Novel screening systems for HIV-1 fusion mediated by two extra-virion heptad repeats of gp41.
Entry of human immunodeficiency virus type 1 (HIV-1) into target cells is mediated by its envelope protein gp41 through membrane fusion. Interaction of two extra-virion heptad repeats (HRs) in the gp41 plays a pivotal role in the fusion, and its inhibitor, enfuvirtide (T-20), blocks HIV-1 entry. To identify agents that block HIV-1 fusion, two screening methods based on detection and quantification by the enzyme-linked immunosorbent assay (ELISA) principle have been established. One method uses an alkaline phosphatase (ALP)-conjugated antibody (Ab-ELISA) and the other uses an ALP-fused HR (F-ELISA) to detect and quantify the interaction of the two HRs. The F-ELISA was more simple and rapid, since no ALP-conjugated antibody reaction was required. Both ELISAs detected all the fusion inhibitors tested except for T-20. Interaction of the two HRs was observed in both ELISAs, even in the presence of 10% dimethyl sulfoxide. Ab-ELISA performed best in a pH ranging from 6 to 8, while F-ELISA performed best at a pH ranging from 7 to 8. These results indicate that both established ELISAs are suitable for the identification of HIV-1 fusion inhibitors.
Nishikawa
Hiroki
H
Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Kodama
Eiichi
E
Sakakibara
Ayako
A
Fukudome
Ayako
A
Izumi
Kazuki
K
Oishi
Shinya
S
Fujii
Nobutaka
N
Matsuoka
Masao
M
D000469:Alkaline Phosphatase / Q000737:chemistry; D000595:Amino Acid Sequence; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000378:metabolism; D023581:HIV Fusion Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology*; D015497:HIV-1 / Q000187:drug effects / Q000472:pathogenicity; D006801:Humans; D008561:Membrane Fusion / Q000187:drug effects*; D020449:Repetitive Sequences, Amino Acid / Q000235:genetics*; D014771:Virion / Q000737:chemistry
D015700:HIV Envelope Protein gp41; D023581:HIV Fusion Inhibitors; D000469:Alkaline Phosphatase