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294 Stabilizing Cleaved Forms of Env in Virus-Like Particles
J. M. Binley*1, A. Master2, L. Schiffner2, D. Schiller2, M. Paluch2, B. Travis3, S.-L. Hu3, W. Olson4, and J. P. Moore2
1The Scripps Res. Inst., La Jolla, CA, USA; 2Weill Med. Coll., Cornell Univ., New York, NY, USA; 3Univ. of Washington, Seattle, USA; and 4Progenics Pharmaceuticals, Tarrytown, NY, USA
Background: In light of the failure of gp120 and gp160 to elicit broadly neutralizing antibodies, modified forms of Env are being investigated. We previously generated a mutant (termed SOS) that encodes Env with a disulfide bridge between gp120 and gp41, stabilizing the cleaved Env complex that is normally prone to dissociation. We are now expressing SOS Env on the surface of virus-like particles (VLPs), in an attempt to generate near-native Env. To facilitate large-scale production of “SOS-VLPs”, we are using poxvirus expression vectors. These produce largely unprocessed Env, suggesting that cellular furin proteases were saturated by overexpression. Since processing is likely to be important for authentic folding, here we attempted to manipulate Env cleavage.
Methods: Approaches toward attaining complete gp120-gp41 cleavage of recombinant Env (isolate JR-FL) included: (1) coexpression of Env with furin, (2) in vitro enzymatic digestion of purified Env with furin, and (3) mutation of Env to improve its properties as a furin substrate.
Results: We found that coexpression of furin with Env leads to inefficient cleavage. In addition, it often leads to a significant reduction in Env expression. In vitro enzymatic digestion of purified Env preparations is also inefficient and insufficient to achieve complete cleavage. Introduction of mutant cleavage sites RRRRRR or RRRKRR in place of wild-type REKR led to enhanced endogenous cellular cleavage. Furthermore, coexpression of furin was able to complete the cleavage without causing a reduction in Env expression.
Conclusions: Here we have determined methods to obtain fully processed Env proteins. Since the natural furin cleavage site of HIV-1 appeared to be an inefficient furin substrate, we found that we could manipulate Env cleavage by introducing mutations to improve the properties of Env as a furin substrate. When combined with disulfide mutations to stabilize Env complexes, this will be useful in obtaining a new generation of VLP immunogens with modified Env proteins.
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