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Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1

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JOURNAL OF VIROLOGY, Aug. 2002, p. 7760–7776 Vol. 76, No. 150022-538X/02/$04.00⫹0 DOI: 10.1128/JVI.76.15.7760–7776.2002Copyright © 2002, American Society for Microbiology. All Rights Reserved.Oligomeric and Conformational Properties of a Proteolytically Mature,Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140Envelope GlycoproteinNorbert Schu¨lke,1Mika S. Vesanen,2Rogier W. Sanders,2† Ping Zhu,3Min Lu,4Deborah J. Anselma,1Anthony R. Villa,1Paul W. H. I. Parren,5James M. Binley,2‡ Kenneth H. Roux,1Paul J. Maddon,1John P. Moore,2and William C. Olson1*Progenics Pharmaceuticals Inc., Tarrytown, New York 105911; Department of Microbiology and Immunology2and Department ofBiochemistry,4Weill Medical College of Cornell University, New York, New York 10021; Department of Biological Science andStructural Biology Program, Florida State University, Tallahassee, Florida 323063; and Department of Immunology,The Scripps Research Institute, La Jolla, California 920375Received 10 December 2001/Accepted 4 May 2002We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized byan intersubunit disulfide bond. HIV-1JR-FLSOS gp140, proteolytically uncleaved gp140 (gp140UNC), and gp120were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structuralproperties before and after purification. Compared with gp140UNC, SOS gp140 reacted more strongly insurface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies(MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140UNCdisplayed the greater reactivity with nonneutralizing anti-gp120 andanti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41ectodomain (gp41ECTO) occludes the “nonneutralizing” face of gp120, consistent with the antigenic propertiesof this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE),a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and otherbiophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140UNCcomprised a mixture ofnoncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conforma-tional properties of SOS gp140 and gp140UNCwere largely unaffected by purification. An uncleaved gp140protein containing the SOS cysteine mutations (SOS gp140UNC) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associ-ated oligomers that were significantly more stable than the full-length protein. Overall, our findings haverelevance for rational vaccine design.The native, fusion-competent form of the human immuno-deficiency virus type 1 (HIV-1) envelope glycoprotein complexis a trimeric structure composed of three gp120 subunits andthree gp41 subunits; the receptor-binding (CD4 and corecep-tor) sites are located in the gp120 moieties, and the fusionpeptides are located in the gp41 components (10, 33, 34, 52, 69,78, 83). In the generally accepted model of HIV-1 fusion, thesequential binding of gp120 to CD4 and a coreceptor inducesa series of conformational changes in the gp41 subunits, lead-ing to the insertion of the fusion peptides into the host cellmembrane in a highly dynamic process (14, 31, 39, 59, 68, 72,81, 84, 91). The associations between the six components of thefusion-competent complex are maintained via noncovalent in-teractions between gp120 and gp41 and between the gp41subunits (52, 84). These interactions are relatively weak, mak-ing the fusion-competent complex unstable. This instabilityperhaps facilitates the conformational changes in the variouscomponents that are necessary for the fusion reaction to pro-ceed efficiently, but it greatly complicates the task of isolatingthe native complex in purified form. Put simply, the nativecomplex falls apart before it can be purified, leaving only thedissociated subunits.One reason it would be desirable to produce the nativeHIV-1 envelope complex is to explore its potential as an im-munogen, perhaps after modification to improve its exposureof critical neutralization epitopes. The limited neutralizing-antibody response to HIV-1 in infected people is directed atthe native complex and is probably raised against it (6, 40, 49,51); P. W. H. I. Parren, D. R. Burton, and Q. J. Sattentau,Letter, Nat. Med. 3:366, 1997). In contrast, the isolated sub-units have not proven efficient at inducing relevant neutralizingantibodies (reviewed in references 6, 49, and 51). We andothers are therefore attempting to make more-stable forms ofthe envelope glycoprotein complex that better mimic the nativestructure. Usually, these efforts have focused on making vari-ous forms of soluble gp140 glycoproteins which contain gp120but only the ectodomain of gp41 (4, 11, 13, 17, 19–21, 57, 66,76, 85–87, 90).* Corresponding author. Mailing address: Progenics Pharmaceuti-cals Inc., 777 Old Saw Mill River Rd., Tarrytown, NY 10591. Phone:(914) 789-2800. Fax: (914) 789-2857. E-mail: [email protected]† Present address: Department of Human Retrovirology, AcademicMedical Center, University of Amsterdam, 1105 AZ Amsterdam, TheNetherlands.‡ Present address: Department of Immunology, The Scripps Re-search Institute, La Jolla, CA 92037.7760An approach to resolving the instability of the native com-plex is to remove the cleavage site that naturally exists betweenthe gp120 and gp41 subunits. Doing so means that proteolysisof this site does not occur, leading to the expression of gp140glycoproteins in which the gp120 subunit is covalently linked tothe gp41 ectodomain (gp41ECTO) by means of a peptide bond(2, 3, 16–18). Such proteins can be oligomeric, sometimestrimeric (11, 16–21, 54, 66, 85–87, 90). However, it is not clearthat they truly represent the structure of the native, fusion-competent complex in which the gp120-gp41 cleavage site isfully utilized. Hence the receptor-binding properties of un-cleaved gp140 (gp140UNC) proteins tend to be impaired, andnonneutralizing antibody epitopes are exposed on them thatprobably are not accessible on the native structure (4, 6, 28, 60,90).We have taken an


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