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Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

ABSTRACT: For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the N-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely understood. Here, we employed NMR techniques to elucidate the molecular determinants of the membrane-binding domain of ASV MA (MA87) to lipids and liposomes. We report that MA87 binds to the polar head of phosphoinositides such as PI(4,5)P2 We found that MA87 binding to inositol phosphates (IPs) is significantly enhanced by increasing the number of phosphate groups, indicating that the MA87-IP binding is governed by charge-charge interactions. Using a sensitive NMR-based liposome-binding assay, we show that binding of MA87 to liposomes is enhanced by incorporation of PI(4,5)P2 and phosphatidylserine. We also show that membrane binding is mediated by a basic surface formed by Lys-6, Lys-13, Lys-23, and Lys-24. Substitution of these residues to glutamate abolished binding of MA87 to both IPs and liposomes. In an accompanying paper, we further report that mutation of these lysine residues diminishes Gag assembly on the PM and inhibits ASV particle release. These findings provide a molecular basis for ASV Gag binding to the inner leaflet of the PM and advance our understanding of the basic mechanisms of retroviral assembly.

SUBMITTER: Vlach J

PROVIDER: S-EPMC6295720 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

Vlach Jiri J Eastep Gunnar N GN Ghanam Ruba H RH Watanabe Susan M SM Carter Carol A CA Saad Jamil S JS

The Journal of biological chemistry 20181011 49

For most retroviruses, including HIV-1, binding of the Gag polyprotein to the plasma membrane (PM) is mediated by interactions between Gag's N-terminal myristoylated matrix (MA) domain and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P<sub>2</sub>) in the PM. The Gag protein of avian sarcoma virus (ASV) lacks the <i>N</i>-myristoylation signal but contains structural domains having functions similar to those of HIV-1 Gag. The molecular mechanism by which ASV Gag binds to the PM is incompletely ...[more]

PMID: 30309983

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Project description:Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.

Dataset Information

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

Publications

Structural basis for targeting avian sarcoma virus Gag polyprotein to the plasma membrane for virus assembly.

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