Figure 4

Molecular analysis of the HIV-1 related products in 18-4s cells and VLPs. (A). Western blot analysis for the cell expression and VLP incorporation of both HIV-1 Gag-related products and NefG3C-GFP in control and/or induced 18-4s and parental 293/RGP cells. As control, both cell and viral lysates from HIV-1 pNL4-3 transfected 293T cells were used. Fifty μg of total proteins from each cell lysate or 50 ng of VLPs were assayed using a 1:1000 dilution of pooled strongly HIV-1 positive human sera, or a 1:1000 dilution of the ARP444 anti-Nef sheep antiserum. NaB: sodium butyrate. (B) Western blot analysis of subtilisin A treated 18-4s VLPs. Hundred ng of (VSV-G) NefG3C-GFP VLPs were treated with 10 μg of subtilisin A or with an equal amount of bovine serum albumine (Ctrl) and, after 2 hours of incubation at 37°C, the reaction was stopped with phenylmethylsulfonyl fluoride, and the samples analyzed for both NefG3C-GFP and VSV-G contents by Western blot using the ARP444 anti-Nef polyclonal Abs or anti-VSV-G polyclonal Abs recognizing the VSV-G intracytoplasmic tail. The lowest signals overlaps the front-dye of the SDS-PAGE where degraded protein products are expected to accumulate. (C) Anti-Nef and anti-CAp24 Western blot analysis of relevant fractions from an iodixanol gradient loaded with 18-4s VLPs. Fifteen ml of supernatant from induced 18-4s cells containing about 50 μg of VLPs were concentrated by ultracentrifugation on a 20% sucrose cushion, and loaded on a 6 to 35% discontinuous iodixanol gradient. The Western blot analysis of the eight most relevant fractions out of the fifteen harvested is reported. (D) Quantitation of NefG3C-GFP virion incorporation. Two-fold serial dilutions of 18-4s VLPs were analyzed by Western blot for the presence of NefG3C-GFP as compared with serial dilutions of rNef. The nanograms of rNef and 18-4s VLPs, the latter measured by quantitative anti-CAp24 ELISA, are indicated on the top. Data are representative of 8 (A), 3 (B), and 2 (D) independent experiments. For all panels, the migration of major viral products are indicated on the left side, whereas the molecular marker sizes are reported on the right.