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Host cell infection with HIV-1 requires fusion of viral and cell membranes. Sifuvirtide (SFT) is a peptide-based HIV-1 fusion inhibitor approved for phase III clinical trials in China. Here, we focused on characterizing HIV-1 variants highly resistant to SFT to gain insight into the molecular resistance mechanism. Three primary substitutions (V38A, A47I, and Q52R) located at the inhibitor-binding site of HIV-1's envelope protein (Env) and one secondary substitution (N126K) located at the C-terminal heptad repeat region of the viral protein gp41, which is part of the envelope, conferred high SFT resistance and cross-resistance to the anti-HIV-1 drug T20 and the template peptide C34. Interestingly, SFT's resistance profile could be dramatically improved with an M-T hook structure-modified SFT (MTSFT) and with short-peptide inhibitors that mainly target the gp41 pocket (2P23 and its lipid derivative LP-19). We found that the V38A and Q52R substitutions reduce the binding stabilities of SFT, C34, and MTSFT, but they had no effect on the binding of 2P23 and LP-19; in sharp contrast, the A47I substitution enhanced fusion inhibitor binding. Furthermore, the primary resistance substitutions impaired Env-mediated membrane fusion and cell entry and changed the conformation of the gp41 core structure. Importantly, whereas the V38A and Q52R substitutions disrupted the N-terminal helix of gp41, a single A47I substitution greatly enhanced its thermostability. Taken together, our results provide crucial structural insights into the mechanism of HIV-1 resistance to gp41-dependent fusion inhibitors, which may inform the development of additional anti-HIV drugs. [ABSTRACT FROM AUTHOR]