Evolutionary repair of HIV type 1 gp41 with a kink in the N-terminal helix leads to restoration of the six-helix bundle structure.

The HIV-1 envelope glycoprotein complex (Env) can be stabilized by the introduction of a disulfide bond between the gp120 and gp41 subunits. The resulting protein is monomeric, but trimerization can be improved by the introduction of a single helix-breaking residue at the conserved Ile559 site in the N-terminal heptad repeat region of gp41. To provide more insight into how such a substitution in gp41 affects Env structure and function, we evaluated the effect on the wild-type Env in the context of replicating virus. The Ile559Gly and Ile559Pro mutations adversely affect Env biosynthesis and Env incorporation into virions. Biophysical studies show that the Ile559Pro mutation essentially disrupts the folding of a recombinant gp41 ectodomain core into a six-helix bundle structure. Viruses containing the Ile559Gly and Ile559Pro substitutions replicate poorly, but an evolutionary route is described that restores replication competence. In the escape virus, which contains a Pro559Leu first-site pseudoreversion, the local helical structure and, as a consequence, Env biosynthesis and function are restored.