Abstract
Putative cell surface human immunodeficiency virus (HIV) gp41 receptor proteins of 45 and 80 kDa (p45 and p80, respectively) were identified on human cells using a 17-amino acid peptide, referred to as CS3. In contrast, murine P815 cells expressed a peptide binding protein of 80 kDa only. A segment of 8 amino acids within CS3 contains the minimum sequence able to inhibit binding of radiolabeled CS3 to p80 and p45, as shown by competitive binding studies. Human p45 was purified from CD4+ RH9 cells by CS3 peptide affinity chromatography. Human p80 was partially purified from RH9 cell lysates by size exclusion chromatography followed by SDS-polyacrylamide gel electrophoresis; a rabbit polyclonal antibody was raised against this preparation. Anti-p80 antibody inhibited HIV infection in a dose-dependent manner. The CS3 region of gp41 has been been shown previously to be exposed on viral particles and envelope-expressing cells predominately after conformational changes in the HIV envelope occur due to the interaction of CD4 with gp120. These results, together with those from previous studies, suggest that following the interaction of gp120 with CD4, there may be a second receptor interaction necessary for virus entry/fusion.
Highlights
From the $GuthieResearch Institute, Sayre, Pennsylvania 18840 and the %Department of Pathology and Laboratory Medicine, Beth Israel Medical Center, New York, New York 10003
Putative cellsurface human immunodeficiency virus site may play some role in viral entry [11,12,13,14,15]
A (HIV) gp41 receptor proteins of 4 5 and 80 kDa (p45 putative receptor interaction site at a defined region within and p80, respectively) were identified on human cells thetransmembrane glycoprotein,gp41 [16],as well asseusing a 17-amino acid peptide, referred to as CS3. In quences at both the amino [17] ancdarboxyl [18,19] termini contrast, murine P815 cells expresseda peptide bind- of gp41, may be important in viral entry, the formation of ing protein of 80 kDa only.A segment of 8 amino acids syncytia, or as part of the virus within CS3 contains the minimum sequence able to inhibit binding of radiolabeled CS3 to p80 and p45, as shown by competitive binding studies
Summary
With an aliquot of the peptide taken prior to coupling. One million RH9 cells were radiolabeled with 1 mCi of NalZ5Iin tubes precoated. It is possible that the relative detection of p45 or p80 may be influenced by association with the cross-linking moiety APG, rather thanwith the peptide CS3 To determine on human cells cultured inmedium alone or medium containwhether free and carrier-conjugated CS3 interacted with the ing mitogen to learn more about theregulation of CS3 binding same cell surface proteins, unlabeled CS3 or CS3-HSA was sites during lymphocyte activation. Human peripheral blood used to competitively inhibit thebinding of '*'II-CS3.CS3 and lymphocytes were activated with PHA or PMA, harvested, CS3-HSA blocked binding of'*'I-CS3 to both the p45 and and analyzed by flowcytometry for the amount of CS3 binding p80 proteins (Fig. 2).
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