Determining the co-receptor usage of C19 and C27 Env clones introduced into HIV-1 NL4-3 backbone via yeast-based recombination. The 26 C19 and 21 C27 env regions (same as those sequenced for Fig. 1) were cloned into pREC_env/URA3 via yeast-based recombination/gap repair [81, 82]. A 132 and 118 amino acid sequence of the C2-V3 region was used to group the C19 (a) and C27 (b) based on sequence identity/difference. The pREC_env expression vector was then used to pseudotype virus produced from 293T cells co-transfected with the pNL luc-AM vector . Equal virus titers (based on RT activity) were used to infect U87.CD4 cells expressing either CCR5 or CXCR4. The infecting virus is reverse transcribed and integrated to express luciferase but is incapable of subsequent rounds of infection . Luciferase activity (relative light units) was measured from lysed cells collected 72 h post infection with both the C19 (c) and C27 (d) Env pseudotyped virus infections. All assays were performed in triplicate. Background was subtracted from results and dotted lines on c, d represent the value three times the standard deviation of the background.