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 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have 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 reveal 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.
Highlights
Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1
We have demonstrated that IP6 has a potent effect on the assembly and morphology of the mature Rous sarcoma virus (RSV) capsid protein (CA) lattice, and that depletion of IP6 from cells inhibits virus release and spread
We have determined the structure of the mature RSV CA hexamer and pentamer at the highest resolution to date, using Cryo-electron tomography (cryo-ET) and cryo-EM, respectively, to reveal structural mechanisms underlying RSV mature core formation
Summary
Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. In vitro assembly of Gag and CA protein into immature virus-like particles (VLPs) and mature capsid-like particles (CLPs) has facilitated study of the lattice structure of many retroviruses[16]. In this methodology, the viral proteins are purified after expression in Escherichia coli, incubated under defined conditions of pH, ionic strength, temperature, and in some cases with an oligonucleotide or other additions, and observed by negative stain electron microscopy. Our subtomogram averaging analysis of pleomorphic polyhedral CASPNC CLPs reveals that a remarkable, previously unobserved flexibility of mature CA hexamers accommodates the wide range of curvatures intrinsic to mature RSV particles, and that CA pentamers represent rigid building blocks determining local lattice geometry
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