HIV-1 gp160 traffics to the Golgi network in Ubc9 knockdown cells.
(a) Env trafficking through the secretory pathway. 293T cells were transfected with pNL4-3 alone, or in combination with either Ctr. siRNA or Ubc9 siRNA, or left untransfected (Un). Unlike the previous and subsequent experiments, cells were pulse (P) labeled with [35S] methionine/cysteine for a shorter period of time (30 minutes) and chased for 2 and 4 hours. Cell and media associated viral proteins were solublized and immunoprecipitated with pooled AIDS patient sera, split equally, and incubated for 3.5 hours at 37°C in the presence, or absence of Endo H f. Samples were separated by SDS PAGE and visualized by phosphorimaging using The Discovery Series Quantity One software. A representative, over-exposed gel is shown so that partially Endo Hf resistant Env can be more easily visualized. Viral proteins and their positions in the gel are labeled on the left. The identity of Endo Hf, untreated viral proteins and their positions in the gel are labeled on the right. Deglycosylated Endo Hf sensitive forms of gp160 residing in the ER are labeled as gp160s. Partially deglycosylated, Endo Hf resistant forms of gp160 and gp120 that have undergone glycan modification in the TGN are labeled as gp160r and gp120r. gp160r in Endo Hf untreated samples is labeled as gp160*. (b) The gp160 trafficking to TGN. The ratio of gp160r/total gp160 during the 2-hour chase period, normalized to NL4-3.
