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125 Identification of a Fusion Intermediate Conformation of Soluble HIV Envelope gp41
M. Alam*1, H.-X. Liao1, C. Paleos1, S. Stauffer1, R. Scearce1, P. Ear2, and B. Haynes1
1Duke Med. Ctr., Durham, NC, USA and 2 NIAID, NIH, Bethesda, MD, USA
Background: An unresolved problem in HIV vaccine development is the design of an immunogen that will induce antibodies that broadly neutralize HIV. One strategy is to design immunogens that mirror envelope structures that are transiently present following envelope binding by CD4 and/or coreceptor, but before viral-host membrane fusion. One such conformation has been postulated to be that of an "uncoiled" coiled-coil gp41 region, wherein the 2 heptad repeat regions, HR-1 and HR-2, are unopposed and are available for binding by soluble HR-1 or HR-2.
Methods: We have used surface plasmon resonance (SPR) measurements on the BIAcore as well as immunoprecipitation experiments with biotinylated HR-2 peptides to determine if "uncoiled" conformations of gp41 are present in vaccinia-expressed HIV 89.6 gp140.
Results: In SPR assays, we found that gp41 in cleaved gp140 (a mixture of gp41, gp140, and gp120) bound HR-2 peptides in the presence of soluble (s) CD4. As controls, HR-2 peptides bound at background levels to purified HIV 89.6 gp120 bound to sCD4, and scrambled HR-2 peptides did not bind to gp140 bound to sCD4. Immunoprecipitation of complexes of sCD4 and vaccinia-produced HIV 89.6 cleaved gp140 with biotinylated HR-2 peptide demonstrated reactivity of the HR-2 peptide with both gp41 and uncleaved gp140.
Conclusions: Thus, in SPR assays, gp140 can be induced by sCD4 to bind HR-2 peptide, and is postulated to assume an "uncoiled" conformation in sCD4-triggered vaccinia-produced cleaved gp140 preparations.
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