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Journal of Virology, March 2004, p. 2265-2276, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2265-2276.2004

Promoting Trimerization of Soluble Human Immunodeficiency Virus Type 1 (HIV-1) Env through the Use of HIV-1/Simian Immunodeficiency Virus Chimeras

Rob J. Center,1* Jacob Lebowitz,2 Richard D. Leapman,2 and Bernard Moss1*

Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases,1 Division of Bioengineering and Physical Science, Office of Research Services, National Institutes of Health, Bethesda, Maryland 208922

Received 11 July 2003/ Accepted 14 November 2003

The envelope proteins (Env) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) form homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained, but is less stable, after cleavage in a Golgi compartment and transport to the surface of infected cells. Functional, virion-associated HIV-1 and SIV Env have an almost exclusively trimeric structure. In addition, a soluble form of SIV Env (gp140) forms a nearly homogeneous population of trimers. Here, we describe the oligomeric structure of soluble, uncleaved HIV-1 gp140 and modifications that promote a stable trimeric structure. Biochemical and biophysical analyses, including sedimentation equilibrium and scanning transmission electron microscopy, revealed that unmodified HIV-1 gp140 purified as a heterogeneous range of oligomeric species, including dimers and aggregates. Deletion of the V2 domain alone or, especially, both the V1 and V2 domains reduced dimer formation but promoted aggregation rather than trimerization. Expressing gp140 with mannose-only oligosaccharides did not eliminate heterogeneity. Replacement of the entire gp41 segment of HIV-1 gp140 or just the N-terminal half (85 amino acids) of this segment with the corresponding region of SIV was sufficient to confer efficient trimerization for gp140 derived from clade B and C isolates. Importantly, the relatively small segment of the HIV Env replaced by SIV sequences contains no known targets of neutralizing antibody. The soluble trimeric form of HIV-1 Env should prove useful for assessment of antigenic structure and immunogenicity.

* Corresponding author. Mailing address for B. Moss: Laboratory of Viral Diseases, National Institutes of Health, Building 4, Room 229, 9000 Rockville Pike, Bethesda, MD 20892. Phone: (301) 496-9869. Fax: (301) 480-1147. E-mail: bmoss{at}nih.gov. Mailing address for R. J. Center: Department of Microbiology and Immunology, University of Melbourne, Parkville, Victoria 3010, Australia. Phone: 613-8344-9779. Fax: 613-9347-1540. E-mail: rcenter{at}unimelb.edu.au.

Journal of Virology, March 2004, p. 2265-2276, Vol. 78, No. 5
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.5.2265-2276.2004




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