RNA-induced Allosteric Coupling Drives Viral Capsid Assembly

Understanding the mechanisms by which single-stranded RNA viruses regulate capsid assembly around their RNA genomes has become increasingly important for the development of both antiviral treatments and drug delivery systems. In this study, we investigate the effects of RNA-induced allostery in a single-stranded RNA virus—Levivirus bacteriophage MS2 assembly—using the computational methods of the Dynamic Flexibility Index and the Dynamic Coupling Index. We demonstrate that not only does asymmetric binding of RNA to a symmetric MS2 coat protein dimer increase the flexibility of the distant FG-loop, inducing a conformational change to an asymmetric dimer, but also RNA binding reorganizes long-distance communications, making all the other positions extremely sensitive to the fluctuation of the ordered FG-loop. Additionally, we find that a point mutation in the FG-loop, W82R, leads to the loss of this asymmetry in communications, likely being a leading cause for assembly-deficient dimers. Lastly, this dominant communication that enhances its dynamic coupling with all the distal positions is not only a property of the dimer but is also exhibited by all the observed capsid intermediates. This strong dynamic coupling allows for unidirectional signal transduction that drives the formation of the experimentally observed capsid intermediates and fully assembled capsid.

Figure 1

DFI profile and RNA-induced allostery in bacteriophage MS2 coat protein. (a) Cartoon structure of the MS2 capsid (PDB: 1ZDH) and its coat protein dimers. FG-loop B of the asymmetric dimers form the fivefold axis shown in the capsid on the left. FG-loops C and A alternate to form the threefold axes that surround the fivefold axis. (b) Cartoon diagram of the RNA-induced allosteric pathway for a single coat protein dimer (chain A and C in blue; chain B and C* in red). The symmetric dimer bound to RNA represents a transition state that induces a conformational change in the FG-loop farthest away from the binding site (FG-loop C*). (c) DFI profile for each of the coat protein monomers in the unbound symmetric dimer (dashed lines) and the bound symmetric dimer (solid lines). Between the dotted black lines are the FG-loop residues (66–82), which also have the highest dynamic flexibility. (d) Change in DFI from the unbound symmetric dimer to the bound symmetric dimer. RNA-binding enhances the flexibility of the FG-loop that is distal from the binding interface (FG-loop C*), indicating allosteric communication between the two domains.

Figure 2

DFI profiles of asymmetric dimer and fully assembled capsid. (a) DFI profile for each of the coat protein monomers in the unbound asymmetric dimer (dashed lines) and the bound asymmetric dimer (solid lines). One of the flexible FG-loops has now become rigid, with little to no change upon binding of RNA. (b) Color-coded DFI profile of the fully assembled $T=3$ capsid in the absence of RNA. A closeup is shown of an asymmetric dimer, a symmetric dimer, and the threefold/fivefold axes. Even in the fully assembled capsid, the ordered FG-loop A/C retains its flexibility while the disordered FG-loop B remains rigid.

Figure 3

DCI profiles for the unbound symmetric and bound asymmetric wildtype MS2 coat protein dimers. (a) DCI profile for the unbound symmetric dimer. The FG-loops are symmetrically coupled, where a perturbation at one end of the dimer produces a strong response at the other end. (b) DCI profile for the bound asymmetric dimer. The symmetric coupling of the two FG-loops is broken, such that FG-loop A now drives the communication to FG-loop B. All cartoon structures are colored with DCI for perturbations at residue 74 (shown as a sphere).

Figure 4

The proposed assembly pathway for bacteriophage MS2 capsid. Higher-order capsid intermediates are formed by the alternating 1:1 association of asymmetric-symmetric dimers. Threefold rings and fivefold rings form independently, with an RNA-bound asymmetric dimer acting as the nucleation site of capsid assembly. Crescent and horseshoe conformations are two possible intermediates that arise during the formation of the fivefold ring structure. In the full $T=3$ capsid, five threefold rings converge at one fivefold ring.

Figure 5

DCI for perturbations at FG-loop residue 74 in intermediate capsid structures. (a) DCI spectrum for a flexible FG-loop on an outer dimer of the intermediate. These perturbations have rotational symmetry (see Fig. S3) and are highly coupled, indicating long-range communication among these FG-loops during the formation of the threefold and fivefold axes seen in the full capsid. (b) DCI spectrum for an FG-loop on an inner dimer of the intermediate. In contrast to the outer FG-loops, perturbations of inner FG-loops have low dynamic coupling beyond the localized region at the center of the ring, suggesting structural stability of the FG-loop contacts at the fivefold and threefold axes during assembly.

Figure 6

DCI profile of a fully assembled capsid upon perturbation of residue 74 in various FG-loops. Perturbations at the threefold axis propagate further than perturbations at the fivefold axis. Overall, the asymmetric coupling between the two FG-loop conformers is still observed. Perturbations at the flexible threefold axis propagate to the neighboring threefold axes, while perturbations at the rigid fivefold axis are highly localized. Thus, the flexible FG-loops continue to be the primary communicators.