HIV gp41 Trans-Membrane Domain Promotes both Stalk and Fusion Pore Formation in Poly(Ethylene-) Glycol Mediated Membrane Fusion

Abstract

The trans-membrane domain (TMD) of gp41 is essential for efficient fusion between HIV-1 and its host cell in vivo. HIV virus with gp41 mutated by R696L is reported to be defective in infectivity and fusion (Helseth, J Vir, 1990, 6314), so we have examined both native and R696L gp41 TMDs’ effects on PEG-mediated fusion of PC/PE/SM/CH (35/30/15/20) SUVs. Lipid mixing (LM), contents mixing (CM) and leakage (L) time courses were fitted globally to a 3-state sequential model (Weinreb & Lentz, BJ, 2007, 4012), from which we obtained estimates of rate constants for conversion between states as well as probabilities of LM, CM and L for each state. The WT peptide increased the rates of stalk (k1) and fusion pore (k3) formation in a cooperative fashion (maximum effect seen at ∼ 0.25mol% peptide or 6 peptides/vesicle). R696L peptide had no effect on k3 and a very minor effect on k1. At this concentration, CD spectroscopy showed the WT peptide to be ∼ 39% helix, 39% unordered, and 22% β-sheet, but to increase in β- and decrease in α-content at high peptide/lipid ratios up to 1/50. Neither peptide affected the extent of content mixing, but the R696L mutant actually inhibited the extent of lipid mixing. The native but not the mutant peptide increased the probability of content mixing in the stalk intermediate. Studies with hexadecane as a space-filling agent showed that the TMD peptide was unlikely to promote fusion by this mechanism. Analysis of transition state thermodynamics suggests that the TMD may disrupt interfacial packing so as to promote penetration of lipid acyl chains into the inter-bilayer space as a means of catalyzing stalk formation. Supported by NIGMS grant 32707 to BRL.