Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 278, Issue 22, Pages 20278-20285Publisher
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M301627200
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We have engineered two soluble, covalently linked, trimeric polypeptides, N35(CCG)-N13 and N34(CCG) comprising only the internal trimeric coiled-coil of the ectodomain of HIV-1 gp41. Both trimers inhibit human immunodeficiency virus, type 1 ( HIV-1) envelope (Env)mediated cell fusion at nanomolar concentrations by targeting the exposed C-terminal region of the gp41 ectodomain in the prehairpin intermediate state. The IC50 values for N35(CCG)-N13 and N34(CCG) are similar to15 and similar to95 nM, respectively, in a quantitative vaccinia virus-based reporter gene assay for HIV-1 Env-mediated cell fusion using Env from the T cell tropic strain LAV. Polyclonal antibodies were raised against N35(CCG)-N13 and a tightly binding fraction of anti-N35(CCG)-N13 inhibits T cell and macrophage tropic HIV-1 Env- mediated cell fusion with respective IC50 values of similar to0.5 and similar to1.5 mug/ml at 37 degreesC. The tightly binding anti-N35(CCG)-N13 antibody fraction targets the exposed internal trimeric coiled-coil in the prehairpin intermediate state of gp41 in a manner analogous to peptides derived from the C region of the gp41 ectodomain. The potency of the tightly binding anti-N35(CCG)-N13 antibody fraction in the fusion assay is comparable with that of the broadly neutralizing monoclonal antibody 2G12. These results indicate that N35(CCG)-N13 is a potential anti-HIV therapeutic agent and represents a suitable immunogen for the generation of neutralizing monoclonal antibodies targeted to the internal trimeric coiled-coil of gp41. The data on the tightly binding anti-N35(CCG)-N13 antibody fraction demonstrate that the internal trimeric coiled-coil of gp41 in the prehairpin intermediate state is accessible to antibodies and that access is not restricted by either antibody size or the presence of a kinetic barrier.
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