Transmission electron microscopy (TEM) analysis

293T cells were transfected with plasmids containing proviral clones encoding WT HIV-1 89.6 using polyethylenimine (PEI). After 6 hrs, cells were rinsed with DPBS gently, and then cells were resuspended in a fresh media with or without the inhibitor (1 μM, final). At 96 hrs post-transfection, cell cultures were centrifuged (1,800 xg) and filtered through 0.45 μm filter to remove cells and any cell debris, and viruses were harvested by centrifugation at 100,000 xg for 1.5 hrs in the presence of TNE sucrose buffer (5% sucrose, final). The pellets were fixed in a buffer containing 0.1 M Na cacodylate at pH 7.4 and 2.5% Glutaraldehyde, and submitted to Emory Integrated Core Facilities for sectioning, followed by TEM. Virus pellets were dehydrated in a graduated ethanol series and embedded in Epon resin. Ultrathin sections were stained using uranyl acetate and observed under a transmission electron microscope (JEOL JEM-1400), equipped with Gatan CCD camera at the Emory Integrated Electron Microscopy Core. Total of approximately 1,000 virions each (with or without the inhibitor treatment) were captured in the images obtained for comparisons in virion morphology.