Figure 4.

BST-2 dependent down-regulation of SHIV virion release from cells transfected with hBST-2 and ptBST-2. Panel A. p27 release assay. 293 cells were co-transfected with vectors expressing proviral DNA from one of four SHIV (SHIV_KU-2MC4, SHIV_ΔVpu, SHIV_ΔNef or SHIV_ΔVpu/ΔNef) and a vector expressing either the hBST-2 or ptBST-2 protein. At 48 hours post-transfection, the supernatants and cell lysates were collected and cleared of cellular debris and nuclei by centrifugation. The p27 content of both the supernatant and cell lysate from each sample was quantified using a p27 antigen capture assay (Zeptometrix) and the percent p27 release calculated. All conditions were run at least three separate times, normalized to their respective SHIV empty vector controls, and the average percent p27 release and standard error calculated. Significance in the restriction of p27 release for the SHIV_KU-2MC4 samples was determined with respect to the parental SHIV_KU-2MC4 empty vector control using a Student's t-test (▲). Significance in the restriction of p27 release for the SHIV_ΔVpu, SHIV_ΔNef, and SHIV_ΔVpu/ΔNef samples was calculated with respect to the SHIV_KU-2MC4 in the presence of each respective BST-2 using a Student's t-test (*). Panel B. Infectious units assay using TZM-bl indicator cells. Supernatants from transfections described above were added to the TZM-bl cells and serially diluted. At 48hrs post-infection, cells were washed, fixed, and stained for 2 hours. The TCID₅₀ for each supernatant was calculated based on wells containing cells expressing β-galactosidase. All conditions were run at least three times and the TCID₅₀ calculated. The average TCID₅₀ and standard error were calculated. Significance in the restriction of infectivity was determined with respect to the parental SHIV empty vector control using a Student's t-test with p<0.05 considered significant (●).