Amino Acid Side Chains Buried along Intersubunit Interfaces in a Viral Capsid Preserve Low Mechanical Stiffness Associated with Virus Infectivity

Abstract

Single-molecule experimental techniques and theoretical approaches reveal that important aspects of virus biology can be understood in biomechanical terms at the nanoscale. A detailed knowledge of the relationship in virus capsids between small structural changes caused by single-point mutations and changes in mechanical properties may provide further physics-based insights into virus function; it may also facilitate the engineering of viral nanoparticles with improved mechanical behavior. Here, we used the minute virus of mice to undertake a systematic experimental study on the contribution to capsid stiffness of amino acid side chains at interprotein interfaces and the specific noncovalent interactions they establish. Selected side chains were individually truncated by introducing point mutations to alanine, and the effects on local and global capsid stiffness were determined using atomic force microscopy. The results revealed that, in the natural virus capsid, multiple, mostly hydrophobic, side chains buried along the interfaces between subunits preserve a comparatively low stiffness of most (S2 and S3) regions. Virtually no point mutation tested substantially reduced stiffness, whereas most mutations increased stiffness of the S2/S3 regions. This stiffening was invariably associated with reduced virus yields during cell infection. The experimental evidence suggests that a comparatively low stiffness at S3/S2 capsid regions may have been biologically selected because it facilitates capsid assembly, increasing infectious virus yields. This study demonstrated also that knowledge of individual amino acid side chains and biological pressures that determine the physical behavior of a protein nanoparticle may be used for engineering its mechanical properties.

Structural basis for biologically relevant mechanical stiffening of a virus capsid by cavity-creating or spacefilling mutations

“…In contrast, in the L172I capsid the S5 regions were not stiffened, and the pore-associated transition was observed 19 . An inextricable linkage between capsid S5 stiffening, loss of this transition and impaired virus infectivity at physiological temperature has been observed also for alanine mutations of other residues at or near the base of the pores 19, 60 . When some of these virus mutants were tested, additional defects, including premature DNA externalization in the virion, were observed, as for the N170A mutant 46 .…”

“…In fact, the spacefilling, deleterious L172W mutation led to similar changes in conformation and rigidification of specific structural elements scattered over the MVM capsid, and to a subtle overall structural compaction of the viral capsid. Many other single mutations at the pores or elsewhere in the MVM capsid also increased capsid stiffness, while virtually no tested mutation led to stiffness decreases 19, 60 . Further structure-mechanics-dynamics studies may ascertain whether the natural MVM capsid has evolved to a state of lowest stiffness compared to close variants in the sequence space, and whether this local minimum in stiffness may be biologically advantageous.…”

Structural determinants of mechanical resistance against breakage of a virus-based protein nanoparticle at a resolution of single amino acids

“…[26][27][28][29][41][42][43] In particular, our group is using AFM to provide insights into the relationships between the fine structure of model virus particles, their mechanical properties, and their function. [44][45][46][47][48][49][50] By exploring the effects on a certain mechanical property (such as stiffness) of small structural alterations in the virus particle by single mutations introduced by protein engineering, knowledge about the specific chemical groups and interactions that determine that property can be acquired. [45][46][47][48][49][50] A particularly suited model for these studies is the icosahedral capsid of the minute virus of mice (MVM).…”

“…[44][45][46][47][48][49][50] By exploring the effects on a certain mechanical property (such as stiffness) of small structural alterations in the virus particle by single mutations introduced by protein engineering, knowledge about the specific chemical groups and interactions that determine that property can be acquired. [45][46][47][48][49][50] A particularly suited model for these studies is the icosahedral capsid of the minute virus of mice (MVM). 51,52 The MVM capsid ( Fig.…”

“…53,54 Using the MVM capsid, we have recently uncovered at high resolution some structural determinants of mechanical stiffness in a model virus particle. 48 Specific groups of amino acid side chains located at interfaces between capsid building blocks (CBBs) were individually removed through site-directed mutagenesis; and changes in the elastic constant of the viral particle upon mutation were determined by indentation of different capsid regions with the AFM tip under the elastic regime, without breaking the particle. It was found that, in general, hydrophobic side chains in secondary structure elements buried along the interfaces between CBBs contribute to maintain most regions of the viral capsid in a state of comparatively low stiffness; their individual removal led to a large, frequently anisotropic stiffening of different capsid regions.…”