The Assembly Pathway of an Icosahedral Single-Stranded RNA Virus Depends on the Strength of Inter-Subunit Attractions

Abstract

The strength of attraction between capsid proteins (CPs) of cowpea chlorotic mottle virus (CCMV) is controlled by the solution pH. Additionally, the strength of attraction between CP and the single-stranded RNA viral genome is controlled by ionic strength. By exploiting these properties, we are able to control and monitor the in vitro co-assembly of CCMV CP and single-stranded RNA as a function of the strength of CP–CP and CP– RNA attractions. Using the techniques of velocity sedimentation and electron microscopy, we find that the successful assembly of nuclease-resistant virus-like particles (VLPs) depends delicately on the strength of CP–CP attraction relative to CP–RNA attraction. If the attractions are too weak, the capsid cannot form; if they are too strong, the assembly suffers from kinetic traps. Separating the process into two steps—by first turning on CP–RNA attraction and then turning on CP–CP attraction—allows for the assembly of well-formed VLPs under a wide range of attraction strengths. These observations establish a protocol for the efficient in vitro assembly of CCMV VLPs and suggest potential strategies that the virus may employ in vivo.

Role of Electrostatics in the Assembly Pathway of a Single-Stranded RNA Virus

“…Two-step in vitro assembly reactions. Assembly reactions were performed by following the previously optimized two-step protocol (19). Specifically, 30 nM BMV RNA1 and various concentrations of CP were mixed under conditions that prevent their interaction (neutral pH and high ionic strength; 1 M NaCl, 20 mM Tris [pH 7.2], 1 mM EDTA, 1 mM DTT, 1 mM PMSF).…”

“…Recent progress in our ability to control the attractive forces that drive assembly has allowed us to trap stable (i.e., long-lived) assembly intermediates and characterize their coarse-grained structure by cryoelectron microscopy (cryo-EM) (19). These intermediates, formed under conditions of weak CP-CP attraction found at neutral pH and low ionic strength, appear as disordered complexes consisting of an undetermined number of CP molecules bound to single RNA molecules through strong electrostatic attraction.…”

Orientational phase transitions and the assembly of viral capsids

“…Though there are instances for which this is a reasonable assumption, such as the Herpes Simplex virus, this premise is questionable for most cases. The observations on the CCMV precursor state [14] indicates that the RNA-protein condensate, though roughly spherical, fluctuates strongly. Including deformability of the surface is also important because of the relationship between Landau theory and the coarse-grained description of viral shells that is based on application of thin-shell elasticity theory [45].…”

The confinement of an annealed branched polymer by a potential well

“…In solution, these molecules form rather elongated "branched backbone" structures while under confinement they resemble dendrimers with the endpoints extending outwards [7]. One would expect that for larger well radii R , one should encounter more branched backbone configurations with a more uniform branching function profile, since that might provide a higher configurational entropy.…”

“…These vRNA molecules can adopt a very large number of branched "secondary structures" that are within B k T of the groundstate, as demonstrated by microscopy studies of vRNA molecules in solution [6]. Other studies demonstrated the flexibility of the secondary structure: if vRNA molecules are confined then their secondary structure becomes increasingly branched [7]. The physics of the confinement of vRNA molecules inside a potential well, including the manner their secondary structure adapts to a confining potential, is an important issue for a physical description of the process of viral assembly, with the confining potential representing the interaction of the RNA molecule with the capsid proteins [8].…”