Figure 5

Replication-competent HIV-1 with mutation in the isu domain does not induce IL-10 release. (A) Mutations introduced to the isu domain of gp41 HIV-1. The upper numbers started from the first amino acid in Env (accession number AF324493), the lower numbers - with the first amino acid in gp41. The mutated amino acids are underlined. (B) Comparison of the virus-specific proteins in transfected cells and (C) in the virus centrifuged through a 20% sucrose cushion. 1 - wild-type HIV-1_pNL4-3; 2 - mutated HIV-1_pNL4-3 (Q2A); 3 - double mutated HIV-1_pNL4-3 (Q2A + L6A); 4 - negative control, cells transfected with pcDNA3.1(−). A 4-20% gradient SDS-PAGE/Western blot analysis was performed with the mab 2F5 (B, C lower panel) and with pooled sera from HIV-1 infected individuals (C, upper panel). (D) Infectious titers of wt (pNL4-3) and mutated viruses HIV-1pNL4-3 (Q2A) and HIV-1_pNL4-3 (Q2A + L6A) determined on the reporter TZM-bl cell line. Analysis was performed in triplicates. Mock – supernatant from cells transfected with pcDNA 3.1(−). (E) Estimation of the amount of gp41 in virus preparations by SDS-PAGE/Western blot using serial dilutions of T20 as reference. 1–50 ng/lane; 2–25 ng/lane; 3–12.5 ng/lane. Purified HIV-1pNL4-3 was analyzed in two dilutions equivalent to 4 × 10⁵ (Track 4) and 2 × 10⁶ (Track 5) infectious particles per lane. Western blot was performed using the mab 2F5. (F) IL-10 release from PBMCs of donor 1 that were exposed to the wt and mutated viruses inactivated by freeze-thawing. Viruses were diluted twofold; the initial concentration of gp41 was 5 ng/well. The p values were determined as described in Methods.