 3) required to repress splicing at HIV-1 3’ splice site A2 and allow accumulation of …
Figure 6—figure supplement 2
Regulation of cell cycle by naturally occurring Vif variants and corresponding point mutants (representative data).
CEM-T4s were transduced with lentiviruses encoding EGFP-P2A-Vif at an MOI of 3, then fixed in 90% methanol, stained with 7-AAD and analysed by flow cytometry after 48 hr. Representative data from Fig…
Figure 6—figure supplement 3
Depletion of PPP2R5B, APOBEC3G and APOBEC3F by Vif AYR.
(A–B) 293Ts stably expressing HA-tagged PPP2R5B, APOBEC3G or APOBEC3F were transfected with constructs encoding EGFP-P2A-Vif, then fixed/permeabilised, stained with AF647-conjugated anti-HA antibody …
Figure 7 with 4 supplements
Selective regulation of PPP2R5 family subunits during HIV-1 infection.
(A) Overview of proteomic experiment 3 (viral infections). CEM-T4s were infected with HIV-AFMACS viruses at an MOI of 0.5, then purified using AFMACS (Figure 7—figure supplement 1A–B) and analysed …
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Figure 7—source data 1
Complete data from proteomic experiment 3 (viral infections).
Complete dataset (unfiltered) from TMT-based quantitative proteomic experiment illustrated in Figure 7A. For each protein, normalised, unscaled protein abundances, the number of unique peptides used for protein quantitation, and the protein FDR confidence are shown.
- https://cdn.elifesciences.org/articles/53036/elife-53036-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
AFMACS-based purification of infected cells for proteomic experiment 3 (viral infections).
(A–B) HIV-infected cells from Figure 7A were stained with anti-LNGFR and anti-CD4 antibodies and analysed by flow cytometry before (input) and after (purified vs flow-through) selection using …
Figure 7—figure supplement 2
Regulation of cell cycle in HIV-infected primary human CD4+ T cells.
(A–B) Primary human CD4+ T cells were activated with CD3/CD28 Dynabeads and infected with HIV-AFMACS viruses 2 days post-activation at an MOI of 0.5. After a further 48 hr, cells were stained with …
Figure 7—figure supplement 3
Additional bioinformatics analysis.
(A) Contingency tables showing combinations of key amino acid polymorphisms amongst naturally occurring HIV-1 M group Vif variants (clade B) summarised in Figure 7D. (B) Pair-wise combinations of …
Figure 7—figure supplement 4
Additional phylogenetic trees.
(A) Phylogenetic trees of 795 HIV-1 M group viruses (clade B) with protein sequences available for all of Vif, Gag, Env and Nef (based on relatedness of Gag, Env or Nef). Viruses encoding Vif …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (human) | CEM-T4 T cells (CEM-T4s) | NIH AIDS Reagent Program | Cat. #: 117 | Also known as CEM CD4+ cells |
| Cell line (human) | THP-1 cells (THP-1s) | NIH AIDS Reagent Program | Cat. #: 9942 | Used for cDNA library generation |
| Cell line (human) | HeLa cells (HeLas) | Lehner laboratory stocks | RRID:CVCL_0030 | |
| Cell line (human) | HEK 293T cells (293Ts) | Lehner laboratory stocks | RRID:CVCL_0063 | |
| Antibody | Mouse monoclonal BV421-conjugated anti-CD4 | BioLegend | Cat. #: 317434 | Flow cytometry (1:50) |
| Antibody | Mouse monoclonal PE-conjugated anti-CD4 | BD Biosciences | Cat. #: 561843 | Flow cytometry (1:50) |
| Antibody | Mouse monoclonal AF647-conjugated anti-LNGFR | BioLegend | Cat. #: 345114 | Flow cytometry (1:50) |
| Antibody | Mouse monoclonal FITC-conjugated anti-LNGFR | BioLegend | Cat. #: 345103 | Flow cytometry (1:50) |
| Antibody | Mouse monoclonal BV421-conjugated anti CD4 | BioLegend | Cat. #: 317434 | Flow cytometry (1:50) |
| Antibody | Mouse monoclonal DyLight 650-conjugated anti-HA tag | Abcam | Cat. #: ab117515 | Flow cytometry (1:400) |
| Antibody | Rabbit monoclonal anti-PPP2R5D | Abcam | Cat. #: ab188323 | Immunoblot (1:5000) |
| Antibody | Mouse monoclonal anti-HIV-1 Vif | NIH AIDS Reagent Program | Cat. #: 6459 | Immunoblot (1:2500) |
| Antibody | Rabbit polyclonal anti-FMR1 (FMRP) | Cell Signalling Technology | Cat. #: 4317 | Immunoblot (1:1000) |
| Antibody | Rabbit polyclonal anti-DPH7 | Atlas Antibodies | Cat. #: HPA022911 | Immunoblot (1:1000) |
| Antibody | Mouse monoclonal anti-β-actin | Sigma | Cat. #: A5316 | Immunoblot (1:20000) |
| Antibody | Mouse monoclonal anti-p97 (VCP) | Abcam | Cat. # ab11433 | Immunoblot (1:10000) |
| Antibody | Rabbit polyclonal anti-total AURKB | Cell Signalling Technology | Cat. #: 3094 | Immunoblot (1:500) |
| Antibody | Rabbit monoclonal anti-phospho-AURK | Cell Signalling Technology | Cat. #: 2914 | Immunoblot (1:500) |
| Antibody | Rabbit polyclonal anti-GFP | Thermo Scientific | Cat. #: A-11122 | Immunoblot (1:2500) |
| Recombinant DNA reagent | pHRSIN-SE-P2A-SBP-ΔLNGFR-W | Matheson et al., 2014 | N/A | Used as a control and to express codon optimised Vif variants |
| Recombinant DNA reagent | pHRSIN-SE-W-pSV40-puro | van den Boomen et al., 2014 | N/A | Used as a control |
| Recombinant DNA reagent | pHRSIN-S-W-pGK-puro | Greenwood et al., 2016 | N/A | Used to express HA-tagged PPP2R5B, APOBEC3F and APOBEC3G |
| Recombinant DNA reagent | HIV-AFMACS | Naamati et al., 2019 | GenBank: MK435310.1 | pNL4-3-ΔEnv-Nef-P2A-SBP-ΔLNGFR proviral construct |
| Recombinant DNA reagent | V245 pCEP-4HA B56alpha | Addgene | Cat. #: 14532 | Standard for quantification of PPP2R5A mRNA |
| Recombinant DNA reagent | V245 pCEP-4HA B56beta | Addgene | Cat. #: 14533 | Standard for quantification of PPP2R5B mRNA |
| Recombinant DNA reagent | V245 pCEP-4HA B56gamma1 | Addgene | Cat. #: 14534 | Standard for quantification of PPP2R5C mRNA |
| Recombinant DNA reagent | V245 pCEP-4HA B56delta | Addgene | Cat. #: 14536 | Standard for quantification of PPP2R5D mRNA |
| Recombinant DNA reagent | V245 pCEP-4HA B56epsilon | Addgene | Cat. #: 14537 | Standard for quantification of PPP2R5E mRNA |
| Recombinant DNA reagent | TBP cDNA clone: IRATp970C11110D | Source Bioscience | GenBank: BC110341.1 | Standard for quantification of TBP mRNA |
| Commercial assay or kit | NEBuilder HiFi DNA Assembly Cloning Kit | NEB | Cat. #: E5520S | |
| Commercial assay or kit | Fugene 6 Transfection Reagent | Promega | Cat. #E2691 | |
| Commercial assay or kit | Lipofectamine RNAiMAX Transfection Reagent | Invitrogen | Cat. #: 18080044 | |
| Chemical compound, drug | Lenti-X Concentrator | Clontech | Cat. #: 631232 | |
| Commercial assay or kit | Dynabeads Biotin Binder | Invitrogen | Cat. #: 11047 | |
| Commercial assay or kit | Dynabeads Untouched Human CD4 T Cells kit | Invitrogen | Cat. #: 11346D | |
| Commercial assay or kit | Dynabeads Human T-Activator CD3/CD28 | Invitrogen | Cat. #: 11132D | |
| Commercial assay or kit | S-Trap micro MS Sample Preparation Kit | Protifi | Cat. #: C02-micro | |
| Commercial assay or kit | TMT10plex Isobaric Label Reagent Set | Thermo Scientific | Cat. #: 90110 | |
| Commercial assay or kit | Superscript III First-Strand Synthesis System | Invitrogen | Cat. #: 18080051 | |
| Software, algorithm | PyMOL Molecular Graphics System, Version 2.0 | Schrödinger | RRID:SCR_006054 | https://www. schrodinger.com/pymol |
| Software, algorithm | Proteome Discoverer 2.1 | Thermo Scientific | RRID:SCR_014477 | |
| Software, algorithm | R v.3.5.3 | R Development Core Team, 2019 | RRID:SCR_001905 | https://www.R-project.org/ |
| Software, algorithm | limma | Ritchie et al., 2015 | RRID:SCR_010943 | https://bioconductor.org/packages/limma/ |
| Software, algorithm | WebLogo | Crooks et al., 2004 | RRID:SCR_010236 | http://weblogo.berkeley.edu |
| Software, algorithm | seqinr | Charif and Lobry, 2007 | N/A | https://cran.r-project.org/web/packages/seqinr/ |
| Software, algorithm | ggplot2 | Wickham, 2009 | RRID:SCR_014601 | https://ggplot2.tidyverse.org |
| Software, algorithm | ggtree | Yu et al., 2018 | N/A | https://bioconductor.org/packages/release/bioc/html/ggtree.html |
| Software, algorithm | Clustal Omega | Sievers and Higgins, 2014 | RRID:SCR_001591 | https://www.ebi.ac.uk/Tools/msa/clustalo/ |
| Software, algorithm | Prism 7.0 | GraphPad | RRID:SCR_002798 |
Additional files
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Supplementary file 1
DNA and RNA sequences.
Sequences of PCR primers for Vif mutant library construction, codon-optimised Vif variants synthesised as gBlocks, Vif coding sequences in HIV-AFMACS viruses, the C-terminal 4xHA-tagged APOBEC3F coding sequence in pHRSIN-S-W-pGK puro, oligonucleotides for RNAi and primers for qRT-PCR.
- https://cdn.elifesciences.org/articles/53036/elife-53036-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/53036/elife-53036-transrepform-v2.pdf
