The HIV fusion inhibitor Siamycin I, a 21-mer tricyclic peptide, was isolated from a Streptomyces culture using a cell fusion assay involving cocultivation of Hela-CD4 + cells and monkey kidney cells (BSC-1) infected with a vaccinia vector expressing gp160 (Ref. Tsunakawa, Hoshino, Detlefson, Hill, Furumai, Nishio, Kawano, Yamamoto, Fukagawa, Oki, submitted to J. Antibiotics). Siamycin I is effective against acute HIV-1 and HIV-2 infections in a cell protection assay with ED 5 0 s of 0.1-0.6 μM and a CC50 of 150 μM in CEM-SS cells. Inhibition appears to be specific, since Siamycin I will inhibit fusion between HIV chronically-infected CEM-SS and CD4 + -C8166 cells (ED 5 0 of 0.08 μM), while having no effect on Sendai virus-induced fusion or murine myoblast fusion. Siamycin I seems to bind non-covalently, since inhibition of HIV-induced fusion by this compound is reversible. Siamycin I does not inhibit gp120-CD4 binding in either gp120 or CD4 capture ELISAs or I 1 2 5 I-gp120-CD4 binding assays. To determine the anti-viral target of this compound, a variant of HIV resistant to Siamycin I was selected by in vitro passage of virus in the presence of increasing concentrations of the compound. Drug sensitivity studies of chimeric virus and pseudovirus containing the envelope gene from resistant virus suggest that the gp160 domain may be responsible for resistance. Further DNA sequencing of the envelope gene from the resistant and parent viruses revealed a total of 6 amino acid changes in the gp160 domain. Confirming the significance of these substitutions in resistance development using site-directed mutagenesis is in progress.