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Blood, Vol. 113, Issue 15, 3443-3452, April 9, 2009
The F12-Vif derivative Chim3 inhibits HIV-1 replication in CD4+ T lymphocytes and CD34+-derived macrophages by blocking HIV-1 DNA integration
Blood Porcellini et al.
113: 3443
Supplemental materials for: Porcellini et al
Plasmids
Chim1 and Chim2 pBluescript plasmids were kindly provided by Dr. M. Federico (Laboratory of Virology, ISS, Roma, Italy). Chim3 gene was generated by DNA synthesis (Primm s.r.l, Milano, Italy). The lentiviral vectors (LVs) Chim1PΔN, Chim2PΔN and Chim3PΔN, carrying chimeric WT-vif/F12-vif genes, were generated from the empty PΔN LV1 (Fig. S1A and B). The three chimeras were cloned into the ClaI site of the PΔN by PCR amplification (Fig. S1C). The F12-VifPΔN and the WT-VifPΔN were previously de-scribed.1 The pCEM15:HA/pCMV42 plasmid expressing HA-tagged human APOBEC3G (hA3G-HA) was a kind gift from Dr. M. Malim (King’s College, London, UK), whereas the pCEM15/pCMV4 plasmid, expressing hA3G was generated by re-moving the 110-bp HA sequence by XbaI and XmaI digestion. We con-structed Chim3- and WT-Vif-LVs expressing at the C-terminus either the HA or the 3-Myc tag. The WT-Vif-HAPΔN and Chim3-HAPΔN were obtained by substituting the hA3G gene with Vif genes from the pCEM15:HA/pCMV4 plasmid. The tagged genes were removed from the pCEM15:HA/pCMV4 plasmid and then cloned into the ClaI site of the PΔN. The WT-Vif-3MycPΔN and Chim3-3MycPΔN were constructed by am-plifying the 3-Myc sequence from the pcDNA3.1-Sfrp1-3Myc plasmid,3 a kind gift from Dr. P. Bovolenta (Cajal Institute, CSIC, Madrid, Spain) and then inserting the tag into the XcmI and EcoRI sites of the Chim3-PΔN and WT-Vif-PΔN. The pR9ΔenvHIV-1 and the Δvif-HIV-1 molecu-lar clones were a gift from Drs. D. Trono (Ecole Polytechnique Fédérale de Lausanne, Lausanne-Dorigny, Switzerland) and M. Federico (ISS, Rome, Italy), respectively. The molecular clone R9Δenv-Δvif-HIV-1 was obtained by substituting the 4.27-kb SpeI-SalI fragment of the R9Δenv HIV-1 with the corresponding sequence of the Δvif-HIV-1. The lack of expression of both Env and Vif proteins was verified by Western blot analysis (Fig. S2). Production of LVs and cell transduction by LVs
VSV-G pseudotyped LV stock production and cell transduc-tion were performed as previously described.1 Ten days after spinoculation, percent of transduction was evaluated by FACS analysis using the anti–NGFR-PE Ab (C40-1457, BD Pharmingen™). Transduction efficiency was >90% for HEK-293T and SupT1 cells, 80% for CEM A3.01 and CD4+ T lymphocytes. To transduce HSCs, concentrated LV stocks were used at an MOI of 100 in the presence of polybrene (4 µg/ml). A mean±SEM value of 59%±5.5 (n=32) transduction efficiency was obtained. All transduced cells were immune-selected with the anti-human p75-NGFR monoclonal antibody 20.4 (ATCC, Rockville, MD, USA) as previously described.1 In vi-tro differentiation of cord blood-derived CD34+ HSCs to macro-phages
CB-derived CD34+ cells were differentiated in vitro into macrophages following a liquid-culture differentiation protocol adapted from the semisolid methylcellulose MethoCult® culture medium-based protocol previously de-scribed by Anderson et al.4 Briefly, CB-derived CD34+ cells were seeded at 1 × 106 cells/ml in medium A containing complete Iscove’s Modi-fied Dulbecco's Medium (IMDM) supplemented with the BIT 9,500 serum substitute BSA 10 mg/ml, recombinant human (rh) insulin 10 µg/ml, iron-saturated human transferrin 200 µg/ml, (StemCell Technology, Vancouver, Canada) and the following growth factors and cytokines: human stem cell factor (h-SCF) 100 ng/ml (R&D Systems, Minneapolis, MN, USA), h-Flt3L 100 ng/ml (Peprotech, Rocky Hill, NJ, USA), h-IL-6 20ng/ml (R&D Systems) and hu-man thrombopoietin (h-Tpo) 20 ng/ml (Peprotech). In the experimental set-up, after 2–3 days in medium A, cells were cultivated in medium B containing complete IMDM supplemented with FCS 10%, h-SCF 100 ng/ml, h-IL-3 20 ng/ml and rh-GM-CSF 20 ng/ml (Immunotools). Follow-ing 7–10 days of culture in medium B, cells were plated at 1 × 106 cells/ml in medium C containing complete DMEM, FCS 10%, rh-M-CSF 20 ng/ml (Immunotools), and rh-GM-CSF 20 ng/ml. After 8–10 days of further culture, during which fresh medium was replaced every 2–3 days, fully differentiated macrophages were obtained. In vitro differen-tiation of PBMC-derived monocytes to macrophages
Human peripheral blood mononucleated cells (PBMC) were isolated from buffy coats of normal healthy donors by cen-trifugation on a Ficoll-Hypaque gradient and then seeded at the concentration of 1 × 106 cells/ml in 15-cm plates in complete RPMI supplemented with 10% FCS and PSG. One × 106 PBMC/ml were incubated in complete RPMI supplemented with 10% FCS in 15-cm plates. After 2 hours non-adherent cells (mostly lymphocytes) were dis-carded, whereas adherent cells (mostly monocytes) were gently detached from the plate and then seeded in 6-well plates at the concentration of 1 × 106 cells/ml in complete RPMI supplemented with 10% FCS and 10% human serum. After 2 days, medium was changed and mature macrophages were obtained in further 4–7 days. Western blot analy-sis
The lack of Env and Vif expression in the R9ΔenvΔvif HIV-1 molecular clone was confirmed by Western blot analysis using whole cell extracts obtained from 293T cells transfected with the described mo-lecular clones and the following specific antibodies. The HIV-1 gp120 MoAb (ID6) and the HIV-1 gp41 Hybridoma (Chessie 8) were obtained through the AIDS Research and Reference Reagent Program (Division of AIDS, NIAID, NIH, Bethesda, MD, USA) from Dr. K. Ugen and Dr. D. Weiner5 and Dr. G. Lewis,6 respectively, and both used at 1:1,000 di-lution. In the experiment shown in Fig. S3C, CEM A3.01 cells transduced with either WT-Vif-LV or Chim3-LV were stimulated for 18 hours or left untreated with the proteasome in-hibitor MG132 (Calbiochem®, San Diego, California, USA) at the concentra-tion of 12.5 µM. Vif and actin expression was sequentially tested in the same nitrocellulose filter. Phenotypic characterization of monocytes and macrophages derived from buffy coats and CD34+ HSCs.
Phenotypic characterization of monocytes and macrophages was performed by FACS analysis using the following Abs against specific surface markers: CD34-PE or CD34-FITC, MIP1β-Biot + SA-FITC (R&D Systems) to detect CCR5, CD4-PE, CD14-PE, CD16-PE, HLA-DR-FITC, CD13-FITC, CD3-PE or CD3-FITC Abs (BD Pharmingen™). REFERENCES
1. Vallanti G, Lupo R, Federico M, Mavilio F, Bovolenta C. T Lymphocytes Transduced with a Lentiviral Vector Expressing F12-vif Are Protected from HIV-1 Infection in an APOBEC3G-Independent Manner. Mol Ther. 2005;12:697–706.
2. Sheehy AM, Gaddis NC, Choi JD, Malim MH. Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein. Nature. 2002;418:646–650.
3. Lopez-Rios J, Esteve P, Ruiz JM, Bovolenta P. The Netrin-related domain of Sfrp1 interacts with Wnt ligands and antagonizes their activity in the anterior neural plate. Neural Develop. 2008;3:19.
4. Anderson JS, Bandi S, Kaufman DS, Akkina R. Derivation of normal macrophages from human embryonic stem (hES) cells for applications in HIV gene therapy. Retrovirology. 2006;3:24.
5. Dickey C, Ziegner U, Agadjanyan MG, et al. Murine monoclonal antibodies biologically active against the amino region of HIV-1 gp120: isolation and characterization. DNA Cell Biol. 2000;19:243–252.
6. Aba-cioglu YH, Fouts TR, Laman JD, et al. Epitope mapping and topology of baculovirus-expressed HIV-1 gp160 determined with a panel of murine monoclonal antibodies. AIDS Res Hum Ret-roviruses. 1994;10:371–381.
Files in this Data Supplement:
- Figure S1. Schematic illustration of Vif protein sequences and LVs expressing Vifs (JPG, 111 KB)
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(A) Alignment of amino acid sequences of the WT-Vif derived from the pNL4-3 molecular clone (accession No. M19921), F12-Vif (accession No. Z11530) and the three chimera deriva-tives (Chim1–3). The unique amino acid substitutions for F12-Vif are in bold underlined. (B) Schematic structure of the F12-Vif and Chim1–3 proteins. Black dots indicate the unique amino acid substitutions of F12-Vif. (C) Schematic representation of the provirus configuration of the HIV-1-based PΔN and Chim1-3PΔN LVs. Abbreviations denote wild-type HIV-1 long terminal repeat (LTR), packaging signal (ψ), splice donor and acceptor sites (SD and SA), rev responsive element (RRE).
- Figure S2. Lack of Env and Vif protein expression in R9ΔenvΔvif HIV-1 molecular clone (JPG, 30.7 KB)
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Western blot analysis of HIV-1 RT–activity-equivalent virions produced from HEK 293T cells transiently transfected with the indicated plasmids. The filter was sequentially probed with a mixture of anti-gp120 and anti-gp41 Abs (upper panel), an anti-Vif Ab (middle panel) and finally an anti–HIV-1 Ab (lower panel).
- Figure S3. Analysis of Vif proteins expression on transduced cells (JPG, 54.5 KB)
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(A) Whole cell extracts obtained from CEM A3.01 cells either mock- or Vif-LV transduced were analyzed for the basal expression of Vif protein by Western blot analysis using an anti-Vif Ab. Filter was stripped and reprobed with anti-actin Ab to normalize the amount of proteins loaded into the SDS-PAGE. (B) Comparison of basal expression of Chim3 and WT-Vif in CEM A3.01 cells, CD4+ T lymphocytes, and CD34+-derived macrophages by Western blot following the same conditions of panel A. (C) Western blot assay of cell extracts derived from WT-Vif- and Chim3-transduced CEM A3.01 cells treated for 18 hours with the proteasome inhibitor MG132. The filter was probed sequentially with anti-Vif and anti-actin Abs. The fold of increment of Vif protein accumulation following MG132 treatment was calcu-lated by measuring the intensity of Vif bands normalized with the intensity of actin bands. Densi-tometric examination revealed that Chim3 is accumulated 3.5-fold more than WT-Vif after MG132 treatment, indicating that Chim3 is degraded into the proteasome faster and/or more effi-ciently than WT-Vif. (D) Western blot assay of cell extracts derived from Chim3-transduced CEM A3.01 and SupT1 cells either left uninfected or infected with R9Δenv-Δvif-HIV-1 at an MOI of 4. WCEs were prepared 4 days after infection. The filter was probed sequentially with anti-Vif and anti-actin Abs. The fold of increment of Vif protein accumulation following HIV-1 infection was calculated by measuring the intensity of Vif bands normalized with the intensity of actin bands.
- Figure S4. Immune phenotypic and functional features of CD34+-derived macrophages (JPG, 141 KB)
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(A) Expression of cell surface molecules of macrophages obtained from either the CB-derived CD34+ HSCs (left panels) or BC-derived mononuclear cells (right panels) using specific antibodies directly conjugated with FITC or PE. Samples were processed in a FACS Calibur instrument and results were analyzed by the FlowJo software. Values on the right upper corner represent mean fluorescence intensity (mfi). (B) Kinetic of infection of CD34+-derived macrophages infected with the lab-adapted R5 HIV-1 BaL strain and with the X4 NL4-3 molecular clone at the indicated MOI. Supernatants of infected cells were collected every 3 days, stored at −20°C, and then assessed for RT activity. Values represent mean±SEM of quintuplicate cultures. As expected, in contrast to X4 NL4-3, R5 BaL HIV-1 strain was productively infectious at all MOI tested, reaching a peak of HIV-1 production be-tween 9 and 15 days following viral challenge depending on the MOI used.
- Figure S5. F12-Vif expressed in producer cells does not reduce HIV-1 infectiv-ity/integration of both WT-HIV-1 and Δvif-HIV-1 (JPG, 46.9 KB)
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(A) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of the corresponding plasmids in either empty- or F12-Vif–transduced HEK-293T cells, in the presence of hA3G expression plasmid. SupT1 cells were infected at an MOI of 4. Seventy-two hours after viral challenge, in-tracellular p24Gag expression was evaluated by FACS analysis (FACSCalibur, BD Bioscience and FlowJo software, Tree Star) using an anti-p24Gag Ab on fixed and then permeabilized cells. Values represent the mean±SEM percent of p24Gag content of each condition relative to that of wild type HIV-1 (HIV-1 black bar) (n=6). (B) Western blot analysis of the level of the in-tracellular (left panel) and intravirion (right panel) hA3G and Vif proteins. Forty µg of WCE and 1 µg of p24Gag HIV-1 virion equivalent were loaded in each condi-tions.
- Figure S6. Infectivity /Integration assay normalized by p24Gag determination (JPG, 38.2 KB)
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(A) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of HEK-293T cells. One hun-dred ng p24Gag/106 cells were used to infect either empty-LV or Chim3-LV trans-duced SupT1 cells. Seventy-two hours after viral challenge, intracellular p24Gag level was evaluated by FACS analysis using an anti-p24Gag Ab on fixed and permeabilized cells. Values express mean±SEM percent of the p24Gag expression of each condition (n=5) relative to that of wild type HIV-1 on empty-transduced cells (HIV-1 black bar). (B) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of HEK-293T cells. Either 100 ng p24Gag/106 cells or an MOI of 4, as indicated, were used to infect mock-transduced SupT1 cells. Seventy-two hours af-ter viral challenge, intracellular p24Gag level was evaluated by FACS analysis using an anti-p24Gag Ab. Values express percent of the p24Gag expression relative to that of wild type HIV-1 (black bars).
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