Fig. 5
Maintenance of HIV-1 Nef function in 1N and GU1N infected AGMs. a Alignment of Nef sequences derived from 1N (upper three) and GU1N (lower three) infected AGMs. The HIV-1 NA7 sequence is shown on top and dashes indicate gaps introduced to optimize the alignment. Numbers in parentheses indicate the total number of nef alleles analyzed per time point. Black indicates changes in all, blue in ≥50% and gray in at least two sequences. Sites mutated in at least three AGMs are highlighted by orange dots. b Expression of selected SIVagm and HIV-1 Nef proteins. HEK293T cells were transfected with expression plasmids encoding the indicated AU1-tagged Nefs and eGFP. GAPDH and eGFP expression levels were analyzed to control for loading and transfection efficiency, respectively. c Quantitative assessment of Nef-mediated downmodulation of the indicated cellular receptors on PBMCs (MHC-I, CD3, CD28, CXCR4, and CD74), CD4+ T cells (CD4) and THP-1 cells (CD74). Shown are mean (+SD) fluorescence intensities (MFIs) of receptor expression relative to the nef-defective control (100%) derived from three experiments. d HIV-1 Nefs did not evolve activity against AGM tetherin. HEK293T cells were cotransfected with HIV-1 NL4-3 nef-vpu- constructs, expression plasmids for the indicated nef alleles and different amounts of AGM tetherin. Infectious virus yield in the culture supernatants two dpi was determined by triplicate infection of TZM-bl reporter cells. Shown is the mean of three experiments. e Antagonism of AGM SERINC5 by SIVagm and HIV-1 Nef proteins. HEK293T cells were cotransfected with HIV-1 NL4-3 proviral constructs containing the indicated nef alleles and an empty vector control or AGM SERINC5 expression vector. Viral supernatants were obtained 3 days later and infectious HIV-1 yield determined by triplicate infection of TZM-bl cells. Shown are average values of three experiments + SD (n = 3) relative to those obtained in the absence of SERINC5 expression vector (100%)
