The Use of Nanotrap Particles Technology in Capturing HIV-1 Virions and Viral Proteins from Infected Cells
Figure 4
Capture of infectious HIV-1 virions by nanotrap particles.
(A) HIV-1 infected J1.1 supernatants (1, 10, 100 µl) diluted to 1 ml in complete media, were either untreated or added to 50 µl of 30% slurry of NT086 nanotrap particles. The particles were incubated for one hour with gentle rotation at room temperature. The unbound virus was removed by centrifugation for 10 min before incubating the nanotrap particles with TZM-bl cells at 37°C in microtiter plates. Virus samples were also incubated with TZM-bl cells without prior treatment with nanotrap particles. After 48 hr post incubation, cells were lysed and HIV-1 transactivation analyzed by luciferase assay. The 2 asterices (p<0.01) and 1 asterix (p<0.05) represent the level of statistical significance between virus captures from 1 and 10 µl, and 10 and 100 µl supernatants, respectively. The NT073 nanoparticles were similarly used to capture infectious virions from J1.1 supernatants and analyzed by TZM-bl system as above (B) Exosomes (15 µl) collected from J1.1 cell supernatant were spiked into PBS (85 µl) and then incubated with a 30% slurry of five different nanotrap particles (30 µl) for 30 min. Nanotrap particles were washed and subjected to Trizol buffer for total RNA extraction. Evaluation of TAR-RNA contents of exosomes captured by nanotrap particles was performed via qRT-PCR using specific TAR-RNA primers. The five different nanotrap particles were similarly incubated with infected J1.1-derived exosomes and washed as in panel B. The nanoparticles-bound exosomes were then suspended in Laemmli buffer, separated on SDS-PAGE gel and analyzed by western blot using antibody to CD63 and Alix (standard markers for exosomes).
doi: https://doi.org/10.1371/journal.pone.0096778.g004