Figure 3

HIV vEnv induced viral transcription in HIV-infected CD4 T cell line (J-Lat 6.3), HIV-infected non-stimulated PBMCs, and latent-infected PBMCs in ART-treated patients. (A) Increased transcription of HIV gag in J-Lat 6.3 T cells treated with Env(X4)-VLP. J-Lat 6.3 T cells were treated or non-treated with Env(X4)-VLP for 24 hrs. HIV gag mRNA was detected by RT-PCR, normalized against the housekeeping gene GAPDH (n = 3), and expressed as comparative transcription level. (B) J-Lat 6.3 T cells were treated with Env(X4)-VLP for 0, 6, and 24 hrs (as indicated) and after 24 hrs, cells were lysed and HIV comparative transcription levels (gag/GAPDH) and the reporter gene GFP comparative transcription levels (GFP/GAPDH) were detected by RT-PCR (left panel). Also, the Env(X4)-VLP treated or untreated J-Lat 6.3 T cells were lysed and the expression of GFP was detected by western blot with corresponding antibody (right panel). (C) HIV comparative transcription levels in HIV-infected resting PBMCs treated with Env(X4)-VLP or untreated (n = 3). PBMCs were isolated from five donors and infected with HIV virus for 24 hrs without stimulation and the infected PBMCs were washed and kept in culture medium for 2 days. Then, cells were treated with Env(X4)-VLP or untreated. Meanwhile, the untreated HIV-infected PBMCs, untreated and uninfected PBMCs or Env(X4)-VLP-treated uninfected PBMCs were used as controls. After 24 hours of treatment, HIV comparative transcription levels (gag/GAPDH) were detected by RT-PCR (left panel). Meanwhile, the infected resting PBMCs treated or untreated with Env-VLP, (as described in left panel) were co-cultured with C8166 T cells for three days. Then, the HIVp24 levels in the supernatant of co-cultures were measured by anti-HIVp24 ELISA (right panel). (D) HIV comparative transcription levels were detected in PHA or anti-CD3/CD28 stimulated HIV-infected PBMCs followed by the treatment with Env-VLP or not (n = 3). (E) HIV comparative transcription levels in non-stimulated PBMCs isolated from seven HIV latent patients, that were treated or non-treated with Env(X4)-VLP for 24 hrs, and then the HIV gag mRNA was detected by RT-PCR (n = 3). Data are the mean and sd. Ns, not significant p > 0.05. (two-tailed unpaired t-test)