Figure 2
Generation of HIV(CD4) particles. (A) HDPack1. Three deletions were made in pNL4-3 DNA, an infectious clone of HIV-1 [62], and the SV40 polyadenylation site was added to create the defective HIV-1 helper virus construct, HDPack1. HIV-1-Neo is a defective HIV-1 DNA construct derived from HIV-1 HXB2 DNA with part of env replaced by the SV40 promoter and neomycinr gene [63]. (B) and (C) Sucrose gradient fractionation of HIV(CD4) particles. HIV particles without CD4 or carrying either CD4 or CD4/gpi were isolated after transfection of HeLa, HeLaT4 and HeLaS2 cells with pHDPack1 DNA. The particles were concentrated by centrifugation onto a 65 % sucrose cushion followed by separation through a 15–60 % sucrose gradient. A portion of the particles isolated from HeLa cells was pretreated with Triton X-100 prior to centrifugation to solubilize the viral membrane. The p24 (B) and CD4 (C) concentrations of each gradient fraction were determined by ELISA. HIV(w/o CD4) particles (●), Triton X-100 treated HIV(w/o CD4) particles (X), HIV(CD4) (□) and HIV(CD4/gpi+) particles (△). (D) Immunoprecipitation of HIV(CD4) particles with anti-CXCR4 antibody. Pseudotype virions were isolated after co-transfections of HDPack1 and pCR3-CD4 DNA in either NIH 3T3 (□) and COS-7 (●) cells or human embryonic kidney 293 (△) and HeLa (X) cells. The amounts of immunoprecipitated p24 antigen were determined by ELISA and are shown as a percentage of the total amount of input p24 antigen.