HIV-1 Nef protein is secreted into vesicles that can fuse with target cells and virions

Abstract

HIV-1 Nef is a major determinant in HIV-1 pathogenicity. However, its properties have mainly been associated with its biochemical activities within the producer cell. Nef is also secreted from infected and transfected cells. Our primary objective was to determine the nature of secreted Nef protein and its effect on target cells. We determined that HIV-1 Nef is secreted in the form of exosome-like vesicles. Nef protein present in these vesicles is largely protected from protease digestion, which suggests that most of the protein is present on the lumenal side of the vesicles. We observed that HEK293 cells, transfected with a Nef-GFP expression vector, can secrete vesicles containing Nef-GFP fusion protein into the extracellular medium. When the conditioned medium was used to treat Jurkat cells, we found that the cells can take up the Nef fusion protein. The pattern of distribution of the Nef-GFP fusion protein within the target cells is mainly cytoplasmic and results in a punctate staining pattern. We also observed that Nef (-) virions treated with Nef-conditioned medium have their infectivity restored to near wild-type levels. This implies that the Nef contained within vesicles has the ability to fuse with HIV-1 virions and deliver functional Nef to these virions. It also demonstrates that Nef protein delivered in trans can restore infectivity even after virion maturation has occurred. These studies suggest that secreted Nef could play a role in HIV-1 pathogenesis by inducing effects in noninfected bystander cells.

Fig 1

Conditioned tissue culture supernatant was collected from HEK293 cells that were transfected with pNef expression plasmid or a G2A mutation in the Nef sequence. The supernatants were spun in a TLA100 rotor at 400,000 × g for 1 h to create the pellets. Medium, supernatants, and pellets were each separated on an 12.5% SDS PAGE and Nef was detected by immunoblot using anti-Nef antiserum. Note that the majority of Nef present in these blots is actually in the form of a dimmer.

Fig 2

Pellets from high-speed spins were treated with equal volumes of subtilisin (1mg/mL final concentration) for 16 h at 37°C. The reaction was then terminated by adding PMSF (5μg/mL final concentration). The products were then separated on an 12.5% gel and either stained with Coomasie blue or immunoblot using anti-Nef antiserum.

Fig 3

Pellets from high-speed spins were immobilized in 1% agar and then fixed, embedded and stained with 2% uranyl acetate. They were then visualized using a JEOL 1200EX transmission microscope. A control using cells that were mock transfected is also included. The size bar in the transfected sample is 200nm in size.

Fig 4

Conditioned medium was collected from Nef-GFP transfected cells after 48h. Fresh Jurkat cells were then added and allowed to come in contact with the medium for 30min. The cells were then observed by confocal microscopy for the localization of Nef-GFP. Cells were counterstained with DRAQ5. (A) Note that the small dots in this high magnification field are thought to be individual Nef vesicles. (B) A lower magnification image shows that some cells have taken up significant quantities of the fusion protein.