FIG. 2.

Schematic representation of envelope chimeras in which multiple Env domains were swapped. (A) Domain organization of parental Envs and chimeras. Open and solid boxes represent domains derived from amphotropic 4070A MLV Env (denoted A) and ecotropic MoMLV Env, respectively. The cytoplasmic sequences are shown as grey boxes and consist of the cytoplasmic tails and the p2R peptides. PRO (or PRR), proline-rich region; C, SU carboxy-terminaldomain; TM, transmembrane subunit; Anc, anchor domain. The first amino acids of each domain are indicated. (B) Results of cell-cell and virus-cell fusion assays. Cell-cell fusion activity was determined after transfection of the envelope expression vectors in TELac2 cells and cocultivation for 24 h with CHO-PiT2 (grey bars) or XC (black bars) indicator cells. The fusion index is defined in the legend to Fig. 1. Infectivity was tested with supernatants harvested from stably transfected packaging cells on different target cells (XC, Cear13, NIH 3T3, and TE671 cells) and is expressed as the number of LacZ infectious units per milliliter of viral supernatant. The values show the means ± standard deviations of up to six independent experiments performed on XC target cells (open bars). Identical results were obtained on the other target cells tested. As discussed previously (33, 51), the wild-type MoMLV glycoprotein and the BDPROMO chimera exhibited high cell-cell fusion (data not shown), associated with recognition of the ecotropic receptor, despite the stability of their Env complexes. na, not applicable. (C) Detection of envelope glycoproteins in pellets of retroviruses generated with the indicated Envs by immunoblotting in reducing and denaturing conditions with anti-SU and anti-TM antisera. Equivalent loading of viral samples was demonstrated by immunoblotting with an anti-capsid (CA) antiserum. The positions of the molecular size markers are shown. Expression of Env glycoproteins in producer cells is shown in the bottom blot by immunoblotting of cell lysates of Env-transfected cells with anti-SU antibodies.