Structures of immature EIAV Gag lattices reveal a conserved role for IP6 in lentivirus assembly.

Robert A Dick,Marc C Johnson,Volker M Vogt,Juan R Perilla,Vladyslav Kravchuk,Clifton L Ricana,Arianna M Broad,John A G Briggs,Florian K M Schur,Terri D Lyddon,Dustin R Morado,J Ryan Feathers,Chaoyi Xu,Félix A Rey

doi:10.1371/journal.ppat.1008277

Abstract

Retrovirus assembly is driven by the multidomain structural protein Gag. Interactions between the capsid domains (CA) of Gag result in Gag multimerization, leading to an immature virus particle that is formed by a protein lattice based on dimeric, trimeric, and hexameric protein contacts. Among retroviruses the inter- and intra-hexamer contacts differ, especially in the N-terminal sub-domain of CA (CANTD). For HIV-1 the cellular molecule inositol hexakisphosphate (IP6) interacts with and stabilizes the immature hexamer, and is required for production of infectious virus particles. We have used in vitro assembly, cryo-electron tomography and subtomogram averaging, atomistic molecular dynamics simulations and mutational analyses to study the HIV-related lentivirus equine infectious anemia virus (EIAV). In particular, we sought to understand the structural conservation of the immature lentivirus lattice and the role of IP6 in EIAV assembly. Similar to HIV-1, IP6 strongly promoted in vitro assembly of EIAV Gag proteins into virus-like particles (VLPs), which took three morphologically highly distinct forms: narrow tubes, wide tubes, and spheres. Structural characterization of these VLPs to sub-4Å resolution unexpectedly showed that all three morphologies are based on an immature lattice with preserved key structural components, highlighting the structural versatility of CA to form immature assemblies. A direct comparison between EIAV and HIV revealed that both lentiviruses maintain similar immature interfaces, which are established by both conserved and non-conserved residues. In both EIAV and HIV-1, IP6 regulates immature assembly via conserved lysine residues within the CACTD and SP. Lastly, we demonstrate that IP6 stimulates in vitro assembly of immature particles of several other retroviruses in the lentivirus genus, suggesting a conserved role for IP6 in lentiviral assembly.

Highlights

Of the retrovirus particle typically takes place at the inner leaflet of the plasma membrane (PM) and involves the formation of a curved lattice by the structural multidomain protein Gag
SP is similar to domains in the Gag proteins of the alpha-retrovirus Rous sarcoma virus (RSV) [6,7,8,9,10] and of the gamma retrovirus murine leukemia virus (MLV) [11] in that it folds into a six-helix bundle (6HB) at the base of the CA domain of the Gag hexamer
Equine infectious anemia virus (EIAV) is only the second retrovirus, after HIV-1, to have its immature Gag lattice determined to this level of resolution by Cryo-electron tomography (cryo-ET) and subtomogram averaging

Summary

Introduction

Of the retrovirus particle typically takes place at the inner leaflet of the plasma membrane (PM) and involves the formation of a curved lattice by the structural multidomain protein Gag. EIAV Gag and HIV-1 Gag share only 30% amino acid sequence identity but have an overall similar domain arrangement (S1A and S1B Fig), with the canonical Gag domains MA, capsid (CA), nucleocapsid (NC), and an unstructured C-terminal domain mediating the late stages of budding These two retroviral Gag proteins include a short segment of polypeptide, in HIV-1 termed SP1 (“spacer”, here generically called “SP”), that is critical for formation of the immature lattice [4,5]. After the viral protease ablates this immature binding site, IP6 is inferred to be released and to interact with a ring of six arginine residues in the CANTD, thereby enhancing the formation of the mature hexameric HIV-1 CA lattice and promoting infectivity [2,17,18].

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Conclusion