Abstract
The membrane proximal external region (MPER) is a highly conserved membrane-active region located at the juxtamembrane positions within class I viral fusion glycoproteins and essential for membrane fusion events during viral entry. The MPER in the human immunodeficiency virus type I (HIV-1) envelope protein (Env) interacts with the lipid bilayers through a cluster of tryptophan (Trp) residues and a C-terminal cholesterol-interacting motif. The inclusion of the MPER N-terminal sequence contributes to the membrane reactivity and anti-viral efficacy of the first two anti-HIV peptidyl fusion inhibitors T20 and T1249. As a type I transmembrane protein, Env also interacts with the cellular membranes during its biosynthesis and trafficking. Here we investigated the roles of MPER membrane-active sequences during both viral entry and assembly, specifically, their roles in the design of peptidyl fusion inhibitors and the biosynthesis of viral structural proteins. We found that elimination of the membrane-active elements in MPER peptides, namely, penta Trp→alanine (Ala) substitutions and the disruption of the C-terminal cholesterol-interacting motif through deletion inhibited the anti-viral effect against the pseudotyped HIV-1. Furthermore, as compared to C-terminal dimerization, N-terminal dimerization of MPER peptides and N-terminal extension with five helix-forming residues enhanced their anti-viral efficacy substantially. The secondary structure study revealed that the penta-Trp→Ala substitutions also increased the helical content in the MPER sequence, which prompted us to study the biological relevance of such mutations in pre-fusion Env. We observed that Ala mutations of Trp664, Trp668 and Trp670 in MPER moderately lowered the intracellular and intraviral contents of Env while significantly elevating the content of another viral structural protein, p55/Gag and its derivative p24/capsid. The data suggest a role of the gp41 MPER in the membrane-reactive events during both viral entry and budding, and provide insights into the future development of anti-viral therapeutics.
Highlights
The envelope protein (Env) of human immunodeficiency virus type I (HIV-1) is a class I fusion glycoprotein [1]
Six membrane proximal external region (MPER)-containing peptides (Fig 1) ranging from 19 to 39 a.a were prepared with acetylated N-termini and amidated C-termini and were tested as fusion/entry inhibitors against pseudotyped HIV-1(NL4-3)
The pseudotyped HIV-1 (NL4-3) particles were pre-incubated with each of the six MPER-containing peptides for 1 h and added to the target cells, TZM-bl cells, which stably express a tat-responsive luciferase reporter gene allowing for the monitoring of successful HIV-1 entry and viral protein production
Summary
The envelope protein (Env) of human immunodeficiency virus type I (HIV-1) is a class I fusion glycoprotein [1]. Recognition of the viral receptor and co-receptor by the surface gp120 subunit activates the fusion machinery in the transmembrane (TM) gp subunit (Fig 1) [5,6,7,8], resulting in the insertion of gp N-terminal fusion peptide region (FP) into the target cell membrane This pre-fusion intermediate conformation of gp connects the cellular membrane and the viral envelope, exposing and extending the two heptad repeat (HR) regions, HR1 and HR2 [9,10,11]. The intermediate conformation quickly resolves into a stable six-helix bundle (6-HB) conformation, after HR2 folds back onto the central HR1 to form a coiled-coil trimer-of-dimers [12, 13] This predisposes the opposing membranes into sufficient proximity for subsequent envelope fusion with the plasma membrane and viral content delivery [14]
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