Arrayed ISG expression screening identifies multiple ISGs that inhibit transmitted HIV-1 and its matched CC virus.

(A) A schematic of the SCRPSY lentiviral vector (GenBank accession KT368137.1) used to deliver ISGs, and of the fluorescent GFP-IRES-Nef (GIN) TF and CC viruses used. (B) The infectious titres of CH058 IMC and CH058 GIN viruses in a single cycle of infection in TMZR5 cells. Cells were treated with dextran sulphate 17–18 hours post infection and the percentage of GFP positive cells was determined at 48 hours post-infection using flow cytometry. (C) Western blot analysis of viral antigens present in TMZR5 cell lysates infected with the indicated virus for 48 hours. (D) Growth kinetics of CH058 TF GIN in clonal MT4 CCR5-R126N in the absence and presence of 0.5 pg/μl IFNα14. Cells were treated with IFNα14 24h prior to infection. (E) A schematic of the ISG screening pipeline used in panel F. (F) Normalized infection (median centred) of cells expressing different ISGs (each dot represents the observed infection in the presence of a single ISG). (G) Validation screen, conducted as in E and F, of ISGs ‘hits’ selected that were more inhibitory than human Mx2 in panel F. Empty SCRPSY transduced MT4-R5 cells were used as controls.