The human immuno deficiency virus type 1 (HIV-1) envelope glycoprotein gp41 plays animportant role in the virus entry. During the process of fusion between the viral and target cell membranes, the N-and C-terminal heptad repeat (HR) regions of the gp41 extracellular domain associate to form a 6-helical bundle, corresponding to the fusion-active gp41 core. Any compound that blocks the gp41 6-helix bundle formation between the N- and C-peptides, which are derived from the N- and C-terminal HR regions, respectively, may inhibit HIV-1 mediated membrane fusion. Based on this principle, we previously established a sandwich enzyme-linked immunosorbent assay (ELISA) for drug screening by using the N-peptide N36 and the C-peptide C34 and a monoclonal antibody (NC-1) which specifically recognizes the gp41 6-helix bundle. In the present study, a fluorescence-linked immunosorbent assay (FLISA) was developed by using fluorescein isothiocyanate (FITC)-conjugated C34 to replace C34 and by directly detecting fluorescence intensity instead of more complicated enzymatic reaction. Compared with the sandwich ELISA, this FLISA has similar sensitivity and specificity, but it is much more rapid, economic and convenient. Using an Integrated Robotic Sample Processing System, this assay has been applied for high-throughput screening of organic compounds on a large scale for HIV-1 fusion inhibitors targeting gp41.
References
1.
1. Chan DC, Kim PS: HIV entry and its inhibition. Cell1998;93:681-684.
2.
2. Moore JP, Dragic T: HIV. See a pocket, block it. Nature1999;401:759-759.
3.
3. LaBranche CC, Galasso G, Moore JP, Bolognesi DP, Hirsch MS, Hammer SM: HIV fusion and its inhibition. Antiviral Res2001;50:95-115.
4.
4. Lu M, Blacklow SC, Kim PS: A trimeric structural domain of the HIV-1 transmembrane glycoprotein. Nat Struct Biol1995;2:1075-1082.
5.
5. Sattentau QJ, Moore JP: Conformational changes induced in the human immunodeficiency virus envelope glycoprotein by soluble CD4 binding. J Exp Med1991;174:407-415.
6.
6. Berger EA, Murphy PM, Farber JM: Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism, and disease. Annu Rev Immunol1999;17:657-700.
7.
7. Eckert DM, Kim PS: Mechanisms of viral membrane fusion and its inhibition. Annu Rev Biochem2001;70:777-810.
8.
8. Chan DC, Fass D, Berger JM, Kim PS: Core structure of gp41 from the HIV envelope glycoprotein. Cell1997;89:263-273.
9.
9. Weissenhorn W, Dessen A, Harrison SC, Skehel JJ, Wiley DC: Atomic structure of the ectodomain from HIV-1 gp41. Nature1997;387:426-428.
10.
10. Tan K, Liu J, Wang J, Shen S, Liu M: Atomic structure of a thermostable subdomain of HIV-1gp41. Proc Natl Acad Sci U SA1997;94:12303-12308.
11.
11. Wild C, Oas T, McDanal C, Bolognesi D, Matthews T: A synthetic peptide inhibitor of human immunodeficiency virus replication: correlation between solution structure and viral inhibition. Proc Natl Acad Sci U S A1992;89:10537-10541.
12.
12. Jiang S, Lin K, Strick N, Neurath AR: HIV-1 inhibition by a peptide. Nature1993;365:113-113.
13.
13. Wild CT, Shugars DC, Greenwell TK, McDanal CB, Matthews TJ: Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci U S A1994;91:9770-9774.
14.
14. Jiang S, Zhao Q, Debnath AK: Peptide and non-peptide HIV fusion inhibitors. Curr Pharm Des2002;8:563-580.
16. Lalezari JP, Henry K, O’Hearn M, et al: Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N Engl J Med2003;348:2175-2185.
17.
17. Lalezari JP, Eron JJ, Carlson M, et al: A phase II clinical study of the long-term safety and antiviral activity of enfuvirtide-based antiretroviral therapy. AIDS2003;17:691-698.
18.
18. Lu M, Kim PS: A trimeric structural subdomain of the HIV-1 transmembrane glycoprotein. J Biomol Struct Dyn1997;15:465-471.
19.
19. Jiang S, Lin K, Zhang L, Debnath AK: A screening assay for antiviral compounds targeted to the HIV-1 gp41 core structure using a conformation-specific monoclonal antibody. J Virol Methods1999;80:85-96.
20.
20. Jiang S, Lin K, Lu M: A conformation-specific monoclonal antibody reacting with fusion-active gp41 from the HIV-1 envelope glycoprotein. J Virol1998;72:10213-10217.
21.
21. Zhao Q, Ernst JT, Hamilton AD, Debnath AK, Jiang S: XTT formazan widely used to detect cell viability inhibits HIV type 1 infection in vitro by targeting gp41. AIDS Res Hum Retroviruses2002;18:989-997.
22.
22. Jiang S, Lin K, Neurath AR: Enhancement of human immunodeficiency virus type-1 (HIV-1) infection by antisera to peptides from the envelope glycoproteins gp120/gp41. JExpMed1991;174:1557-1563.
23.
23. Jiang S, Radigan L, Zhang L: A convenient cell fusion assay for rapid screening for HIV entry inhibitors. Proc SPIE2000;3926:212-219.
24.
24. Chou TC, Talalay P: Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul1984;22:27-55.
25.
25. Macarron R, Hertzberg RP: Designand implementation of high throughput screening assays. In Janzen WP (ed): High Throughput Screening: Method and Protocols. Totowa, NJ: Humanan, 2002:1-29.
26.
26. Oldenburg KR, Kariv I, Zhang J, Chung TDY, Lin S: Assay miniaturization: developing technologies and assay formats. In Seethala R, Fernandes PB (eds): Handbook of Drug Screening. New York: Marcel Dekker, 2001;525-562.
27.
27. Debnath AK: A “fragment fitting” approach to model disulfide loops by utilizing homologous peptide fragments from unrelated proteins of known structures: applicationto the V3 loop of the HIV-1 envelope glycoproteingp120. J Mol Modeling1997;3:31-47.
28.
28. Debnath AK, Radigan L, Jiang S: Structure-based identification of small molecule antiviral compounds targeted to the gp41 core structure of the human immunodeficiency virus type 1. J Med Chem1999;42:3203-3209.
29.
29. Murakami T, Zhang TY, Koyanagi Y, et al: Inhibitory mechanism of the CXCR4 antagonist T22 against human immunodeficiency virus type 1 infection. JVirol1999;73:7489-7496.
30.
30. Deen KC, McDougal JS, Inacker R, et al: A soluble form of CD4 (T4) protein inhibits AIDS virus infection. Nature1988;331:82-84.
31.
31. Smith DH, Byrn RA, Marsters SA, Gregory T, Groopman JE, Capon DJ: Blocking of HIV-1 infectivity by a soluble, secreted form of the CD4 antigen. Science1987;238:1704-1707.
32.
32. Okumura N, Aosai F, Yui K, et al: Cellular FITC-linked immunospecific assay (cell-FLISA) for detection of monoclonal antibodies against cell-surface antigens. Hum Immunol1986;15:97-108.
33.
33. Swartzman EE, Miraglia SJ, Mellentin-Michelotti J, Evangelista L, Yuan PM: A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology. Anal Biochem1999;271:143-151.
34.
34. Oelschlaeger P, Srikant-Iyer S, Lange S, Schmitt J, Schmid R: Fluorophor-linked immunosorbent assay: a time- and cost-saving method for the characterization of antibody fragments using a fusion protein of a single-chainantibody fragment and enhanced green fluorescent protein. Anal Biochem2002; 309:27-34.
35.
35. Komatsu N, Shichijo S, Maeda Y, Itoh K: Measurement of interferon-gamma by high-throughput fluorometric microvolume assay technology system. J Immunol Methods2002;263:169-176.
