Fig 10. Effect of SIVcol Nef on viral replication in ex vivo HLT and NF-κB or NF-AT activity.

(A) Replication kinetics of HIV-1 constructs containing the indicated nef alleles in blocks of human lymphoid tissues ex vivo. Panels A to C show mean values (±SEM) obtained using tissues from seven different donors. (B) Cumulative virus production over 15 days of infection by the indicated HIV-1 constructs (see panel A) relative to the replication of the HIV-1 construct containing the NA7 nef allele (100%). (C) Levels of CD4+ T cell depletion in the tissue blocks (left) and cells that migrated in the gel foams (right) at the end of culture at 15 days post-infection. (D) HEK293T cells were cotransfected with a firefly luciferase reporter construct under the control of three NF-κB binding sites, a Gaussia luciferase construct for normalization, and expression vectors for a constitutively active mutant of IKKβ and the indicated Nef variants. Luciferase activity was determined 40 h post-transfection. The mean value of 9 transfections + SEM is shown. (E) Jurkat cells stably transfected with an NF-AT-dependent luciferase reporter gene were transduced with the indicated HIV-1 Nef-IRES-eGFP variants. The levels of luciferase activity were determined at 16 h post-stimulation. Shown are average values (+SD) derived from triplicate transductions relative to the nef-defective control HIV-1 construct (100%). Similar results were obtained in two independent experiments. *, p < 0.05; ***, p < 0.001.