A novel stabilization mechanism accommodating genome length variation in evolutionarily related viral capsids

Abstract

Tailed bacteriophages are one of the most numerous and diverse group of viruses. They store their genome at quasi-crystalline densities in capsids built from multiple copies of proteins adopting the HK97-fold. The high density of the genome exerts an internal pressure, requiring a maturation process that reinforces their capsids. However, it is unclear how capsid stabilization strategies have adapted to accommodate the evolution of larger genomes in this virus group. Here we characterized a novel capsid reinforcement mechanism in two evolutionary-related actinobacteriophages that modifies the length of a stabilization protein to accommodate a larger genome while maintaining the same capsid size. We used cryo-EM to reveal that capsids contained split hexamers of HK97-fold proteins with a stabilization protein in the chasm. The observation of split hexamers in mature capsids was unprecedented, so we rationalized this result mathematically, discovering that icosahedral capsids can be formed by all split or skewed hexamers as long as their T-number is not a multiple of three. Our results suggest that analogous stabilization mechanisms can be present in other icosahedral capsids, and they provide a strategy for engineering capsids accommodating larger DNA cargoes as gene delivery systems. Significance StatementHow capsids are stabilized and change size is an important part of understanding how to design protein containers and understand viral evolution. We describe a novel capsid stability mechanism that allows the capsid to package a larger genome without changing the capsid architecture and have predicted other capsids using this mechanism. Beyond the evolutionary implications, our findings provide a mechanism to increase the amount of DNA packaged in a capsid, offering a solution to engineer gene delivery systems with larger DNA content, a pressing challenge in gene therapy.