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2-terminus of CCR2B (aa 1 to 44) fused to CCR5 (aa 33 to 352) (108 ± 17%); 2255, CCR2B (aa 1 to 136) fused to CCR5 (aa 124 to 352) (119 ± 33%).
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note
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COS-7 cells were transfected with 2 μg of plasmid DNA per well in a six-well plate as described (19). DNA samples consisted of appropriate combinations of 0.5 μg of a human CD4 expression plasmid [pCD4Neo (19)] or plain vector, and 1.5 μg of a chemokine receptor-expressing plasmid or plain vector. About 30 hours after addition of DNA, the medium in each well was replaced with 1.0 ml of medium containing HIV-1 Ba-L (∼100 to 170 ng of p24 per sample; source: NIH AIDS Reagent Repository, passaged on primary human macrophagesl. About 10 hours later, an additional 1.0 ml of medium was added to each well. After 30 hours, the cells were recovered from the dish as described (19) and analyzed with a FacScan (Becton Dickinson). Staining for intracytoplasmic HIV-1 p24 was carried out with the Fix and Perm reagents (Caltag Laboratories), with a monoclonal antibody to p24 (Coulter Immunology) and goat anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Becton Dickinson). Cells were further stained with phycoerylhrin (PE)-conjugated anti-CD4 (Becton Dickinson]. Appropriate controls indicated that the appearance of double-positive cells (FITC + PE) was dependent on cotransfection with both CD4 and human CCR5 expression plasmids and on the presence of HIV-1 Ba-L.
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27
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12644294998
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note
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We acknowledge the advice of M. Warmerdam (transfection-infection assay), E. Weider (FACS studies), and L. Boring, H. Arai, and R. Speck (scientific interpretation). We appreciate the assistance of J. Carroll and M. Ceniceros in the preparation of this manuscript. Supported in part by NIH grant HL52773 (I.F.C.) and by Pfizer (MAG.).
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