The Structure of Myristoylated Mason-Pfizer Monkey Virus Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Its Membrane Binding

Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

doi:10.1016/j.jmb.2012.07.021

Cited by 37 publications

(67 citation statements)

References 43 publications

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“…PI(4,5)P 2 binds to HIV-2 MA in a manner that is essentially identical to that of HIV-1 MA; however, structural data revealed that the myr group in HIV-2 MA is tightly sequestered and does not exhibit concentration-or PI(4,5)P 2 -dependent exposure (61). Another recent study on Mason-Pfizer monkey virus MA also revealed that the myr group is tightly sequestered and that PI (4,5)P 2 binding to MA does not trigger myr exposure (62). The MA proteins of RSV and equine infectious anemia virus (EIAV) lack myr modification; Gag binding to the PM is mediated by electrostatic interactions (63)(64)(65).…”

Section: Discussionmentioning

confidence: 95%

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Vlach

Saad

2013

Proc. Natl. Acad. Sci. U.S.A.

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Localization of the HIV type-1 (HIV-1) Gag protein on the plasma membrane (PM) for virus assembly is mediated by specific interactions between the N-terminal myristoylated matrix (MA) domain and phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P 2 ]. The PM bilayer is highly asymmetric, and this asymmetry is considered crucial in cell function. In a typical mammalian cell, the inner leaflet of the PM is enriched in phosphatidylserine (PS) and phosphatidylethanolamine (PE) and contains minor populations of phosphatidylcholine (PC) and PI(4,5)P 2 . There is strong evidence that efficient binding of HIV-1 Gag to membranes is sensitive not only to lipid composition and net negative charge, but also to the hydrophobic character of the acyl chains. Here, we show that PS, PE, and PC interact directly with MA via a region that is distinct from the PI(4,5)P 2 binding site. Our NMR data also show that the myristoyl group is readily exposed when MA is bound to micelles or bicelles. Strikingly, our structural data reveal a unique binding mode by which the 2′-acyl chain of PS, PE, and PC lipids is buried in a hydrophobic pocket whereas the 1′-acyl chain is exposed. Sphingomyelin, a major lipid localized exclusively on the outer layer of the PM, does not bind to MA. Our findings led us to propose a trio engagement model by which HIV-1 Gag is anchored to the PM via the 1′-acyl chains of PI(4,5)P 2 and PS/PE/PC and the myristoyl group, which collectively bracket a basic patch projecting toward the polar leaflet of the membrane.

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Section: Discussionmentioning

confidence: 95%

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Vlach

Saad

2013

Proc. Natl. Acad. Sci. U.S.A.

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“…For example, on the one hand, the specificity of the HIV-1 MA binding pocket for short-chain PI(4,5)P2 is well established (7), and depletion of PI(4,5)P2, the major PIP species at the PM, compromises HIV-1 Gag PM association (8,9,11). But on the other hand, HIV-1 Gag shows only very modest specificity for PI (4,5)P2 by flotation analysis (8,9). It remains to be established to what degree PIP stimulation of membrane binding in vitro is due only to electrostatics.…”

Section: Amentioning

confidence: 99%

“…Examples include human immunodeficiency virus type 1 (HIV-1) and HIV-2, Mason-Pfizer monkey virus (MPMV) (4), and EIAV (5). Other viruses, such as RSV, murine leukemia virus (MLV), and human T-lymphotropic virus (HTLV), apparently do not (6,7).…”

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confidence: 99%

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

Dick

Kamynina

Vogt

2013

J Virol

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In most retroviruses, plasma membrane (PM) association of the Gag structural protein is a critical step in viral assembly, relying in part on interaction between the highly basic Gag MA domain and the negatively charged inner leaflet of the PM. Assembly is thought to begin with Gag dimerization followed by multimerization, resulting in a hexameric lattice. To directly address the role of multimerization in membrane binding, we fused the MA domains of Rous sarcoma virus (RSV) and HIV-1 to the chemically inducible dimerization domain FK506-binding protein (FKBP) or to the hexameric protein CcmK4 from cyanobacteria. The cellular localization of the resulting green fluorescent protein (GFP)-tagged chimeric proteins was examined by fluorescence imaging, and the association of the proteins with liposomes was quantified by flotation in sucrose gradients, following synthesis in a reticulocyte extract or as purified proteins. Four lipid compositions were tested, representative of liposomes commonly reported in flotation experiments. By themselves, GFP-tagged RSV and HIV-1 MA proteins were largely cytoplasmic, but both hexamerized proteins were highly concentrated at the PM. Dimerization led to partial PM localization for HIV-1 MA. These in vivo effects of multimerization were reproduced in vitro. In flotation analyses, the intact RSV and HIV-1 Gag proteins were more similar to multimerized MA than to monomeric MA. RNA is reported to compete with acidic liposomes for HIV-1 Gag binding, and thus we also examined the effects of RNase treatment or tRNA addition on flotation. tRNA competed with liposomes in the case of some but not all lipid compositions and ionic strengths. Taken together, our results further underpin the model that multimerization is critical for PM association of retroviral Gag proteins. In addition, they suggest that the modulation of membrane binding by RNA, as previously reported for HIV-1, may not hold for RSV.

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“…Mutating these residues results in reduction of Gag membrane association and of virus release (5)(6)(7). In vitro, the negatively charged lipids phosphatidylserine (PS) or phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] are crucial for Gag or MA membrane association (8)(9)(10)(11)(12)(13). Second, the Gag proteins of most retroviruses, like HIV-1, are modified by addition of the 14-carbon fatty acid myristate at the N-terminal glycine residue.…”

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confidence: 99%

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

Wen

Dick

Feigenson

et al. 2016

J Virol

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The retroviral structural protein Gag binds to the inner leaflet of the plasma membrane (PM), and many cellular proteins do so as well. We used Rous sarcoma virus (RSV) Gag together with membrane sensors to study the principles governing peripheral protein membrane binding, including electrostatics, specific recognition of phospholipid headgroups, sensitivity to phospholipid acyl chain compositions, preference for membrane order, and protein multimerization. We used an in vitro liposome-pelleting assay to test protein membrane binding properties of Gag, the well-characterized MARCKS peptide, a series of fluorescent electrostatic sensor proteins (mNG-KRn), and the specific phosphatidylserine (PS) binding protein Evectin2. RSV Gag and mNG-KRn bound well to membranes with saturated and unsaturated acyl chains, whereas the MARCKS peptide and Evectin2 preferentially bound to membranes with unsaturated acyl chains. To further discriminate whether the primary driving force for Gag membrane binding is electrostatic interactions or preference for membrane order, we measured protein binding to giant unilamellar vesicles (GUVs) containing the same PS concentration in both disordered (Ld) and ordered (Lo) phases. RSV Gag and mNG-KRn membrane association followed membrane charge, independent of membrane order. Consistent with pelleting data, the MARCKS peptide showed preference for the Ld domain. Surprisingly, the PS sensor Evectin2 bound to the PS-rich Ld domain with 10-fold greater affinity than to the PS-rich Lo domain. In summary, we found that RSV Gag shows no preference for membrane order, while proteins with reported membrane-penetrating domains show preference for disordered membranes. IMPORTANCERetroviral particles assemble on the PM and bud from infected cells. Our understanding of how Gag interacts with the PM and how different membrane properties contribute to overall Gag assembly is incomplete. This study examined how membrane charge and membrane order influence Gag membrane association. Consistent with previous work on RSV Gag, we report here that electrostatic interactions provide the primary driving force for RSV Gag membrane association. Using phase-separated GUVs with known lipid composition of the Ld and Lo phases, we demonstrate for the first time that RSV Gag is sensitive to membrane charge but not membrane order. In contrast, the cellular protein domain MARCKS and the PS sensor Evectin2 show preference for disordered membranes. We also demonstrate how to define GUV phase composition, which could serve as a tool in future studies of protein membrane interactions.T he retroviral structural protein Gag provides the primary driving force for virus assembly at the inner leaflet of the plasma membrane (PM), and Gag alone is sufficient to assemble into virus-like particles (VLPs) in cells (1) and also in vitro (2). Gag is synthesized in the cytosol and then is targeted to the PM by the N-terminal domain, MA. How Gag interacts with the inner leaflet of the PM and how the biophysical properties of the PM...

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The Structure of Myristoylated Mason-Pfizer Monkey Virus Matrix Protein and the Role of Phosphatidylinositol-(4,5)-Bisphosphate in Its Membrane Binding

Cited by 37 publications

References 43 publications

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Trio engagement via plasma membrane phospholipids and the myristoyl moiety governs HIV-1 matrix binding to bilayers

Effect of Multimerization on Membrane Association of Rous Sarcoma Virus and HIV-1 Matrix Domain Proteins

Effects of Membrane Charge and Order on Membrane Binding of the Retroviral Structural Protein Gag

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