Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Abstract

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 was ∼100-fold more potent at promoting RSV mature CA assembly than observed for HIV-1 and removal of IP6 in vivo reduced infectivity by 100-fold. By cryo-electron tomography and subtomogram averaging, mature virus-like particles (VLPs) showed an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 had opposing effects on CA in vitro assembly, inducing formation of T=1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles revealed that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Inositol Hexakisphosphate (IP6) Accelerates Immature HIV-1 Gag Protein Assembly toward Kinetically Trapped Morphologies

“…Beyond HIV-1, IP6 has also been implicated as an assembly cofactor in other retroviruses, such as Rous sarcoma virus and equine infectious anemia virus. 71,72 It would be interesting to investigate the utility of IP6 and its derivatives for the efficient production of broad VLPs that have been repurposed for biomedical technologies, including vaccine development and biologics delivery. 73−76 Our results will hopefully stimulate future experimental studies as well as the use and development of even more sophisticated CG models.…”

Structural insights into HIV-1 polyanion-dependent capsid lattice formation revealed by single particle cryo-EM

“…Given the relationship between pentamers and lattice curvature, we speculated that pentamers could be enriched by forcing CA to form a high-curvature lattice similar to that permitted by pentamers at the ends of authentic capsid cores (3)(4)(5). Indeed, we and others have shown that CA from other retroviruses can be induced to assemble into highly-curved icosahedral particles enriched in pentamers under specific buffer and assembly conditions (36)(37)(38). For HIV-1, however, this is possible only with the introduction of mutations (39,40).…”

Exploring high-resolution cryo-ET and subtomogram averaging capabilities of contemporary DEDs

“…Additionally, since the crosscorrelation cleaning approach was not applicable when using Relion, we attempted to increase the homogeneity of the subvolumes used for refinement by a more stringent exclusion of subvolumes that deviate from ideal lattice geometry. In order to retain only subvolumes containing the structurally most similar local CA hexamers we employed a cleaning strategy based on local geometry, similar to what has been previously used for mature HIV-1 and RSV CA assemblies (Mattei et al, 2016; Obr et al, 2021) (see also Figure S4 and Materials & Methods for more details).…”

“…For this, we used an approach as employed in previous papers studying structures of retroviral Gag assemblies. This approach is based on tomogram reconstruction with 3D CTF correction using novaCTF and the AV3-derived subtomogram averaging/alignment pipeline (Dick et al, 2020; Förster et al, 2005; Obr et al, 2021; Turoňová et al, 2020, 2017).…”