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CD4-chemokine receptor pseudotyped particles were produced by cotransfecting QT6 cells with three plasmids: (i) 10 μg pNL4-3-Iuc-E-R-(13), (ii) 10 μg of pT4, and (iii) 10 μg of either pCXCR4-cDNA3 or pCCR5-cDNA3. Control particles were produced by substituting pcDNA3 for plasmids encoding CD4 or a chemokine receptor. Forty-eight hours after transfection, medium was harvested, filtered (0.22 μm), aliquoted, and stored at -80°C. Pseudotyped virus was standardized by a p24 assay (Dupont).
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Supematants from transfected cells (7) were titered onto CEMx174 cells that were chronically infected by HIV-1 or SIV isolates (Fig. 1A), and luciferase activity was determined. Viral stocks were concentrated by pelleting conditioned medium for 2 hours at 50,000g.
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This work is dedicated to the memory of B. J. Endres. We thank P. Endres, L. Brass, M. Gardner, and M. Nathanson for support and encouragement; T. Keler, N. Yeilding, A. Tehranian, N. Leung, and M. Hitchcock for helpful discussions; N. Landau, R. Doms, D. Gabuzda, R. Collman, I. Verma, G. Cohen, F. Gonzalez-Scarano, V. Planelles, and P. Luciw for generously providing reagents; and J. Romano for expert technical assistance. M.J.E. was supported with a postdoctoral fellowship (NIH HL 07439). B.J.D. was supported with a predoctoral fellowship (Howard Hughes Medical Institute). Additional work was supported by grants to J.A.H. (AI33854 and AI40880) and D.L.K. (R29 NS35007). J.A.H. is a fellow of Cater-Wallace, Inc.
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