We then cloned ORFs encoding mutant GP1s into plasmids expressing full-length MACV GPC. Retroviral pseudotypes were created as described [17, 26] : HEK 293T/17 cells were transfected by the calcium-phosphate method with plasmid encoding 1) MACV GPC and variants thereof, together with 2) the pQCXIX vector (Clonetech) expressing enhanced green fluorescent protein (eGFP) flanked by the Moloney murine leukemia virus (MoMLV) long terminal repeats, and 3) plasmid containing the MoMLV gag/pol genes. Cell supernatants were cleared and measured using the Retro-X TM qRT-PCR Titration kit (Clonetech). Equivalent amounts of pseudotypes were adsorbed on MACV-permissive cells for 2 h. After 48 h, cells were stained with Hoechst 33342 (Invitrogen) and the percentage of eGFP-positive cells was determined by high-content quantitative image-based analysis using an Opera confocal imager (Perkin Elmer). Cells exposed to mock pseudotypes (media of cells transfected with the pQCXIX vector and the MLV gag/pol plasmid) were used as negative controls. Western blot analysis of MACV GP2 expression in the producer cells (data not shown) and in the various MoMLV pseudotype preparations ( Fig. 3 lower panel) was performed using an antibody against MACV GP2 (New England Peptide) to examine expression, processing, and incorporation levels of wt MACV GPC and variants thereof. In agreement with the co-IP and cell-binding data, MoMLV carrying GPC mutants D114A, S116A, D140A, and K169A had no effect on entry into human (HeLa) and nonhuman primate (Vero) cells (Fig. 3) . Mutants D123A, D155A, P160A, F226A, K167A, and K211A had an intermediate effect on MACV entry, with the first four amino acids playing a more prominent role. Interestingly, MACV GPC mutant F226A had a much more pronounced effect on HeLa cell entry (.80% reduction) than on Vero cell entry (.40% reduction).