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1 Supplementary Information Concerted action of cellular JNK and Pin1 restricts HIV1 genome integration to activated CD4 T lymphocytes Lara Manganaro 1, Marina Lusic 1,#, Maria Ines Gutierrez 1, Anna Cereseto 2, Giannino Del Sal 3 and Mauro Giacca 1 1 Molecular Medicine Laoratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Area Science Park, Padriciano 99, Trieste, Italy 2 Molecular Biology Laoratory, Scuola Normale Superiore, 561 Pisa, Italy 3 Laoratorio Nazionale CIB (LNCIB), Area Science Park, Padriciano 99, Trieste and Dipartimento di Scienze della Vita, Universita' degli Studi di Trieste, Via Giorgieri 1, Trieste, Italy Nature Medicine: doi:1.138/nm.212
2 1 Luciferase activity (% of 48hinduced) Time after PHA/IL2 (h): Suppl. Fig. 1. Primary CD4 T cells are efficiently infected with HIV1 only after prolonged stimulation. Primary CD4 T cells were purified from peripheral lood of three normal donors and either left untreated (>95% resting cells; not shown) or stimulated with PHA/IL2 for 1, 18 or 48 hours prior to infection with VSVGNL4Luc. Luciferase activity of the sample stimulated for 48 hours prior to infection was set as 1% (mean±sem of at least three different experiments). Nature Medicine: doi:1.138/nm.212
3 Numer of copies (% of PHA/IL2 at 7 h) c Numer of copies (% of PHA/IL2 at 7 h) Numer of copies (% of PHA/IL2 at 24 h) Early reverse transcripts Time after infection (h) Late reverse transcripts Time after infection (h) LTR circles Time after infection (h) Not induced PHA/IL2 PHA/IL PHA/IL2SP6125 PHA/IL2AZT Not induced PHA/IL2 PHA/IL PHA/IL2SP6125 PHA/IL2AZT Not induced PHA/IL2 PHA/IL PHA/IL2SP6125 PHA/IL2AZT d e f Integrated HIV DNA (% of activated) VSVGNL4Luc PHA/IL2 SP6125 Integrated HIV DNA (% of infectedactivated) Integrated HIV DNA (% of 48 hinduced) HIV1 BRU PHA/IL2 SP6125 (µm) WB: antijnk WB: antituulin p52 p46 Tuulin 4 Suppl. Fig. 2. JNK activity is required for efficient HIV1 cdna integration in primary lymphocytes. (a, ) JNK inhiition does not impair reverse transcription. PHA/IL2 stimulated PBLs were infected with HIV1BRU with or without treatment with SP6125 or in the presence of AZT. The levels of early (a) and late () reverse transcripts were determined at different time points. Values are mean±sem of infection of three different donors after normalization for the amount of total genomic DNA. (c) JNK inhiition reduces 2LTR circle formation. Time course analysis of 2LTR circles in samples infected with HIV1 BRU with or without treatment with SP6125 or in the presence of AZT (mean±sem of infection of three different donors). (d) JNK inhiition locks integration of pseudotyped HIV1NL4luciferase. PBLs were stimulated or not with PHA/IL2 and treated with or with SP6125 or left untreated for 15 h prior to infection with VSVGNL4Luc; the relative levels of integrated DNA were measured 24 h post infection y real time AluPCR. Each sample was normalized for the amount of total genomic DNA using the Lamin B2 B13 primers and proe (mean±sem of at least three different experiments). (e) JNK inhiition locks HIV1 integration in a dose dependent manner. Primary human CD4 T cells, stimulated with PHA/IL2 as aove, were treated with the indicated concentrations of JNK inhiitor SP6125 for 15 h prior to infection with HIV1BRU. The levels of integrated HIV1 DNA were assayed y real time AluPCR. Each sample was normalized for the amount of total genomic DNA (mean±sem of at least three different experiments). (f) Parallel kinetics of increase of the amount of integrated HIV1 and endogenous JNK levels. CD4 Tlymphocytes were infected with HIV1 BRU either in resting conditions or at different times (1, 18 and 48 h) after activation with PHA/IL2, followed y the measurement of the levels of integrated viral DNA y real time AluPCR after additional 24 h postinfection. Efficient viral integration required 48 h of PHAIL2 stimulation, while it was highly ineffective (>1fold less) upon shorter stimulation. Lysates from the same cells were proed y western lotting with antijnk or antituulin antiodies (lower panel). Of notice, the levels of JNK were arely appreciale in unstimulated cells or in cells stimulated for 1 or 18 h, while the protein was clearly detectale only at 48 h, the same time at which HIV1 integration occurred efficiently. Nature Medicine: doi:1.138/nm.212
4 Pep 49 AMC 65 PPep 49 AMC 65 Peptide (ng) PPep Pep P WB: antip total IgG d Phosphorylated/Total (% of control) PD9859 SP6125 SB2358 c PPep Pep HIV1 BRU λppase WB: antip WB: antiflag IP: antiflag P WB: antip affinitypurified Ratio P: λppase λppase e Total levels of (% of control) PD9859 SP6125 SB2358 Suppl. Fig. 3. A novel antiphospho antiody detects phosphorylated HIV1 in vivo phosphorylation is impaired upon treatment with the JNK inhiitor SP6125. (a) Amino acid sequences of the and phospho peptides used for immunization of raits. Serine 57 is the site of JNK phosphorylation. () IgG polyclonal and affinitypurifed antiphospho antiody (antip) recognizes the phospho peptide. Serial dilutions of phosphos57 and S57, lotted on a Protran BA79 memrane, were proed with anti phospho antiserum purified for IgGs (upper memrane). After an additional purification y affinity chromatography, the phosphorylated peptide was exclusively recognized y the antip antiody (lower memrane). (c) Phosphorylated can e detected in the context of viral infection. 15x16 SupT1 cells were infected with wt HIV1 BRU Flag virus (7.6 µg p24) and the cell lysates were immunoprecipitated with an antiflag antiody, followed y visualization with the same antiody or with our affinitypurified anti P antiody. Prior to western lotting, the precipitate was incuated for 1 hour with PPase at 3 C. Results were quantified densitometrically and presented in the graph on the right. (d) Levels of phosphorylation. The graph shows the percentage of phosphorylated protein in transfected cells after cell treatment with the indicated MAPK inhiitors dissolved in. Values are expressed as a percentage of phosphorylated in cells treated with control, after standardization for the total levels shown in (e); (mean±sd of at least three different experiments). (e) Total levels of. The histogram shows the percentage of total protein in cells treated with MAPK inhiitors as compared to control, treated cells (mean±sd of at least three different experiments). Nature Medicine: doi:1.138/nm.212
5 WB: antijnk WB: antiflag FlagLuc Flag p52 p46 Input (1/5) GST GSTNterm GSTCore GSTCterm GST Luc 35 SJNK IP: antiflag WLC Suppl. Fig. 4. HIV1 inds cellular JNK protein in vitro and in vivo. (a) inds endogenous JNK in vivo. Let panel: extracts from HEK 293T cells transfected with Flag or FlagLuciferase, as a control, were immunoprecipitated with antiflag antiody and the immunoprecipitate was analysed for the presence of endogenous JNK with an antijnk1 antiody; the levels of Flagtagged, immunoprecipitated proteins were controlled y immunolotting with an antiflag antiody. Right panel: western lotting using antijnk and antiflag antiodies, as indicated, on whole cell lysates (WLC) efore ummunoprecipitation, to verify the levels of protein expression. () The core domain is required for interaction with JNK1. In vitro translated, radiolaeled JNK1 p46 was incuated with GST fusion proteins corresponding to full length or to domains 15 (Nterminus), (core) and (Cterminus); ound was resolved y gel electrophoresis. The autoradiograpy strip in the middle shows the levels of recovered at the end of the GST pulldown procedure in a representative experiment; for the same experiment, the Coomassie luestained gel on top shows recominant proteins efore exposure. The histogram at the ottom shows the levels of ound (mean±sd) in three independent experiments, expressed as JNK ound (% of input) Nature Medicine: doi:1.138/nm.212
6 Flag Flag(S57A) FlagLuciferase WB: antip3 p3 c (K3R) (S57A) WB: antiflag Luciferase IgH WB: antip IP: antiflag P IP: antiflag WB: antiflag WB: antip3 p3 WCL (K3R) (S57A) p3 WCL d His pmol His(S57A) WB: antiac Ac P IP: antiflag WB: antiflag WCL S Suppl. Fig. 5. HIV1 S57 phosphorylation is independent from Nterminus acetylation and does not affect enzymatic activity. (a) The (S57A) mutant inds endogenous p3 in vivo similar to wt. Extracts from HEK 293T cells transfected with Flag, Flag(S57A) or Flagluciferase, as indicated, were immunoprecipitated with an antiflag antiody and immunolotted with antip3 (top panel) or antiflag (middle panel) antiodies. Whole cell lysates (WCL) was immunolotted with either antip3 or antiflag antiody (ottom panel) to visualize the levels of endogenous p3. () (S57A) is acetylated on lysines at positions 264, 266 and 273 similar to wt (ref. 8). Lysates of HEK 293T cells transfected with Flag, Flag(K3R) in which lysines 264, 266 and 273 are mutated to arginines, or Flag(S57A), with or without p3, were immunoprecipitated with antiflag antiody and immunolotted with an antiacetylated antiody (antiac). The same lysates were immunolotted with the antiflag antiody to verify protein expression levels (ottom gel). (c) mutant defective for acetylation can still e phosphorylated. Extracts of HEK293T cells transfected with Flag, Flag (K3R) or Flag(S57A) were sujected to immunoprecipitation with antiflag antiody and immunolotting with antip. Protein expression levels were verified y westernlot on total cell lysates with antiflag antiody. (d) enzymatic activity is not affected y the S57A mutation. In vitro strand transfer activity of scalar amounts of wt His or His(S57A) (.2, 1 and 5 pmol). In the first lane, the sustrate without His was incuated with the strand transfer uffer alone as a control. S: DNA sustrate; P: catalytic products. Nature Medicine: doi:1.138/nm.212
7 HIV1 BRU PHA/IL2 IgH IgL WB: antiflag IP: antiflag Suppl. Fig. 6. Detection of upon HIV1 infection of activated ut not resting peripheral lood lymphocytes. 15x1 6 primary PBLs, either stimulated or not with PHA/IL2, were infected with wt HIV1BRUFlag virus (7.6 µg p24). Five hours postinfection, was immunoprecipiated and its total levels were determined y immunolotting with an antiflag antiody. Nature Medicine: doi:1.138/nm.212
8 Reverse transcripts (% of infectednot treated) HIV1 BRU Pi Early RT Late RT Suppl. Fig. 7. Levels of early and late reverse transcripts are not altered y Pin1 inhiition or upon infection with the HIV1 BRU (S57A) virus. (a) Pin1 inhiition does not affect reverse transcription. SupT1 cells were infected with HIV1 BRU after treatment with Pin1 inhiitor dissolved in and analyzed for the levels of early and late reverse transcripts (mean±sem of at least three indipendent experiments). () Mutation of Ser 57 does not impair virion production. Westernlot analysis of HIV1BRU and HIV1 BRU (S57A) viral stocks with antiflag (upper panel) or antip17 (matrix) (lower panel) antiodies is shown. (c) Serine 57 mutation does not affect reverse transcription. SupT1 cells were infected with HIV1BRU or HIV1 BRU (S57A). mutant and analyzed for the levels of early and late reverse transcripts (mean±sem of at least three independent experiments). c 12 HIV1 BRU HIV1 BRU (S57A) WB: antiflag WB: antip17 Virion lysate p17 Reverse transcripts (% of wt HIV1infected) Early RT Late RT HIV1 BRU HIV1 BRU (S57A) Nature Medicine: doi:1.138/nm.212
9 HGQVDCGPGIWQLD HGQVNSDLGTWQMD HGQVDCSPGIWQVD HGQVDASPGVWQMD HGQVNAELGTWQMD HGQVNAELGTWQMD AGCVMRSPNHWQAD HIV1 Sutype A HIV1 Sutype B HIV1 Sutype C HIV1 Sutype D HIV1 Sutype F HIV1 Sutype G HIV1 Sutype H HIV1 Sutype K HIV1 Sutype 1 HIV1 Sutype 2 HIV2 SIV(CPZ.CD.9.ANT) SIV(VER.DE.x.AGM3) SIV(MAC.US.x.251_1A11) SIV(SMM.US.x.PGM3) EIAV GGQLKIGPGIWQMD 2. FIV 3. Luciferase activity (normalized) Luciferase activity (normalized) VSVGpNL4Luc SP6125 VSVGpRodLuc SP6125 Nature Medicine: doi:1.138/nm.212 Suppl. Fig. 8. Conservation of the SP motif and effects of JNK inhiition on HIV2 infection. (a) Conservation of the serineproline sequence (position 5758 in HIV1, in red) in the proteins of different HIV1 sutypes and other Lentiviruses. () HIV2 is insensitive to JNK inhiition. HOS CCR5 cells treated with SP6125 dissolved in were infected with VSVGNL4Luciferase virus or with VSVGRODLuciferase. Luciferase activity was assessed 24 hours after infection and normalized for the levels of ATP present in the cells and the amount of virus used for infection. (mean±sd of at least three independent experiments).
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Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
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IP: Flag a Mascot PTM Modified Mass Error Position Gene Names Score Score Sequence m/z [ppm] 81 MAT2A;AMS2;MATA2 35.6 137.28 _AAVDYQK(ac)VVR_ 595.83-2.28 b Pre-immu After-immu Flag- WT K81R WT K81R / Flag
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glpx Rv1100 Probe 4.4 kb 75 kda 50 kda 37 kda 25 kda 20 kda
PvuI 2.0 k PvuI PvuI Rv1101c Rv1100 glpx fum 4.4 k PvuI PvuI ΔglpX Rv1101c Rv1100 HygR fum ΔglpX 5 k 4 k 3 k c 75 kda 50 kda 37 kda GLPX Δ Δ/C GLPX 2 k 1.5 k 25 kda 20 kda PRCB 1 k 0.5 k Supplementary
Ctrl CCT7 CCT2 GAPDH * * ** ** * Ctrl Size of LC3 dots (a.u.) mrfp GFP mrfp-gfp-lc3 CCT5 KD CCT7 KD
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p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
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Time after injection (hours) ns ns
Platelet life span (% iotinylated platelets) 1 8 6 4 2 4 24 48 72 96 Time after injection (hours) 6 4 2 IgG GPIα GPIβ GPII GPVI Receptor expression (GeoMean, fluorescence inteity) Supplementary figure
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