Analysis of the Interaction between the HIV-Inactivating Protein Cyanovirin-N and Soluble Forms of the Envelope Glycoproteins gp120 and gp41
- Barry R. O'Keefe1,
- Shilpa R. Shenoy1,
- Dong Xie2,
- Wentao Zhang3,
- Jeffrey M. Muschik1,
- Michael J. Currens1,
- Irwin Chaiken3 and
- Michael R. Boyd1
- 1Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis (B.R.O., S.P.S., J.M.M., M.J.C., M.R.B) and 2Structural Biochemistry Program (D.X.), SAIC Frederick, Frederick Cancer Research and Development Center, Frederick, Maryland; and 3Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania (W.Z., I.C.)
Abstract
The novel virucidal protein cyanovirin-N (CV-N) binds with equally high affinity to soluble forms of either H9 cell-produced or recombinant glycosylated HIV-1 gp120 (sgp120) or gp160 (sgp160). Fluorescence polarization studies showed that CV-N is also capable of binding to the glycosylated ectodomain of the HIV-envelope protein gp41 (sgp41) (as well as SIV glycoprotein 32), albeit with considerably lower affinity than the sgp120/CV-N interaction. Pretreatment of CV-N with either sgp120 or sgp41 abrogated the neutralizing activity of CV-N against intact, infectious HIV-1 virions. Isothermal calorimetry and optical biosensor binding studies showed that CV-N bound to recombinant sgp120 with a Kd value ranging from 2 to 45 nM and to sgp41 with a Kd value of 606 nM; furthermore, they indicated an approximate 5:1 stoichiometry for CV-N binding to sgp120 and a 1:1 stoichiometry for CV-N binding to sgp41. Circular dichroism studies additionally illuminated the binding of CV-N with both sgp120 and sgp41, providing the first direct evidence that conformational changes are a consequence of CV-N interactions with both HIV-1 envelope glycoproteins.
Footnotes
-
Send reprint requests to: Dr. Michael R. Boyd, Laboratory of Drug Discovery Research and Development, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, Frederick Cancer Research and Development Center, Bldg. 1052, Rm. 121, Frederick, MD 21702-1201. E-mail: boyd{at}dtpax2.ncifcrf.gov
-
The work of W.Z. and I.C. was supported by Grant P01-GM56550-01 from the National Institutes of Health. J.M.M. was a member of the National Institutes of Health Student Research Training Program, 1997. This article is number 65 in the NCI Laboratory of Drug Discovery Research and Development series “HIV-Inhibitory Natural Products”; for part 64, see Meragelman et al. (2000).
- Abbreviations:
- Env
- HIV envelope glycoproteins
- gp120
- glycoprotein 120 from HIV
- gp41
- glycoprotein 41 from HIV
- gp160
- glycoprotein 160 from HIV
- CV-N
- cyanovirin-N
- ELISA
- enzyme-linked immunosorbent assay
- SIV
- simian immunodeficiency virus, FIV, feline immunodeficiency virus
- sCD4
- soluble CD4
- gp32
- glycoprotein 32 from SIV
- CD
- circular dichroism
- BODIPY
- 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene
- TPBS
- PBS augmented with 0.05% Tween 20
- ITC
- isothermal titration calorimetry
- XTT
- 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide
-
- Received April 8, 2000.
- Accepted July 24, 2000.
- U.S. Government
