The Six-Helix Bundle of Human Immunodeficiency Virus Env Controls Pore Formation and Enlargement and Is Initiated at Residues Proximal to the Hairpin Turn

Abstract

Residues that create the grooves of the human immunodeficiency virus type 1 (HIV-1) Env triple-stranded coiled coil (HR1) and the residues that pack into the grooves (HR2) to complete the formation of the six-helix bundle (6HB) were mutated. The extent and kinetics of fusion as well as pore enlargement were measured for each mutant. Mutations near the hairpin turns of each monomer of the 6HB were more important than those far from the turn, for both HR1 and HR2. This result is consistent with the idea that binding of HR2 to the HR1 grooves is initiated near the hairpin turn of each monomer. Mutations at the distal portions also reduced fusion, albeit to a smaller extent. An intermediate of fusion (temperature-arrested state [TAS]) was formed, and the consequences of mutation were compared; a mutant that exhibited less fusion also showed slower kinetics from TAS. This suggests that formation of the bundle is a rate-limiting step downstream of the intermediate state. The rate of enlargement of a fusion pore also correlated with the extent and kinetics of fusion. The rate of pore enlargement was severely reduced by mutation. This supports our prior conclusion that formation of the 6HB occurs after pore creation and strongly suggests that the free energy released by bundle formation is directly used to promote pore growth.All class I viral fusion proteins are composed of three identical monomers that together fold into a six-helix bundle (6HB) in the protein's final, postfusion state (33, 50). Each monomer of human immunodeficiency virus (HIV) Env (which is a class I fusion protein) consists of two subunits, gp120 and gp41. The three gp41 subunits of each Env trimer form the 6HB. The crystallographic structure of gp41 has been determined for its final state; its 6HB reveals that the three N-terminal segments of gp41-each segment referred to as heptad repeat 1 (HR1)-have folded into a central triple-stranded coiled coil of ␣-helices, and the three C-terminal segments (HR2) have packed, antiparallel, as ␣-helices into the three grooves of the coiled coil. The 6HB of HIV Env is thermally stable (43) and can confidently be considered a final structure.The crystallographic structures for both the initial and final states have been obtained for several other class I viral fusion proteins (2,7,22,26,43,54). They reveal that the protein must undergo large-scale conformational changes in transiting from their initial state to final 6HB state, including changes in secondary structure. Although the initial structure of gp41 has not been determined, it is known that grooves are not exposed in its initial conformation; they become exposed as part of the gp41 conformational changes during fusion (13). The configurations of Env during which grooves are exposed are referred to as prebundles. It is assumed that gp41 undergoes large-scale reconfigurations during the fusion process.It is certain that formation of the 6HB is a central event in infection; 6HBs form in all class I proteins; peptides that prevent formation of the bundle b...

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