FIG. 6.
PPXY-mediated viral particle production is specifically reduced upon transient expression of ART proteins. (A) Analysis of infectious particle production. 293T cells were transfected with MLV provirus expression vectors containing its natural PPXY L domain (MLV) or a mutant depending on a PTAP motif (MLV hPTAP) as well as expression constructs encoding YFP fusion proteins as indicated. Infectious virus release was analyzed using a chemiluminescent assay after infection of TZM-bl reporter cells with supernatants harvested from the transfected 293T cells. In these experiments, absolute infectivity values of 2.4 × 106 for MLV and 1.0 × 106 for MLV hPTAP were obtained on average for cells expressing unfused YFP, and these values were set to 100% to calculate relative infectious virion release for either MLV or MLV hPTAP. Error bars indicate the standard deviation from the mean of at least three independent experiments. (B) Analysis of virus-like particle release. Virus-like particles (VLPs) were isolated from 293T cells transiently expressing HA-tagged MLV Gag featuring either a PPXY (MLV) or a mutant PTAP L domain (MLV hPTAP) together with the indicated YFP fusion proteins. VLPs and cellular lysates were analyzed by Western blotting (WB) against the MLV HA tag; signal intensities were measured using Odyssey, version 3.0, software, and budding efficiency was determined. The depicted Western blots are representative of three independent experiments; the graph illustrates the budding efficiency gained upon expression of YFP-ARRDC1NC. Error bars indicate standard deviations from three independent experiments. α, anti.