sdAb19 inhibits Nef-mediated enhancement of virus infectivity and the Nef-positive effect on virus replication. (A) Single-round GFP reporter viruses were produced in 293T cells in the absence or presence of increasing amounts of the plasmid expressing sdAb19. Forty-eight hours later, viruses were pelleted from cell culture supernatants and were used to infect HeLa-CD4 cells. The percentages of GFP-positive infected cells were then measured by flow cytometry 60 hours later. Viral infectivity was normalized to that of viruses produced in the absence of Nef. (B) WT (black curves) or ΔNef (gray curves) replication-competent viruses were produced in 293T cells in the absence (plain lines) or the presence of sdAb19 (dashed lines) in a 1:1 ratio and were used to infect PBMCs. Aliquots of cell culture supernatant were collected 2, 4, 6, and 8 days after infection for CAp24 quantification.