Volume 7 issue 3 March 2006
The Pericentriolar Recycling Endosome Plays a Key Role in Vpu-mediated Enhancement of HIV-1 Particle Release
Vasundhara Varthakavi, Rita M. Smith, Kenneth L. Martin, Aaron Derdowski, Lynne A. Lapierre, James R. Goldenring and Paul Spearman

SUPPLEMENTARY MATERIALS:

Fig. S1. Subcellular localization of Vpu in permissive Cos-7 cells. (A) GM130 (green) and Vpu-HcRed. (B) TGN46 (green) and Vpu-HcRed. (C) Rab11a (green) and Vpu-HcRed. (D) CD63 (green) and Vpu-HcRed. Vpu-HcRed in each case is shown in red. (E-H) Alex 488-transferrin pulse-chase in Cos-7 cells expressing Vpu-HcRed and fixed at the indicated timepoints following the pulse. (I) Cos-7 cells cells expressing EGFP-myosin Vb tail and HIV-2 ROD envelope (red) were immunostained for envelope expression using Rabbit polyclonal antiserum against HIV-2_ST gp120. (J) Colocalization of Myosin Vb tail-CFP (green) with Vpu-HcRed and Gag-YFP (blue) in Cos-7 cells. (K) Colocalization of EGFP-Rab11a (green) with Vpu-HcRed and Gag-CFP (blue) in Cos-7 cells.

Figure S1 (.eps)

Fig. S2. Rab11aS25N and Myosin Vbtail affect Vpu mediated Gag release but not its synthesis. Hep-2 cells expressing indicated proteins were analysed by Western blot for Pr55^Gag expression (Top panel). Gag virus like particles in supernatants from these cells were pelleted and analyzed by Western blot (Lower Panel). HIV-positive patients' serum was used for the detection of Gag. Note that under these conditions using a limited number of cells, released Gag was detected by Western primarily only from wells in which Gag and Vpu were expressed in the absence of inhibitors.

Figure S2 (.eps)

Fig. S3. Quantitative image analysis of Vpu colocalization with TGN and CD63. (A) Percent colocalization of VpuHcRed with TGN46 from 30 cells (Hep-2 and Cos-7). (B) Percent colocalization of Vpu with CD63. The letter “n” represents the number of cells analysed in each of the following groups: no apparent colocalization (white bar), partial colocalization (orange bar) and extensive colocalization (yellow bar); quantitative analysis was performed using MetaMorph imaging software. (C) Images of cells representing each of the groups described in the S3B, in indicated single color channels and as well as overlay. Arrow in lowest panel indicates cell with extensive Vpu-HcRed/CD63 colocalization.

Fgure S3 (.eps)