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, Env-activating 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 coreceptor-binding 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 i...