Structural Model of the Tubular Assembly of the Rous Sarcoma Virus Capsid Protein

Abstract

The orthoretroviral capsid protein (CA) assembles into polymorphic capsids, whose architecture, assembly, and stability are still being investigated. The N-terminal and C-terminal domains of CA (NTD and CTD, respectively) engage in both homotypic and heterotypic interactions to create the capsid. Hexameric turrets formed by the NTD decorate the majority of the capsid surface. We report nearly complete solid-state NMR (ssNMR) resonance assignments of Rous sarcoma virus (RSV) CA, assembled into hexamer tubes that mimic the authentic capsid. The ssNMR assignments show that, upon assembly, large conformational changes occur in loops connecting helices, as well as the short 3 helix initiating the CTD. The interdomain linker becomes statically disordered. Combining constraints from ssNMR and cryo-electron microscopy (cryo-EM), we establish an atomic resolution model of the RSV CA tubular assembly using molecular dynamics flexible fitting (MDFF) simulations. On the basis of comparison of this MDFF model with an earlier-derived crystallographic model for the planar assembly, the induction of curvature into the RSV CA hexamer lattice arises predominantly from reconfiguration of the NTD-CTD and CTD trimer interfaces. The CTD dimer and CTD trimer interfaces are also intrinsically variable. Hence, deformation of the CA hexamer lattice results from the variable displacement of the CTDs that surround each hexameric turret. Pervasive H-bonding is found at all interdomain interfaces, which may contribute to their malleability. Finally, we find helices at the interfaces of HIV and RSV CA assemblies have very different contact angles, which may reflect differences in the capsid assembly pathway for these viruses.

Curvature of the retroviral capsid assembly is modulated by a molecular switch

“…A 2D 15 N- 13 C correlation spectrum with cross peaks created by 50 ms dipolar assisted rotational resonance 35 is shown in Figure 2A. The typical linewidth is about 0.7 ppm along the 13 C dimension and 1.2 ppm along the 15 N dimension, comparable to that of our prior RSV CA tubular assembly under moderate magic angle spinning (MAS), 32 and similar to that of other highly ordered protein structures [36][37][38][39] and HIV CA assemblies. [24][25][26][27][28][29][30][31] It indicates that RSV CA adopts highly order structure at the molecular level, consistently with our TEM evaluation.…”

“…As shown in Figure 4A to D, close inspection confirms that the same set of residues are employed to stabilize each interface in both pentamers in the RSV CA T=1 capsid assembly, and hexamers in the tubular assembly. 32 Thus, the molecular differences in torsion angles do not cause large variations of assembly interfaces in the CA pentameric assembly compared to its hexameric assembly. It shows the remarkable efficiency of the viral CA to achieve assembly versatility with minimum modulation.…”

“…Based on the assignments, the secondary structure composition and local dynamics were derived with TALOS-N, 41 shown in Figure 2B. Overall, the RSV CA in the T=1 capsid assembly adopts a folding nearly identical to that in the tubular assembly, 32 with the exception for a small number of residues. This notable difference can be better appreciated by overlay of the 2D 15 N- 13 C correlation spectra, as shown in Figure 2A: the RSV CA demonstrates similar resonances in either T=1 or tubular assembly, except for about a dozen residues exhibiting shifted resonances in both C and side chain regions.…”

“…The results agree remarkably well with those predicted by TALOS-N 41 derived directly from NMR chemical shifts ( Figure 3A). 32 The thick color lines at the top highlight segments of secondary structured units, blue for strands and red for -helices. (C).…”

Virus Structures and Dynamics by Magic-Angle Spinning NMR

“…MAS frequencies of at least 8-10 kHz are necessary for recording well-resolved spectra for 13 C, 15 N, and 31 P nuclei. While this range of frequencies is sufficient for basic biological MAS NMR experiments, in practice the faster the specimen is spun, the higher the resolution and the sensitivity for the same rotor diameter.…”