USE OF A GREEN FLUORESCENT PROTEIN AS A MARKER FOR HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 INFECTION

We constructed a recombinant human immunodeficiency virus type 1 (HIV- 1) provirus called R7-GFP that expresses a modified form of a green fl uorescent protein (GFP) from the jellyfish Aequorea victoria by substi tuting GFP-coding sequences for Nef-coding sequences, Alanine was subs tituted for serine at amino acid position 65 in the modified GFP, resu lting in markedly increased fluorescence at an excitation wavelength o f 488 nm as compared to wild-type GFP, The replication kinetics of R7- GFP were identical to that measured with an isogenic, nef-negative str ain lacking GFP, Expression of GFP by replication-competent HIV-1 allo wed simultaneous quantitation of viral infection and cell surface CD4 levels, revealing rapid and nearly complete CD4 downregulation on R7-G FP-infected PBMCs.