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Viral entry of HIV-1 is mediated by the envelope glycoprotein (Env), which consists of gp120 and gp41 trimer subunits. Entry begins when gp120 binds the CD4 receptor on the host cell. This induces conformational changes that expose the binding site for the CCR5 or CXCR4 chemokine co-receptors. Subsequently, heptad repeat 1 (HR1) and heptad repeat 2 (HR2) of gp41 self-assemble to form a 6-helix bundle (6HB) that drives membrane fusion needed for viral entry. Previously, we identified HR1 peptide resistant Envs with key resistance mutations in HR1 or HR2 of gp41 that impact 6HB stability. These key gp41 resistance mutations defined 2 resistance pathways that were each associated with additional mutations in gp120 and gp41. Here, we further characterized the relative contribution of individual gp120 and gp41 mutations on Env conformational structure and sensitivity to CD4-induced conformational changes. Mutant Envs were assessed using a panel of conformation-dependent broadly neutralizing antibodies, temperature sensitivity studies, and soluble CD4-mediated entry into CD4-CCR5+ cells. Our data show that Envs from both resistance pathways have relaxed (more open) trimer conformations in their native state that is primarily mediated by individual mutations in gp120. Despite increased sensitivity to CD4 neutralization, gp41 mutations decrease conformational reactivity to CD4 binding by altering the transition of Envs from a fusion-competent to the inactive form. Our findings identify gp41 residues, particularly those in HR1, as important regulators of Env conformational transitions.