(A) Alignment of tetherin amino acid sequences from humans (HUM), chimpanzees (CPZ), rhesus macaques (MAC) and colobus monkeys (COL). Amino acid identity is indicated by dots and gaps by dashes. Differences between COL and other tetherins in the TM domain are highlighted by yellow boxes. Some known domains, the serine GPI anchor site (red box), and two potential glycosylation sites (underlined) are indicated. (B, C) Release of p24 capsid antigen (B) and infectious virus yield (C) from HEK293T cells following transfection with a ΔvpuΔnef proviral HIV-1 NL4-3 construct, SIVcol Nef or GFP only (nef*) expression constructs, and varying amounts of plasmids expressing human or Colobus tetherin. Virus release was determined by ELISA of cell-free and cell-associated p24 antigen and infectious virus was determined by infection of TZM-bl indicator cells. Both are shown as percentages of those detected in the absence of tetherin (100%) and all values were derived from triplicate experiments ± SEM.