Engineering Thermal Stability in RNA Phage Capsids via Disulphide Bonds

Abstract

The RNA bacteriophages, a group that includes phages Qbeta and MS2, have a number of potential bionanotechnological applications, including cell specific drug delivery and as substrates for the formation of novel materials. Despite extensive sequence identity between their coat protein subunits, and an almost identical three-dimensional fold, Qbeta and MS2 capsids have dramatically different thermal stabilities. The increased stability of Qbeta has been correlated with the inter-subunit disulphide bonds present in that capsid and not present in MS2. We have tested this hypothesis directly using mass spectrometry. Analysis of the dissociated coat protein subunits suggests that inter-molecular disulphides are formed at the capsid five-fold but may not be at the three-fold axes. This conclusion has been tested by engineering disulphide cross-links into either the five-fold or three-fold positions of the recombinant MS2 capsid. Five-fold cross-linking results in a mutant with stability properties similar to those of Qbeta. Three-fold cross-linking results in a mutant unable to assemble T = 3 shells, implying that five-fold structures are on pathway to capsid assembly in these phages. The results demonstrate how it is possible to redesign the physical properties of phage shells and may be of general relevance to future applications of viruses and virus-like particles.

Disulfide Linkage and Structure of Highly Stable Yeast-derived Virus-like Particles of Murine Polyomavirus

“…Instead, we suppose that the N-terminal peptides intertwine to form defined globular domains with intermonomeric disulfide linkages. Disulfide bonds are known to increase the thermal stability of viral capsids (26,27). In this study, reduction of disulfide bonds only slightly decreased thermal stability of yVLPs compared with the original yVLPs.…”

Engineering Viable Foot-and-Mouth Disease Viruses with Increased Thermostability as a Step in the Development of Improved Vaccines

“…Engineered disulfide bonds would be formed in an oxidizing extracellular medium (e.g., a vaccine formulation), but not in the reducing intracellular environment; thus, we expected those bonds to stabilize the virion with no adverse effects on its infectivity, even if there was an inverse stability/infectivity relationship. Natural disulfide bonds are indeed involved in stabilizing some virus capsids (3,27,50). Unfortunately, our disulfide approach was not successful.…”

Plasma Clearance of Bacteriophage Qβ Particles as a Function of Surface Charge

“…The lightlyloaded particle 5a, as well as particles bearing only an innocuous label (SelMet on 9) or a label on the particle interior (10), have the longest circulation times. Those particles that were heavily acylated were cleared approximately 10 times faster, whether all of the acyl linkages were tipped with Gd complexes (particle 5c) or not (particle 6).…”

“…Coordinates for the capsid structure were generated using the VIPER database (http://viperdb.scripps.edu/). 16 Qβ particles labeled with selenium (9) and interior-surface attachment of Gd(DOTA) (10), and their plasma clearance measured as in Figure 2. For clarity, averaged values are plotted from two or more independent measurements, with error bars showing the standard deviation.…”