Intracellular trafficking of HIV-1 Gag via Syntaxin 6-positive compartments/vesicles: Involvement in tumor necrosis factor secretion

HIV-1 Gag protein is synthesized in the cytosol and is transported to the plasma membrane, where viral particle assembly and budding occur. Endosomes are alternative sites of Gag accumulation. However, the intracellular transport pathways and carriers for Gag have not been clarified. We show here that Syntaxin6 (Syx6), a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) involved in membrane fusion in post-Golgi networks, is a molecule responsible for Gag trafficking and also for tumor necrosis factor-alpha (TNF alpha) secretion and that Gag and TNF alpha are cotransported via Syx6-positive compartments/vesicles. Confocal and live-cell imaging revealed that Gag colocalized and cotrafficked with Syx6, a fraction of which localizes in early and recycling endosomes. Syx6 knockdown reduced HIV-1 particle production, with Gag distributed diffusely throughout the cytoplasm. Coimmunoprecipitation and pulldown show that Gag binds to Syx6, but not its SNARE partners or their assembly complexes, suggesting that Gag preferentially binds free Syx6. The Gag matrix domain and the Syx6 SNARE domain are responsible for the interaction and cotrafficking. In immune cells, Syx6 knockdown/knockout similarly impaired HIV-1 production. Interestingly, HIV-1 infection facilitated TNF alpha secretion, and this enhancement did not occur in Syx6-depleted cells. Confocal and live-cell imaging revealed that TNF alpha and Gag partially colocalized and were cotransported via Syx6-positive compartments/vesicles. Biochemical analyses indicate that TNF alpha directly binds the dence that both Gag and TNF alpha make use of Syx6-mediated trafficking machinery and suggest that Gag expression does not inhibit but rather facilitates TNF alpha secretion in HIV-1 infection.