Figure 2

Experimental setup of the screen and the different measurements involved. (A) In orthogonal screens, 841 human genes were identified to be under positive selection, of which only 30 were retained as candidates (11 were upregulated during HIV-1 infection, 14 reported to interact with viral proteins, and 5 fulfilled both criteria). (B) Schematic presentation of the bi-cistronic constructs coexpressing the gene candidate together with the blue fluorescent protein (BFP) under the control of a CMV promoter and the HIV-1 NL4-3-based IRES-eGFP proviral reporter construct. The position of premature stop codons in the vpr, vpu and nef genes of the Δ3 derivative are indicated by stars. (C) Overview of the experimental outline to determine the effect of cotransfected gene candidates on early viral gene expression (detection of eGFP by FACS) and late viral gene expression (cell-associated p24 antigen ELISA), virion release (cell-free p24 antigen ELISA) and viral infectivity (TZM-bl reporter assay). (D) Examples of primary FACS data. 293T cells were transfected with the indicated proviral constructs and expression vectors and analyzed by flow cytometry 48 h later.