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 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.

A Structural Perspective of the Role of IP6 in Immature and Mature Retroviral Assembly

“…As we and others have hypothesized [ 5 ], HIV selectively recruits the cytosolic molecule IP6 into virions via its immature Gag IP6-binding site, thus increasing the local concentration of IP6 within virions and promoting formation of a mature lattice following Gag cleavage. Consistent with the absence of basic residues near or at the MHR and the putative 6HB of RSV, IP6 did not enhance immature RSV assembly [ 37 ]. This observation suggests that IP6 is not selectively recruited into immature RSV particles, and also agrees with the finding that RSV requires significantly lower IP6 concentrations than does HIV-1 to promote mature assembly [ 37 ].…”

“…In immature Gag assembly, the quaternary Gag protein arrangement can vary because of different CA NTD interactions, while in mature CA assembly the CA–CA interactions appear well conserved in different retrovirus genera [ 1 , 31 , 34 , 35 , 36 , 37 , 38 ]. However, mature CA cores display a remarkable polymorphism, where the architectures from different virus species (and even within one species) can vary significantly ( Figure 3 , morphology panel).…”

“…Proof for such a conserved role of IP6 outside the lentiviruses genus was recently provided by a study on the mature alpharetrovirus RSV. A structure of the mature RSV CA hexamer solved from CANC tubes, which were assembled in vitro in the presence of IP6, showed a density in the center of the hexamer pore [ 37 ], where it is coordinated by a ring of six lysines (K17 in RSV CA) and six arginines (R21 in RSV CA) ( Figure 4 B–D right panel). The density suggested that the IP6 molecule is oriented “on edge” with the equatorial phosphates pointing up and down in the pore, which contrasts with the “flat” orientation of IP6 in HIV-1 CA.…”

“…One explanation for the different orientation of IP6 in mature RSV could be that the RSV CA hexamer is more flexible than the HIV CA hexamer, and the central pore is wider than in HIV-1 ( Figure 4 D). The implementation of classification and alignment workflows to address the increased structural pleomorphism of RSV cores [ 37 ], which can form polyhedrons, cones, or tubes [ 51 ], showed that the RSV CA hexamer substantially deviates from C6 symmetry. This results in a deformation of the central pore, manifested as increased and uneven separation of helices 1 from different CA molecules ( Figure 4 D).…”

“…Experiments in RSV also showed that phosphate promotes pentamer formation, while inositol phosphates promote hexamer formation. Such differential mechanisms of charge compensation in RSV could be related to the varying flexibility of hexamers over pentamers, where the latter have been found to be structurally rigid organization centers within the mature CA lattice [ 37 ].…”