A Covalent Inhibitor Targeting an Intermediate Conformation of the Fusogenic Subunit of the HIV-1 Envelope Complex

Jacobs, Amy; Quraishi, Omar; Huang, Xicai; Bousquet-Gagnon, Nathalie; Nault, Geneviève; Francella, Nicholas; Alvord, W. Gregory; Pham, Nga; Soucy, Chantal; Robitaille, Martin; Bridon, Dominique; Blumenthal, Robert

doi:10.1074/jbc.m705577200

Cited by 17 publications

(36 citation statements)

References 48 publications

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“…After the interaction with target cell surface-bound CD4, a substantial increase in immunoreactivity to certain antibodies is observed, reflecting conformational changes in gp120, resulting in exposure of the CR-binding site (gp120 CR ), and in gp41, resulting in the prehairpin conformation (gp41 PHP ) (Fig. 4, step 1) (76). The increased reactivity has been observed in the absence of gp120 dissociation (77), indicating that subunit dissociation is not absolutely required for these CD4-induced conformational changes to occur.…”

Section: Conformational Changes Of Hiv Env Proteins In the Course Of mentioning

confidence: 99%

HIV Entry and Envelope Glycoprotein-mediated Fusion

Blumenthal

Durell

Viard

2012

Journal of Biological Chemistry

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183

203

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HIV entry involves binding of the trimeric viral envelope glycoprotein (Env) gp120/gp41 to cell surface receptors, which triggers conformational changes in Env that drive the membrane fusion reaction. The conformational landscape that the lipids and Env navigate en route to fusion has been examined by biophysical measurements on the microscale, whereas electron tomography, x-rays, and NMR have provided insights into the process on the nanoscale and atomic scale. However, the coupling between the lipid and protein pathways that give rise to fusion has not been resolved. Here, we discuss the known and unknown about the overall HIV Env-mediated fusion process. The Virus and Its TargetHIV virions exist as roughly spherical nanoparticles ϳ100 nm in diameter and are coated by the viral envelope membrane (1). The viral membrane is a lipid bilayer ϳ4 nm thick, interspersed with membrane-embedded glycoproteins. The HIV matrix protein Gag Pr55 drives viral assembly by recruiting all the building blocks, which include both viral and cellular components required for the formation of a fully infectious virion (2). A typical HIV viral membrane contains ϳ300,000 lipids, with a rather unique distribution of lipids compared with the lipid composition of cells from which it is derived (3,4). In addition to the virally encoded envelope glycoprotein gp120/ gp41, the HIV membrane also incorporates a plethora of cell membrane proteins in the process of assembly and budding (5). The HIV envelope proteins are expressed in the endoplasmic reticulum as a precursor protein, gp160, which transits the Golgi apparatus, where glycosylation is completed (6, 7). The precursor is cleaved in the trans-Golgi by the cellular protease furin into two proteins, gp120 and gp41 (8). These proteins are present on the cell surface as the envelope complex, a mushroomshaped trimer of heterodimers of gp120 and gp41, incorporated into the viral envelope through the transmembrane region of gp41 as virus particles bud from the cell surface (9).The number of gp120/gp41 trimers per virion ranges between 10 and 100 depending on the isolate (10). HIV gp120 is ϳ50% carbohydrate and is one of the most glycosylated proteins known (11). The number of accessory HIV membrane proteins has, in general, not been determined, with the exception of HLA class II in HIV-1 MN derived from H9 cells, which has ϳ50 native HLA class II complexes (12). The lipids and proteins are glycoconjugated, providing an additional shield for HIV against immune and environmental challenges. The viral genome is thus comfortably ensconced in a wrapper of lipid and protein covered by carbohydrate. However, mannose-type carbohydrates on HIV are recognized by DC-SIGN (dendritic cellspecific intercellular adhesion molecule-3-grabbing non-integrin), which is a C-type lectin receptor present on dendritic cells (13). The dendritic cells internalize HIV-DC-SIGN complexes and migrate to the lymph nodes, where they interact with T cells. The HIV-DC-SIGN complexes are then recycled to the cell periphery, and ...

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Section: Conformational Changes Of Hiv Env Proteins In the Course Of mentioning

confidence: 99%

HIV Entry and Envelope Glycoprotein-mediated Fusion

Blumenthal

Durell

Viard

2012

Journal of Biological Chemistry

Self Cite

183

203

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“…designed a covalent inhibitor that specifically targets residue Lys 574. Using a temperature-arrested state prime wash in vitro assay, they provided evidence for the trapping of a pre-six-helix bundle fusion intermediate conformation by a covalent reaction of Lys 574 with a specific anti-HIV peptide composed of a chemical spacer and a reactive moiety positioned strategically to facilitate covalent attachment (26). Therefore, the salt bridge between the NHR and CHR of the gp41 core is critical for the conformation and stability of the six-helix bundle structure and thereby can serve as a potential target for designing anti-HIV peptides and small molecules.…”

mentioning

confidence: 99%

“…Therefore, the salt bridge-forming residue Lys 574 may serve as a key determinant of the target of the compounds, and the breakage of the salt bridge formed by Lys 574 and Asp 632 may be their mechanism of action. Recently, Jacobs et al (26). designed a covalent inhibitor that specifically targets residue Lys 574.…”

mentioning

confidence: 99%

Conserved Salt Bridge between the N- and C-Terminal Heptad Repeat Regions of the Human Immunodeficiency Virus Type 1 gp41 Core Structure Is Critical for Virus Entry and Inhibition

Liu

et al. 2008

J Virol

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The fusogenic human immunodeficiency virus type 1 (HIV-1) gp41 core structure is a stable six-helix bundle formed by its N-and C-terminal heptad repeat sequences. Notably, the negatively charged residue Asp 632 located at the pocket-binding motif in the C-terminal heptad repeat interacts with the positively charged residue Lys 574 in the pocket formation region of the N-terminal heptad repeat to form a salt bridge. We previously demonstrated that the residue Lys 574 plays an essential role in six-helix bundle formation and virus infectivity and is a key determinant of the target for anti-HIV fusion inhibitors. In this study, the functionality of residue Asp 632 has been specifically characterized by mutational analysis and biophysical approaches. We show that Asp 632 substitutions with positively charged residues (D632K and D632R) or a hydrophobic residue (D632V) could completely abolish Env-mediated viral entry, while a protein with a conserved substitution (D632E) retained its activity. Similar to the Lys 574 mutations, nonconserved substitutions of Asp 632 also severely impaired the ␣-helicity, stability, and conformation of six-helix bundles as shown by N36 and C34 peptides as a model system. Furthermore, nonconserved substitutions of Asp 632 significantly reduced the potency of C34 to sequestrate six-helix bundle formation and to inhibit HIV-1-mediated cell-cell fusion and infection, suggesting its importance for designing antiviral fusion inhibitors. Taken together, these data suggest that the salt bridge between the N-and C-terminal heptad repeat regions of the fusion-active HIV-1 gp41 core structure is critical for viral entry and inhibition.Infection with human immunodeficiency virus type 1 (HIV-1) is mediated by its envelope glycoprotein (Env), a type I transmembrane protein which is originally synthesized as the single, glycosylated, polyprotein precursor gp160 and subsequently cleaved by a cellular protease to yield gp120 and gp41 subunits (13,14,20,46,48). Upon binding of the HIV-1 Env surface subunit gp120 to the cell receptor CD4 and subsequently to a coreceptor (CCR5 or CXCR4), its transmembrane subunit gp41 is released to mediate fusion of viral and cellular membranes (20,25,54). Structurally, HIV-1 gp41 consists of extracellular, transmembrane, and cytoplasmic domains (Fig. 1A). Its extracellular domain (ectodomain) contains four major functional regions: a hydrophobic, glycine-rich fusion peptide; an N-terminal heptad repeat (NHR) (also called HR1), a C-terminal heptad repeat (CHR) (also called HR2), and a tryptophan-rich region. In the early 1990s, several peptides derived from the NHR (N peptides) and CHR (C peptides) were found to have potent anti-HIV activity (30,43,68,69). Although their mechanism of action was not known at that time, the unprecedented anti-HIV activity of these peptides opened a new avenue for developing antiviral drugs. A C peptide known as T20 (brand name, Fuzeon) has been successfully developed as a novel class of anti-HIV drugs for clinical use (36,37,50).The findin...

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“…In this study, we sought to address these shortcomings by engineering CHR peptides (C34 and T-20) to bond covalently to cysteine 34 of human albumin. Given the steric block associated with the NHR-trimer region of gp41 (11) and given the current dearth of neutralizing antibodies against this target, it was impossible to predict whether such conjugates could access the hydrophobic grooves of the NHR of gp41 that are exposed only transiently once the viral and target membranes are in close contact (35), i.e. within the contact zone referred to as the "entry claw" (36).…”

Section: Discussionmentioning

confidence: 99%