Dose-dependent reduction in the accumulation of SIV p27 in the cell culture supernatant relative to p55 Gag in cells at increasing expression levels of tetherin.

The accumulation of SIV p27 capsid in the cell culture supernatant relative to p55 Gag in cell lysates was compared for wild-type SIV, SIV Δenv and SIV Δnef at increasing expression levels of human and rhesus tetherin. 293T cells were transfected with proviral DNA for wild-type SIV, SIV Δenv or SIV Δnef together with 0, 2, 10 and 50 ng of plasmid DNA for hBST2 (A) or rBST2 (B). Forty-eight hours post-transfection, virus was recovered from the culture supernatant by centrifugation and cell lysates were prepared. Proteins were separated on a 10% SDS-polyacrylamide gel, transferred to a PVDF membrane and probed with monoclonal antibodies to p27/p55 Gag (183-H12-5C) and to BST2 (HM1.24). The blots were then probed with an HRP-conjugated goat anti-mouse secondary antibody, developed in chemiluminescent substrate and visualized using a Fujifilm Image Reader LAS 3000. Band intensities for p27 in virions and p55 in cell lysates were determined using the Image J Software (Rasband, W.S., Image, U.S. NIH, Bethesda, MD, http://rsb.info.nih.gov/ij, 1997–2008) and compared as p27/p55 ratios. ‘ND’ indicates that the p27 band was below the limit of detection.