Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

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Abstract
We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated. Significance Trimeric forms of HIV-1 envelope glycoproteins are being used for structural and vaccine studies. The most common way to make these proteins is to eliminate the cleavage site between the glycoprotein (gp)120 and gp41 subunits. We show that doing so creates trimers that adopt irregular, nonnative configurations. Cleaved, stabilized trimers, in contrast, resemble the native spikes on the HIV-1 virus. Our findings will help structural and vaccine programs by showing how to make native-like trimers. The rationale for vaccine trials based on the use of uncleaved gp140 trimers should be reevaluated.

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