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Journal of Virology, August 2002, p. 7760-7776, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7760-7776.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Oligomeric and Conformational Properties of a Proteolytically Mature, Disulfide-Stabilized Human Immunodeficiency Virus Type 1 gp140 Envelope Glycoprotein

Norbert Schülke,1 Mika S. Vesanen,2 Rogier W. Sanders,2,{dagger} Ping Zhu,3 Min Lu,4 Deborah J. Anselma,1 Anthony R. Villa,1 Paul W. H. I. Parren,5 James M. Binley,2,{ddagger} Kenneth H. Roux,1 Paul J. Maddon,1 John P. Moore,2 and William C. Olson1*

Progenics Pharmaceuticals Inc., Tarrytown, New York 10591,1 Department of Microbiology and Immunology,2 Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021,4 Department of Biological Science and Structural Biology Program, Florida State University, Tallahassee, Florida 32306,3 Department of Immunology, The Scripps Research Institute, La Jolla, California 920375

Received 10 December 2001/ Accepted 4 May 2002

We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1JR-FL SOS gp140, proteolytically uncleaved gp140 (gp140UNC), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140UNC, SOS gp140 reacted more strongly in surface 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, gp140UNC displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41ECTO) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of 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 other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140UNC comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140UNC were largely unaffected by purification. An uncleaved gp140 protein 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 associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.

* Corresponding author. Mailing address: Progenics Pharmaceuticals Inc., 777 Old Saw Mill River Rd., Tarrytown, NY 10591. Phone: (914) 789-2800. Fax: (914) 789-2857. E-mail: olson{at}progenics.com.

{dagger} Present address: Department of Human Retrovirology, Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands.

{ddagger} Present address: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037.

Journal of Virology, August 2002, p. 7760-7776, Vol. 76, No. 15
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.15.7760-7776.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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