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.
2013-02-21T05:56:01Z (GMT) by
<p>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 <i>Δenv</i> and SIV Δ<i>nef</i> at increasing expression levels of human and rhesus tetherin. 293T cells were transfected with proviral DNA for wild-type SIV, SIV <i>Δenv</i> or SIV Δ<i>nef</i> 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, <a href="http://rsb.info.nih.gov/ij" target="_blank">http://rsb.info.nih.gov/ij</a>, 1997–2008) and compared as p27/p55 ratios. ‘ND’ indicates that the p27 band was below the limit of detection.</p>