The uncaptured preparation of wild type virus contained five-fold more HIV-1 p24 capsid antigen than that of vpu, consistent with the restriction of virion-release by endogenous BST-2 and counteraction by Vpu (data not shown)

The uncaptured preparation of wild type virus contained five-fold more HIV-1 p24 capsid antigen than that of vpu, consistent with the restriction of virion-release by endogenous BST-2 and counteraction by Vpu (data not shown). the ultrastructural level and in relation to nascent HIV virions, cells stained in an identical manner to those shown in Physique 1A-C were processed for transmission electron microscopy. In uninfected cells, BST-2 was found in foci along the plasma membrane (Figures 1D and S2), which likely correspond to the puncta seen using immunofluorescence. Some of these foci were associated with endocytic pits, which appeared either coated or uncoated, whereas other foci were not associated with any apparent structure. In cells expressing the Entacapone sodium salt complete HIV-1 genome including (vpu; B, D, F, and G), then stained for cell surface BST-2 and processed for electron microscopy as described in the legend of Physique 1. Panels A-F are images of thin sections obtained using a JEOL 1200C microscope operated at 80 keV. Panel G is a projection view of a thick section (approximately 250 nm) derived from a tomographic tilt series of images obtained using an FEI Titan electron microscope operated at Entacapone sodium salt 300 keV. BST-2 is usually incorporated into infectious HIV-1 viral particles To determine whether BST-2 is usually incorporated into virions, we looked for profiles of budding virions and for profiles of virions distant from the cell surface. Surprisingly, wild type virions were not infrequently labeled for BST-2 (Physique 2C and E; see Physique S3 for control stain). This result is usually consistent with functional data indicating that Vpu is not a fully effective antagonist of BST-2 [5], and it is consistent with the virion-capture and immunoblot experiments described below. In rare cases, label for BST-2 was found directly between virions that appeared linked to each other (Physique 2E). In the case of (vpu). Forty-eight hours later, cell-free culture supernatants were collected, incubated with antibody to CD44, BST-2, CD45, or an IgG2a isotype control, then captured using magnetic microbeads coated with staphylococcal protein G. Immuno-captured virions were resuspended in the original culture volumes. Virions, uncaptured or captured using the indicated antibodies, were used to infect CD4-positive HeLa cells made up of an LTR–galactosidase indicator construct. Two days later, infectious centers were visualized by incubation with X-gal to generate blue colored foci. Images of individual wells were acquired using a CCD camera. The wells shown are the result of contamination using undiluted preparations, which were each equivalent to 200 l of the original, uncaptured cell-free culture supernatants. The uncaptured preparation of wild type virus contained five-fold more HIV-1 p24 capsid antigen than that of vpu, consistent with the restriction of virion-release by endogenous Rabbit Polyclonal to HSL (phospho-Ser855/554) BST-2 and counteraction by Vpu (data not shown). B and C. An independently prepared set of virions was produced from HeLa cells, subjected to immuno-capture, and infectivity was measured as described for panel A. Each preparation was serially diluted as shown (0 indicates undiluted sample) and used to infect CD4-positive HeLa cells in duplicate. Infectious centers were counted using image analysis software; error bars are the actual values of duplicate wells and in some cases are too small to see. The data shown are representation of three impartial experiments. D. Virions (wild type) were produced by transfection of HEK293T cells lacking endogenous BST-2, subjected to immuno-capture using antibody to BST-2 or an antibody isotype control, and infectivity was measured as in panels B and C. Data are the averages of duplicate Entacapone sodium salt wells; the error bars are too small to see. E. The fraction of the total viral p24 capsid antigen that was recovered in these immuno-capture experiments was measured using an ELISA. Error bars are the standard deviation of values obtained from three capture experiments using independent preparations of viruses made from HeLa cells. Immunoblot analysis also supported the conclusion that BST-2 is usually incorporated into virions and further suggested that Vpu inhibits this (Physique 4, in which virions produced from HeLa cells and concentrated by centrifugation were analyzed). Remarkably, when normalized by the volume of the original culture supernates (Physique 4A, left panel), preparations of wild type virions contained more BST-2, as Entacapone sodium salt well as more p24 capsid protein, than virions produced by detected in this assay (an apparent 40-fold increase in virion output).