Volume 12, Issue 6, Pages 1185-1196 (December 2005) Cell Death Induced by the Herpes Simplex Virus-1 Thymidine Kinase Delivered by Human Immunodeficiency Virus-1-Based Virus-like Particles  Silvia Peretti, Ilaria Schiavoni, Katherina Pugliese, Maurizio Federico  Molecular Therapy  Volume 12, Issue 6, Pages 1185-1196 (December 2005) DOI: 10.1016/j.ymthe.2005.06.474 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Nef7 is strongly and specifically incorporated in both lenti- and retroviral particles. (A) Western blot analysis of the expression and virion incorporation of Nef7 expressed either in cis (top) or in trans (bottom). 293T cells were transfected with HIV-1 NL4-3 molecular clones expressing either wt Nef or the Nef7 mutant or, as control, with the isoform defective for nef expression (Δnef). Alternatively, cells were transfected with the Δnef NL4-3 alone or with vectors expressing either wt Nef or Nef7. In both cases, nontransfected cells (Ctrl) were used as control. Thirty micrograms of lysates of transfected cells was analyzed for the expression of Nef and actin. At the same time, 20 ng of HIV-1 particles was analyzed for the relative amounts of Nef- and CAp24-related products. (B) Western blot analysis of purified Nef7 HIV-1 particles. Twenty nanograms of purified viral preparations from 293T cells transfected with Δnef NL4-3 molecular clone either alone or together with vectors expressing wt Nef or Nef7 was analyzed by Western blot for the presence of both Nef- and CAp24-related products. (C) Western blot analysis of Nef7-incorporating MLV particles. Thirty micrograms of cell lysates from 293T cells cotransfected with a MLV molecular clone together with wt Nef- or Nef7-expressing vectors was analyzed for the expression of both Nef and actin. Nontransfected cells were used as control (Ctrl). Meanwhile, amounts equivalent to 5 × 106 cpm of MLV particles were analyzed for Nef incorporation. (D) Quantitation of Nef7 virion incorporation. Two-fold serial dilutions of purified NL4-3/Nef7 viral particles were analyzed by Western blot for the presence of Nef7 together with serial dilutions of rNef (top). As control, HIV-1 particles were also analyzed for the CAp24 contents (bottom). Data are representative of (A) 16, (B) 2, (C) 3, and (D) 2 independent experiments. For all blots, the migration of major viral products is indicated on the left side, whereas the molecular marker sizes are reported on the right. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 Wt Nef and Nef7 have similar half-lives. Pulse–chase analysis of 293T cells either mock transfected (Ctrl) or transfected with wt Nef or Nef7. Cells were sampled at the end of the pulse (time 0) and 3, 6, and 9 h after the chase and processed by anti-Nef immunoprecipitation. On the left side, the migration of Nef products is indicated. On the right side, the molecular marker weights are reported. The results are representative of two independent experiments. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 Nef7 accumulates in rafts at high levels. Analysis of fractions recovered from discontinuous sucrose gradients carried out on lysates of 293T cells transfected with NL4-3 or NL4-3/Nef7 HIV-1 molecular clones, (A) with Δnef NL4-3 together with wt Nef or Nef7 expressing vectors or (B) with wt Nef or Nef7 alone. Thirty micrograms of total proteins was analyzed for the Nef expression (top), and the remainder of the cell lysates was used for the raft isolation. Fractions were analyzed by Western blot for the presence of Nef and CD71 and by dot blot for the GM1 contents. Results from the analysis of relevant fractions are reported. For the Western blots, the molecular marker sizes are reported on the right. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Similar amounts of CD8/wtNef and CD8/Nef7 fusion products are incorporated into virions. Western blot analysis of the virion incorporation of CD8/wtNef and CD8/Nef7. Both Nef-related products and CAp24 were revealed by Western blot performed on 500 ng of HIV-1 obtained from 293T cells cotransfected with Δnef NL4-3 HIV-1 molecular clone either alone or together with vectors expressing either CD8/wtNef or CD8/Nef7. As control, cells were also transfected with the parental NL4-3 molecular clone. On the left side, the migration of viral products is indicated. On the right side, the molecular marker weights are reported. The results are representative of four independent experiments. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Nef7-based fusion products are incorporated at high efficiency in HIV-1 VLPs. Western blot analysis of the Nef incorporation in VSV-G-pseudotyped HIV-1-based VLPs carrying (A) wt Nef or Nef7/GFP and (B) wt Nef or Nef7/TK. Twenty nanograms of each VLP preparation was analyzed by Western blot for the presence of Nef- and CAp24-related products. (C) Western blot analysis of the Nef incorporation in HIV-1-based VLPs carrying wt Nef/GFP, Nef7/GFP, or GFP/Vpr. Two-fold serial dilutions of each viral preparation were analyzed by Western blot for the presence of both GFP-related products (top) and CAp24 (bottom). Data are representative of (A) 2, (B) 6, and (C) 2 independent experiments. For all blots, the migration of major viral products is indicated on the left side, whereas the molecular marker sizes are reported on the right. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 6 Nef7/GFP is efficiently delivered in cells by VLPs. (A) Effective removal of adsorbed VLPs by trypsin treatment. FACS analysis of CEMss cells challenged for 2 h at 4°C with Nef7/GFP VLPs and treated or not with trypsin for 10 min at 4°C. (B) Delivery of Nef7/GFP molecules upon VLP challenge. FACS analysis of CEMss cells challenged with Nef7/GFP VLPs expressing or not the VSV-G receptor. After 3, 6, and 10 h, cells were harvested, treated with trypsin, and analyzed. In each graph, the percentage of GFP positive cells is indicated. The results are representative of three independent experiments. (C) Cell internalization of Nef7/GFP VLPs pretreated with anti-GFP Abs. Anti-GFP immune-depleted VLPs were used to challenge CEMss cells as described underMaterials and Methods. After 3 h, cells were treated with trypsin and analyzed by FACS. The results are representative of two independent experiments. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 7 Cell killing upon (VSV-G) Nef7/TK VLP challenge of CEMss cells and GCV cultivation. Growth kinetics of CEMss cells left untreated or challenged with VSV-G-pseudotyped Nef7 or Nef/TK VLPs and cultivated in the presence or absence of GCV. At the indicated times, the cell viability was scored by the trypan blue dye exclusion assay. The cell mortality at day 7 of culture (i.e., the time of higher percentages of mortality) is reported in parentheses, except that for (VSV-G) Nef7/TK VLP-treated cells cultivated in GCV, whose values are reported in the pertinent panel. The mean values ±SD from five independent experiments are reported. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 8 Primary human MDM cells are efficiently killed by (VSV-G) Nef7/TK VLPs. Cell viability as evaluated by FACS scatter-plot analysis of MDM from two healthy donors (I and II) after challenge with 1 to 5 ng of (VSV-G) Nef7/TK VLPs and cultivation in the presence or absence of GCV. As a control, cells either unchallenged or challenged with Nef7 (VSV-G) VLPs and cultivated with or without GCV were also analyzed. The regions including apparently intact cells were gated. Percentages of events included in the regions are reported in each graph. Data refer to the analysis performed at day 5 after challenge of MDM from six healthy donors. Molecular Therapy 2005 12, 1185-1196DOI: (10.1016/j.ymthe.2005.06.474) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions