Statistical Learning by 8 - Month - Old Infants Jenny

1. F. Cocchi et al., Science 270, 1811 (1995). 2. Y. Feng, C. C. Broder, P. E. Kennedy, E. A. Berger, ibid. 272, 872 (1996). 3. M. Samson, 0. Labbe, C. Mollereau, G. Vassart, M. Parmentier, Biochemistry 35, 3362 (1996); C. J. Raport, J. Gosling, V. L. Schweickart, P. W. Gray, I. F. Charo, J. Biol. Chem. 271, 17161 (1996). 4. H. Choe et al., Cell 85, 1135 (1996); B. J. Doranz et al., ibid., p. 1 149. 5. T. Dragic et al., Nature 381, 667 (1996); H. Deng et al., ibid., p. 661; G. Alkhatib et al., Science 272,1955 (1996). 6. S. Gartner et al., Science 233, 215 (1986). 7. R. Atchison et al., unpublished observations. 8. L. Boring et al., J. Biol. Chem. 271, 7551 (1996). 9. We cloned cDNAs encoding human or murine CCR5 into the expression vector pcDNA3 (Invitrogen) after engineering the FLAG epitope into the NH2-terminus as described (13). Expression of each construct was determined by FACS with an antibody to FLAG (antiFLAG) (Boehringer Mannheim), and relative expression for each (see below) was calculated as the percentage of cells expressing human CCR5 on the cell surface normalized to the expression of hCCR5 (defined as 100%), with standard errors of the mean. The mean fluorescence intensity of the positive cells from any single sample never varied from the average by more than 30% in a single experiment. Therefore, neither the relative number of positive cells nor the absolute expression levels within transfected cells explains the differences in coreceptor activity. Chimeric receptors were prepared by the overlap polymerase chain reaction (PCR) method (17). hCCR5 (HHHH), human CCR5 (1 00% relative expression); mCCR5 (MMMM), murine CCR5 (126 + 49%); HMMM, NH2-terminus of human CCR5 [amino acids (aa) 1 to 32] fused to murine CCR5 (aa 35 to 354) (77 + 22%); MHHH, NH2-terminus of murine CCR5 (aa 1 to 34) fused to human CCR5 (aa 33 to 352) (73 + 17%); MHMM, extracellular loop 1 and a portion of transmembrane domain 3 of human CCR5 (aa 86 to 118) replacing the corresponding segment of the murine receptor (aa 88 to 120) (37 + 22%); MMHM, extracellular loop 2 and adjacent portions of human CCR5 (aa 134 to 210) replacing the corresponding region of the murine receptor (aa 136 to 212) (81 + 30%); MMHH, NH2-terminal half of mCCR5 (aa 1 to 162) fused to the COOH-terminal half of hCCR5 (aa 161 to 352) (80 + 39%). 10. I. F. Charo et al., Proc. Natl. Acad. Sci. U.S.A. 91, 2752 (1994). 11. C. Franci, L. M. Wong, J. Van Damme, P. Proost, I. F. Charo, J. Immunol. 154, 6511 (1995). 12. We cloned cDNAs encoding human CCR2B or chimeras into the expression vector pCMV4 (18) after engineering the FLAG epitope into the NH2-terminus as described (13). Expression of each construct (see below) was determined as described earlier. Chimeric receptors were prepared by the overlap PCR method (17). 5555, human CCR5 (100% relative expression); 2222, human CCR2B (87 + 2%); 5222, NH2-terminus of CCR5 (aa 1 to 32) fused to CCR2B (aa 45 to 360) (27 + 5%); 2555, NH2-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%). 13. F. S. Monteclaro and 1. F. Charo, J. Biol. Chem. 271, 19084 (1996); F. S. Monteclaro et al., unpublished observations. 14. J. Gosling et al., unpublished observations. 15. P. J. Maddon et al., Cell 47, 333 (1986). 16. P. Lores et al., AIDS Res. Hum. Retroviruses 8, 2063 (1992). 17. S. N. Ho, H. D. Hunt, R. M. Horton, J. K. Pullen, L. R. Pease, Gene 77, 51 (1989). 18. S. Andersson, D. L. Davis, H. Dahlback, H. Jornvall, D. W. Russell, J. Biol. Chem. 264, 8222 (1989). 19. M. A. Goldsmith, M. T. Warmerdam, R. E. Atchison, M. D. Miller, W. C. Greene, J. Virol. 69, 4112 (1995). 20. COS-7 cells were transfected with 2 pLg of plasmid DNA per well in a six-well plate as described (19). DNA samples consisted of appropriate combinations of 0.5 p.g of a human CD4 expression plasmid [pCD4Neo (19)] or plain vector, and 1.5 ,g 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 macrophages). 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 phycoerythrin (PE)-conjugated antiCD4 (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. 21. H. Arai and 1. F. Charo, J. Biol. Chem. 271, 21814 (1996). 22. 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 (M.A.G.).