Abstract
Like most enveloped viruses, HIV must acquire a lipid membrane as it assembles and buds through the plasma membrane of infected cells to spread infection. Several sets of host cell machinery facilitate this process, including proteins of the endosomal sorting complexes required for transport pathway, which mediates the membrane fission reaction required to complete viral budding, as well as angiomotin (AMOT) and NEDD4L, which bind one another and promote virion membrane envelopment. AMOT and NEDD4L interact through the four NEDD4L WW domains and three different AMOT Pro-Pro-x (any amino acid)-Tyr (PPxY) motifs, but these interactions are not yet well defined. Here, we report that individual AMOT PPxY and NEDD4L WW domains interact with the following general affinity hierarchies: AMOT PPxY1>PPxY2>PPxY3 and NEDD4L WW3>WW2>WW1∼WW4. The unusually high-affinity of the AMOT PPxY1–NEDD4L WW3 interaction accounts for most of the AMOT–NEDD4L binding and is critical for stimulating HIV-1 release. Comparative structural, binding, and virological analyses reveal that complementary ionic and hydrophobic contacts on both sides of the WW–PPxY core interaction account for the unusually high affinity of the AMOT PPxY1–NEDD4L WW3 interaction. Taken together, our studies reveal how the first AMOT PPxY1 motif binds the third NEDD4L WW domain to stimulate HIV-1 viral envelopment and promote infectivity.
Highlights
Cells were washed off the plate in 1 ml PBS, pelleted by centrifugation, and lysed for 15 min at 4 C in 150-μl RIPA buffer (10 mM Tris, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl, pH 8.0) supplemented with mammalian protease inhibitor (Sigma-Aldrich)
Insoluble material was removed by centrifugation (10 min, 15,000g, 4 C). 150 μl of 2 × Laemmli SDS-PAGE loading buffer supplemented with 10% 2-mercaptoethanol (Sigma-Aldrich) were added and samples were boiled for 5 min
Virions from 1-ml supernatant were pelleted by centrifugation through a 200-μl 20% sucrose cushion (90 min, 15,000g, 4 C) and denatured by adding 100 μl 1× Laemmli SDSPAGE loading buffer and boiling for 5 min
Summary
Cells were washed off the plate in 1 ml PBS, pelleted by centrifugation, and lysed for 15 min at 4 C in 150-μl RIPA buffer (10 mM Tris, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X100, 0.1% sodium deoxycholate, 0.1% SDS, and 140 mM NaCl, pH 8.0) supplemented with mammalian protease inhibitor (Sigma-Aldrich). HeLa-TZM-bl cells (5000 cells per well, 96-well plate) were infected with three different dilutions of virus-containing culture media, each in triplicate, harvested after 48 h, washed once with 200-μl PBS, and lysed in 50-μl 25 mM Tris phosphate (pH 7.8), 2 mM DTT, 2 mM 1,2diaminocyclohexane N,N,N0,N0-tetraacetic acid, 10% glycerol, and 1% Triton X-100 (10 min, 23 C).
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