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ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Figure 5

HIV Gag-GFP VLP budding from yeast spheroplasts is independent of ESCRT function.

(A) or GFP-Cps1 were transformed into wild type cells or strains deleted for a component of each ESCRT complex, ESCRT-0 (), ESCRT-I (), ESCRT-II (), ESCRT-III () or VPS4 (), and GFP localization was assessed by fluorescence microscopy of live cells. In the wild type background GFP-Cps1 was sorted into the lumen of the yeast vacuole, while loss of ESCRT function, Bro1 function, or Vps4 function resulted in missorting to the vacuolar limiting membrane and accumulation within the aberrant class E compartment. (B) or were expressed in the indicated strains strains and whole cell extracts (W.C.) and VLP fractions were analyzed by Western blotting with anti-GFP antibody. (C) VLPs were collected from cells expressing wild type Gag-GFP and subjected to centrifugation across a sucrose gradient (20–70%, weight to volume). 1 ml fractions were subsequently collected, the density of each fraction was measured, and each fraction was subjected to SDS-PAGE and anti-GFP immunoblotting. (D) cells expressing wild type Gag-GFP were processed to isolate the whole cell and VLP fractions. The VLP fraction was subsequently subjected to incubation with either PBS alone (PBS), PBS+Trypsin (T), or PBS+Trypsin+Triton X-100 (T+TX100) and the reactions were visualized by immunoblotting using anti-GFP antibody. Whole cell lysate (W.C.) from the cells used to generate the VLP fraction was loaded to indicate expression.

doi: https://doi.org/10.1371/journal.pone.0052603.g005