Enhanced Local Symmetry Interactions Globally Stabilize a Mutant Virus Capsid That Maintains Infectivity and Capsid Dynamics

Abstract

Structural transitions in viral capsids play a critical role in the virus life cycle, including assembly, disassembly, and release of the packaged nucleic acid. Cowpea chlorotic mottle virus (CCMV) undergoes a well-studied reversible structural expansion in vitro in which the capsid expands by 10%. The swollen form of the particle can be completely disassembled by increasing the salt concentration to 1 M. Remarkably, a single-residue mutant of the CCMV N-terminal arm, K42R, is not susceptible to dissociation in high salt (salt-stable CCMV [SS-CCMV]) and retains 70% of wild-type infectivity. We present the combined structural and biophysical basis for the chemical stability and viability of the SS-CCMV particles. A 2.7-Å resolution crystal structure of the SS-CCMV capsid shows an addition of 660 new intersubunit interactions per particle at the center of the 20 hexameric capsomeres, which are a direct result of the K42R mutation. Protease-based mapping experiments of intact particles demonstrate that both the swollen and closed forms of the wild-type and SS-CCMV particles have highly dynamic N-terminal regions, yet the SS-CCMV particles are more resistant to degradation. Thus, the increase in SS-CCMV particle stability is a result of concentrated tethering of subunits at a local symmetry interface (i.e., quasi-sixfold axes) that does not interfere with the function of other key symmetry interfaces (i.e., fivefold, twofold, quasi-threefold axes). The result is a particle that is still dynamic but insensitive to high salt due to a new series of bonds that are resistant to high ionic strength and preserve the overall particle structure.Assembly, stability, and disassembly of icosahedral virus particles are coordinated by protein-protein and protein-nucleic acid interactions. Cowpea chlorotic mottle virus (CCMV) provides a model system for examining these interactions. The advantages of this system include in vivo and in vitro assembly assays (4, 7, 12, 55), a reversible transition between distinctly different closed and expanded forms (6), a high-resolution crystal structure of the closed particle (42), an electron cryomicroscopy image reconstruction of the swollen particle (42), and a general understanding of the requirements for assembly and stabilization of the capsid (6,53,55,56).CCMV is a small RNA plant virus that belongs to the Bromovirus genus in the Bromoviridae family. The viral genome is composed of four (positive-sense) single-stranded RNA molecules that are encapsidated in three morphologically identical particles (8,21,27). RNA1 and RNA2 encode proteins involved in RNA-dependent RNA replication and are packaged in separate particles. RNA3, encoding the movement and capsid proteins, and RNA 4, a subgenomic RNA of RNA 3 encoding just the capsid protein, are copackaged in a third particle (2). The structure of the CCMV virion has been determined to a 3.2-Å resolution (42) revealing a Tϭ3 truncated icosahedron having a diameter of 286 Å and composed of 180 identical protein subunits arranged as protruding ...

The Tripartite Virions of the Brome Mosaic Virus Have Distinct Physical Properties That Affect the Timing of the Infection Process

“…2B). SDS-PAGE of the BMV CP treated with the same conditions revealed that less than 20% of the CP migrated to the position expected of the cleaved product and that the RNA was not fragmented (21) (Fig. 2C and 1B).…”

Phosphorylation of the Brome Mosaic Virus Capsid Regulates the Timing of Viral Infection

“…Many other viruses have capsid proteins with positively charged and highly flexible sequences that translocate from within the virion to the external surface of the virion (7,53). It is possible that these sequences are acted on by host proteins to regulate the disassembly of the virions and enable viral gene expression and replication.…”

Conformational Equilibria and Rates of Localized Motion within Hepatitis B Virus Capsids

“…This large-scale motion is expected to be rare compared to the smaller transitions required for amide proton exchange in HDX. Protein dynamics on the nanometer scale are known to be involved in complex formation (53), viral capsid breathing (49,(54)(55)(56) and viral docking and entry events (57).…”