Technical Reference #1714

1714.

Deficiency of Niemann-Pick type C-1 Protein Impairs HIV-1 Release and Results in Gag Accumulation in Late Endosomal/Lysosomal Compartments Yuyang Tang; Ihid Carneiro Leao; Ebony M. Coleman; Robin Shepard Broughton; and James E.K. Hildreth*, Meharry Medical College, Journal of Virology, 83(1714), (2009)
Link To Paper

Abstract:
Human immunodeficiency virus type 1 (HIV-1) relies on cholesterol-laden lipid raft membrane microdomains for entry into and egress out of susceptible cells. In the present study we examine the need for intracellular cholesterol trafficking pathways with respect to HIV-1 biogenesis using Niemann-Pick type C-1 (NPC1)-deficient (NPCD) cells wherein these pathways are severely compromised causing massive accumulation of cholesterol in late endosomal/lysosomal (LE/L) compartments. We have found that induction of an NPC disease-like phenotype through treatment of various cell types with the commonly used hydrophobic amine drug U18666A resulted in profound suppression of HIV-1 release. Further NPCD Epstein-Barr virustransformed B lymphocytes and fibroblasts from patients with NPC disease infected with a CD4-independent strain of HIV-1 or transfected with an HIV-1 proviral clone respectively replicated HIV-1 poorly compared to normal cells. Infection of the NPCD fibroblasts with a vesicular stomatitis virus G-pseudotyped strain of HIV-1 produced similar results suggesting a postentry block to HIV-1 replication in these cells. Examination of these cells using confocal microscopy showed an accumulation and stabilization of Gag in LE/L compartments. Additionally normal HIV-1 production could be restored in NPCD cells upon expression of a functional NPC1 protein and overexpression of NPC1 increased HIV-1 release. Taken together our findings demonstrate that intact intracellular cholesterol trafficking pathways mediated by NPC1 are needed for efficient HIV-1 production.

Materials & Methods:
For cholesterol staining cells were grown on 35-mm glassbottom dishes (MatTek Corporation Ashland MA) under normal growth conditions and then fixed in 2% paraformaldehyde in PBS for 15 min. The cells were then incubated with filipin at 50 g/ml in PBS on ice for 30 min. Analysis of filipin staining was performed using a Nikon TE2000 wide-field microscope (Nikon Instruments Melville NY). All images were acquired with a plain fluor 60 1.3-numerical aperture oil immersion objective with a UV fluorescence filter. The digital image acquisition was performed with a Photometrics CoolSNAP HQ2 cooled charge-coupled device camera (Roper Scientific Tucson AZ) and Nikon Elements Advanced Research software (Nikon Instruments Melville NY). For immunofluorescence studies cells were grown on acid-washed coverslips or 35-mm glass-bottom dishes to a density of 1 to 5 104 cells. The cells were fixed with 2% paraformaldehyde for 20 min and then blocked with 5% normal goat serum and 1% bovine serum in PBS before being permeabilized with 0.1% saponin. Primary antibody anti-Gag (GagM1 or KC57-FITC) anti- LAMP2 or anti-CD63 was incubated with the cells in the blocking solution for 1 h at a final concentration of 10 g/ml. The cells were then washed two times and incubated with secondary antibodies FITC- or Texas Red-conjugated goat anti-mouse and Texas Red-conjugated streptavidin (Jackson Amershan Bioscience Piscataway NJ) for an additional hour. For Lysotracker Red staining cells were incubated with 75 nM LysoTracker Red DND-99 for 1 h at 37°C. The media were refreshed and the cells were visualized using fluorescence microscopy. The coverslips were mounted onto glass slides using ProLong antifade mounting media (Molecular Probes Eugene OR). The slides or glass-bottom dish was examined using a Nikon TE2000 C1 laser scanning confocal microscope (Nikon Instruments Melville NY) with a 60 1.4-numerical aperture oil immersion objective and analyzed using Nikon Elements Advanced Research software.

Microscopic Technique
Confocal Microscopy, Laser Scanning, Electron Microscopy

Cell Type(s)
CEMX174