The identification of the determinants of sensitivity and resistance to broadly neutralizing antibodies is a high priority for human immunodeficiency virus (HIV) research. An analysis of the swarm of closely related envelope protein variants in an HIV-infected individual revealed a mutation that markedly affected sensitivity to neutralization by antibodies and antiviral entry inhibitors targeting both gp41 and gp120. This mutation mapped to the C34 helix of gp41 and disrupted an unexplored structural feature consisting of a ring of hydrogen bonds in the gp41 trimer. This mutation appeared to affect the assembly of the six-helix bundle required for virus fusion and to alter the conformational equilibria so as to favor the prehairpin intermediate conformation required for the binding of the membrane proximal external region-specific neutralizing antibodies 2F5 and 4E10 and the antiviral drug enfuvirtide (Fuzeon). The "swarm analysis" method we describe furthers our understanding of the relationships among the structure, function, and antigenicity of the HIV envelope protein and represents a new approach to the identification of vaccine antigens.