HIV-1 envelope proteins complete their folding into six-helix bundles immediately after fusion pore formation

Fusion proteins of many viruses, including HIV-1 envelope protein (Env), fold into six-helix bundle structures. Fusion between individual Env-expressing cells and target cells was studied by fluorescence microscopy, and a temperature jump technique, to determine whether folding of Env into a bundle is complete by the time fusion pores have formed. Lowering temperature to 4degreesC immediately after a pore opened halted pore growth, which quickly resumed when temperature was raised again. HIV gp41-derived peptides that inhibit bundle formation (C34 or N36) caused the cold-arrested pore to quickly and irreversibly close, demonstrating that bundle formation is not complete by the time a pore has formed. In contrast, lowering the temperature to an intermediate value also halted pore growth, but the pore was not closed by the bundle-inhibiting peptides, and it enlarged when temperature was again elevated. This latter result shows that bundle formation is definitely required for the fusion process, but surprisingly, some (if not all) bundle formation occurs after a pore has formed. It is concluded that an essential function of the bundle is to stabilize the pore against collapse and ensure its growth.