A Cut above the Rest: Characterization of the Assembly of a Large Viral Icosahedral Capsid.

Erin R Reilly,Ru-Ching Hsia,Milky K Abajorga,Nurul Humaira Mohd Redzuan,Zein Haidar,Juliana A Pinzon,Susan T Weintraub,Randy Diaz,Lindsay W Black,Julie A Thomas,André O Hudson,Cory Kiser,Lily E Adams

doi:10.3390/v12070725

Abstract

The head of Salmonella virus SPN3US is composed of ~50 different proteins and is unusual because within its packaged genome there is a mass (>40 MDa) of ejection or E proteins that enter the Salmonella cell. The assembly mechanisms of this complex structure are poorly understood. Previous studies showed that eight proteins in the mature SPN3US head had been cleaved by the prohead protease. In this study, we present the characterization of SPN3US prohead protease mutants using transmission electron microscopy and mass spectrometry. In the absence of the prohead protease, SPN3US head formation was severely impeded and proheads accumulated on the Salmonella inner membrane. This impediment is indicative of proteolysis being necessary for the release and subsequent DNA packaging of proheads in the wild-type phage. Proteomic analyses of gp245- proheads that the normal proteolytic processing of head proteins had not occurred. Assays of a recombinant, truncated form of the protease found it was active, leading us to hypothesize that the C-terminal propeptide has a role in targeting the protease into the prohead core. Our findings provide new evidence regarding the essential role of proteolysis for correct head assembly in this remarkable parasite.

Highlights

The capsids, or heads, of all tailed phages have a thin protein shell of icosahedral symmetry that protects the dsDNA genome packaged to a density of ~500 mg/mL within it [1,2]
Our research focuses on the giant Salmonella enterica myovirus SPN3US which has a 240 kb genome [27] packaged into its head
That the particles represent mature, or close to mature, heads is inferred based on their similarity to the heads of input phages that had not infected a cell (e.g., Figure 5F,G)

Summary

Introduction

The capsids, or heads, of all tailed phages have a thin protein shell of icosahedral symmetry that protects the dsDNA genome packaged to a density of ~500 mg/mL within it [1,2]. The outer shell is composed of the major capsid protein (MCP), and has at one vertex a turbine-shaped portal structure formed by 12 copies of the portal protein. The mature DNA-full head is the product of a remarkable series of steps that initiate with the assembly of a protein-only precursor structure, the procapsid or prohead which is typically spherical is shape [3]. Proheads undergo a series of maturation steps that create internal volume within the head and by doing so, allows the packaging of DNA into the head. Head maturation in tailed phages typically involves an expansion of the outer shell and loss of internal protein. Not all, the loss of internal prohead material is mediated by a prohead protease that is incorporated into the prohead core during its assembly

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Conclusion