Fig. 1

SIVagm constructs expressing functional Vpu and HIV-1 Nef. a Genomic organization of the WT SIVagm construct with the nef gene (yellow) and the GU1N derivative containing the SIVgsn vpu (blue) and the HIV-1 NA7 nef (orange). b HEK293T cells were cotransfected with a vpu-deleted HIV-1 NL4-3 construct, expression plasmids for HIV-1 or SIVgsn Vpu and a vector expressing AGM tetherin (BST-2). Infectious virus yield was measured by infection of TZM-bl cells. Curves represent the average values (n = 3) relative to those obtained in the absence of tetherin (100%). c Levels of AGM BST-2 and human CD4, NTB-A, and CD1d in HEK293T cells cotransfected with SIVgsn or HIV-1 Vpu constructs relative to those measured in cells transfected with the eGFP control vector (100%). Symbols represent the value obtained in one of three experiments. Mean ± SEM are indicated in panels c, d, e, and j. d Effect of Vpu on NF-κB activity. HEK293T cells were cotransfected with a firefly luciferase NF-κB reporter construct, a Gaussia luciferase construct for normalization, a vector for constitutively active IKKβ and constructs expressing eGFP alone or together with Vpu. Luciferase activity was determined two dpi (n = 3 ± SEM). CTRL represents the vector control. e Receptor downmodulation in human PBMCs or CD4+ T cells (CD4) infected with HIV-1 constructs expressing the indicated Nefs relative the nef-defective virus (100%), (n = 3 ± SEM). f Effect of NA7 and SIVagm Nefs on virus release in the presence of AGM tetherin in transfected HEK293T cells. Curves represent averages (n = 3) relative to those obtained in the absence of tetherin (100%). g, h Infectivity and replication of SIVagm constructs in vitro. TZM-bl and MOLT-4 clone 8 cells (g) or AGM PBMCs (h) were infected with the indicated SIVagm constructs. Virus infectivity or production was monitored by β-galactosidase assay or p27 antigen ELISA, respectively. Shown is g mean of triplicate infection or h one representative experiment. i Average infectious virus yield (n = 3) from HEK293T cells following co-transfection with SIVagm constructs and plasmids expressing AGM tetherin relative to that detected in the absence of tetherin (100%). j Effect of SIVagm constructs on receptor expression by PBMCs from three AGM relative to those infected with the nef-defective SIVagm clone (100%). p-values were calculated using the two-tailed unpaired Student's-t-test and significant differences are indicated as: *p < 0.05; **p < 0.01; ***p < 0.001