Constructs used for the expression of reporter proteins in the mammalian system. (A) The expression vector used in this study. The env gene of HXB2 origin was codon-optimized for human genes. The nomenclatures are as follows: pCMV, cytomegalovirus promoter; CT, cytoplasmic tail; HaloTag, Halo7 gene; f1ori, replication origin of f1; Kan/NeoR, kanamycin or neomycin resistant gene; pER322ori, replication origin of pBR322 plasmid. The composition of the fusion protein used in the study was indicated below the plasmid map. The gp41 proteins with different C-terminal truncation points were fused to HaloTag at their C-terminus. The Halo-GPI, and Tac-Halo constructs and their expected membrane topology are shown schematically. (B) The result of immunoblotting with anti-HaloTag antibody. The names of the mammalian expression vector used are indicated above each lane. (C) Analysis of membrane fusion efficiency. The fusion activity of Halo-fused Env was evaluated by the syncytia-forming activity in 293CD4 cells. The percentage of the number of the nuclei included in syncytia was calculated by counting 300 nuclei in total. The constructs tested are indicated at the bottom of each bar; the number indicated the truncation points shown in Fig.1A.