Figure 5.

Inhibition of VLP formation by lactacystin. HeLa cells were transfected with the chimeric proviral construct HXBH10/SIV^gag, which expresses the full-length Gag polyprotein of SIV_mac239 in the absence of a viral protease. The cells were split 48 h posttransfection and kept for 2 h in the presence or absence of 10 μM lactacystin, followed by 4 h of radiolabeling with [³⁵S]methionine in the absence of lactacystin. The cells were then lysed, and cell-associated viral proteins were immunoprecipitated and resolved by SDS/PAGE (Left). VLPs released during the 4-h labeling period were pelleted through sucrose and analyzed directly by SDS/PAGE (Right). The arrows indicate the expected positions of mono- and di-ubiquitinated forms of Gag.