The F12-Vif derivative Chim3 inhibits HIV-1 replication in CD4+ T lymphocytes and CD34+-derived macrophages by blocking HIV-1 DNA integration by Simona Porcellini, Luca Alberici, Francesco Gubinelli, Rossella Lupo, Clelia Olgiati, Gian-Paolo Rizzardi, and Chiara Bovolenta Blood Volume 113(15):3443-3452 April 9, 2009 ©2009 by American Society of Hematology

The C-terminal domain of F12-Vif possesses antiviral activity. The C-terminal domain of F12-Vif possesses antiviral activity. CB-derived CD4+ T lymphocytes transduced with empty, F12-Vif-, Chim1-, or Chim2-LV were infected in triplicate cultures with either the X4 HIV-1 NL4-3 (A) or the R5 HIV-1 AD8 (B) molecular clones at an MOI of 0.1. Supernatants of the kinetic of infection were collected every 4 days, stored at −20°C, and then assessed for RT activity. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Chim3 inhibits HIV-1 replication analogously to F12-Vif. Chim3 inhibits HIV-1 replication analogously to F12-Vif. LV-transduced CB-derived CD4+ T lymphocytes were infected in triplicate cultures with either the X4 NL4-3 (A) or the R5 AD8 (B) and AD1 (C) molecular clones or the R5 laboratory-adapted BaL (D) HIV-1 strains at an MOI of 0.1. Culture supernatants were harvested every 4 days, stored at −20°C, and then assessed for RT activity. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Both F12-Vif and Chim3 protect macrophages from HIV-1 challenge. Both F12-Vif and Chim3 protect macrophages from HIV-1 challenge. CD34+-derived empty, F12-Vif–, and Chim3-transduced macrophages were infected in quintuplicate cultures with 3 different R5 HIV-1 strains, the molecular clone AD1 (A), the laboratory-adapted BaL (B), and the primary isolate TZ97001 (C) at an MOI of 0.01, and the kinetics of infection were followed for 3 to 5 weeks. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Chim3 does not counteract the antiviral action of hA3G, by acting as a true dominant-negative factor. Chim3 does not counteract the antiviral action of hA3G, by acting as a true dominant-negative factor. Kinetic of infections of CEM A3.01 cells (A), CD4+ T lymphocytes (B), and CD34+-derived macrophages (C) infected with either X4 Δvif-HIV-1 (A,B) or R5 Δvif-HIV-1 (C) at an MOI of 0.1. Values represent mean plus or minus SEM of triplicate (A,B) and quintuplicate (C) cultures. (D) Western blot analysis of cell extracts derived from HEK-293T cells transfected with a fixed amount of hA3G-HA and either WT-Vif– or Chim3-expressing plasmids at the indicated amounts of plasmid DNA. Cell extracts were prepared 48 hours after transfection. Membranes were sequentially probed with anti-HA Abs (top panel), anti-Vif (middle panel), and antiactin (bottom panel) Abs. (E) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of the corresponding plasmids in either empty or Chim3-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, intracellular p24Gag expression was evaluated by FACS analysis (FACSCalibur, BD Biosciences; and FlowJo software, TreeStar) using an anti-p24Gag Ab on fixed and then permeabilized cells. Values represent the mean plus or minus SEM percentage of the p24Gag content of each condition relative to that of wild-type HIV-1 (HIV-1 ■) (n = 6). (F) Western blot analysis of the level of the intracellular (left panel) and intravirion (right panel) hA3G and Vif proteins; 40 μg WCE and 1 μg p24Gag HIV-1 virion equivalent, respectively, were loaded in each conditions. The filter was sequentially probed with the different Abs as indicated. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Coimmunoprecipitation of WT-Vif/Chim3 heterodimers. Coimmunoprecipitation of WT-Vif/Chim3 heterodimers. A total of 500 μg cellular lysates derived from HEK-293T cells cotransfected with the tagged Vifs-expressing constructs was immunoprecipitated (IP) with the anti-HA Ab and then immunoblotted with either the anti-myc or the anti-HA Abs, as indicated. The WT-Vif and Chim3 homodimers were loaded as positive controls of the coimmunoprecipitation, and the input WCE (40 μg) as positive control of the Western blot. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Chim3 blocks HIV-1 replication regardless the presence of hA3G. Chim3 blocks HIV-1 replication regardless the presence of hA3G. Empty-, F12-Vif–, and Chim3-transduced permissive SupT1 cells were infected in triplicate cultures with the X4 NL4-3 molecular clone (A) or the Δvif HIV-1 (B) at an MOI of 0.1 and 1, respectively. Supernatants of the kinetic of infection were collected every 3 to 4 days, stored at −20°C, and then assessed for RT activity. Values represent mean plus or minus SEM of triplicate cultures. (C) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of HEK-293T cells and used at an MOI of 4 to infect either empty-LV- or Chim3-LV–transduced SupT1 cells. Seventy-two hours after viral challenge, intracellular p24Gag level was evaluated by FACS analysis using an anti-p24Gag Ab on fixed and then permeabilized cells. Values express mean plus or minus SEM percentage of the p24Gag expression of each condition (n = 5) relative to that of wild-type HIV-1 on empty transduced cells (HIV-1 ■). (D) The same viruses of panel C were used to carry out a kinetic of single round infection for the indicated time points on empty and Chim3-transduced SupT1 cells. (E) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of the corresponding molecular clones in either empty or Chim3-transduced HEK-293T cells. SupT1 cells were infected at an MOI of 4. Seventy-two hours after single round infection, intracellular p24Gag expression was evaluated by FACS analysis (FACSCalibur, BD Biosciences; and FlowJo software, TreeStar) using an anti-p24Gag Ab on fixed and then permeabilized cells. Values indicate mean plus or minus SEM percentage of p24Gag expression of each condition (n = 6) relative to that of wild-type HIV-1 (HIV-1 ■). Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology

Chim3 blocks HIV-1 DNA integration in productive infection of CEM A3 Chim3 blocks HIV-1 DNA integration in productive infection of CEM A3.01 cells, CD4+ T lymphocytes, and CD34+-derived macrophages. Chim3 blocks HIV-1 DNA integration in productive infection of CEM A3.01 cells, CD4+ T lymphocytes, and CD34+-derived macrophages. (A) Empty and Chim3-transduced CEM A3.01 cells were infected with the X4 HIV-1 NL4-3 molecular clone. (B) Empty and Chim3-transduced CD4+ T lymphocytes were infected with the R5 AD8 molecular clone. (C) Empty and Chim3-transduced CD34+-derived macrophages were infected with the laboratory-adapted R5 HIV-1 BaL. All cells were infected at an MOI of 0.1 for 7 days. Total DNA was extracted at the indicated time points, and the copy number of HIV-1 DNA was analyzed by quantitative real-time PCR. Values are representative of 1 of 3 independent experiments. Simona Porcellini et al. Blood 2009;113:3443-3452 ©2009 by American Society of Hematology