Induction of a Tier-1-Like Phenotype in Diverse Tier-2 Isolates by Agents That Guide HIV-1 Env to Perturbation-Sensitive, Nonnative States

Abstract

The envelope glycoproteins (Envs) on the surfaces of HIV-1 particles are targeted by host antibodies. Primary HIV-1 isolates demonstrate different global sensitivities to antibody neutralization; tier-1 isolates are sensitive, whereas tier-2 isolates are more resistant. Single-site mutations in Env can convert tier-2 into tier-1-like viruses. We hypothesized that such global change in neutralization sensitivity results from weakening of intramolecular interactions that maintain Env integrity. Three strategies commonly applied to perturb protein structure were tested for their effects on global neutralization sensitivity: exposure to low temperature, Envactivating ligands, and a chaotropic agent. A large panel of diverse tier-2 isolates from clades B and C was analyzed. Incubation at 0°C, which globally weakens hydrophobic interactions, causes gradual and reversible exposure of the coreceptorbinding site. In the cold-induced state, Envs progress at isolate-specific rates to unstable forms that are sensitive to antibody neutralization and then gradually lose function. Agents that mimic the effects of CD4 (CD4Ms) also induce reversible structural changes to states that exhibit isolate-specific stabilities. The chaotropic agent urea (at low concentrations) does not affect the structure or function of native Env. However, urea efficiently perturbs metastable states induced by cold and CD4Ms and increases their sensitivity to antibody neutralization and their inactivation rates Therefore, chemical and physical agents can guide Env from the stable native state to perturbation-sensitive forms and modulate their stability to bestow tier-1-like properties on primary tier-2 strains. These concepts can be applied to enhance the potency of vaccine-elicited antibodies and microbicides at mucosal sites of HIV-1 transmission.IMPORTANCE An effective vaccine to prevent transmission of HIV-1 is a primary goal of the scientific and health care communities. Vaccine-elicited antibodies target the viral envelope glycoproteins (Envs) and can potentially inhibit infection. However, the potency of such antibodies is generally low. Single-site mutations in Env can enhance the global sensitivity of HIV-1 to neutralization by antibodies. We found that such a hypersensitivity phenotype can also be induced by agents that destabilize protein structure. Exposure to 0°C or low concentrations of Env-activating ligands gradually guides Env to metastable forms that expose cryptic epitopes and that are highly sensitive to neutralization. Low concentrations of the chaotropic agent urea do not affect native Env but destabilize perturbed states induced by cold or CD4Ms and increase their neutralization. The concept of enhancing antibody sen-

Effects of the SOS (A501C/T605C) and DS (I201C/A433C) Disulfide Bonds on HIV-1 Membrane Envelope Glycoprotein Conformation and Function

“…The metastability of Env creates immense challenges for structural studies and for the creation of vaccine immunogens. The pretriggered state 1 Env readily transitions into lower-energy conformational states upon removal from its native membrane environment (39)(40)(41)(42). Soluble Env trimers lacking the transmembrane region and cytoplasmic tail are labile and easily dissociate into monomers and dimers (39,(43)(44)(45)(46).…”

Properties of HIV-1 env and human seminal fluid that determine virus inhibition by antibodies and microbicides

“…The above data demonstrate that Envs of diverse primary HIV-1 isolates have different sensitivities to changes in membrane lipid content. Interestingly, the 89.6 Env, which is the most sensitive of the panel to membrane lipid changes, was also recently shown to be unstable relative to the Envs of other primary isolates (1). Envs of diverse HIV-1 strains differ considerably in their inherent level of stability (77,87,141).…”

“…Stable Envs maintain a 'closed' conformation wherein cryptic epitopes that overlap the coreceptor-binding site (CoR-BS) are not exposed. Additionally, stable Envs are more resistant to the effects of treatments that perturb protein structure, including cold denaturation and inhibitory ligands (85,246). We hypothesized that sensitivity of HIV-1 to membrane lipid changes is affected by stability of Env.…”

“…Two mutations in the 89.6 Env were shown to increase trimer stability: (i) Met to Ile at position 225 in the second conserved (C2) region of gp120, and (ii) Arg to Glu at position 305 of the third variable (V3) loop (247,248). Both changes increase resistance to cold denaturation (246) and global resistance to antibody neutralization (146,246). Combination of these mutations changes the conformation of Env to a closed form, which does not expose the CoR-BS epitope targeted by mAb 17b (Fig.…”

“…Interestingly, we found that the high content of the monosaccharide fructose in human seminal plasma reduces the potency of broadlyacting antibodies and microbicidal agents that target high-mannose glycans on the Env protein. Chapter II is modified from a manuscript accepted for publication (1). Chapters III, IV and the Appendix are revised for publication.…”