Dataset Information

Mass Spectral Analyses of Salmonella Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

ABSTRACT: Salmonella myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the composition of the mature SPN3US head and elucidate how it is modified by proteolysis during assembly, we conducted tandem mass spectrometry analysis of purified virions and tailless heads. Fourteen protease cleavage sites were identified in nine proteins, including eight sites not previously identified in head proteins in vivo. Among these was the maturation cleavage site of gp245 which was identical to the autocleavage site we had previously identified in purified recombinant gp245. Our findings underscore the value of employing multiple mass spectrometry-based experimental strategies as a way to enhance the detection of head protein cleavage sites in tailed phages. In addition, our results have identified a conserved set of head proteins in related giant phages that are similarly cleaved by their respective prohead proteases, suggesting that these proteins have important roles in governing the formation and function of large icosahedral capsids.

SUBMITTER: Scheuch A

PROVIDER: S-EPMC10052503 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Publications

Mass Spectral Analyses of <i>Salmonella</i> Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

Scheuch Aaron A Moran Sara A M SAM Faraone Julia N JN Unwin Sophia R SR Vu Gialinh G Benítez Andrea Denisse AD Mohd Redzuan Nurul Humaira NH Molleur Dana D Pardo Sammy S Weintraub Susan T ST Thomas Julie A JA

Viruses 20230310 3

<i>Salmonella</i> myovirus SPN3US has a T = 27 capsid composed of >50 different gene products, including many that are packaged along with the 240 kb genome and ejected into the host cell. Recently, we showed that an essential phage-encoded prohead protease gp245 is responsible for cleavage of proteins during SPN3US head assembly. This proteolytic maturation step induces major changes in precursor head particles, enabling them to expand and undergo genome packaging. To comprehensively define the ...[more]

PMID: 36992431

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Project description:Spherical viruses are remarkably well characterized by the Triangulation (T) number developed by Casper and Klug. The T-number specifies how many viral capsid proteins are required to cover the virus, as well as how they are further subdivided into pentamer and hexamer subunits. The T-number however does not constrain the orientations of these proteins within the subunits or dictate where the proteins should place their protruding features. These protrusions often take the form of loops, spires and helices, and are significant because they aid in stability of the capsid as well as recognition by the host organism. Until now there has be no overall understanding of the placement of protrusions for spherical viruses, other than they have icosahedral symmetry. We constructed a set of gauge points based upon the work affine extensions of Keef and Twarock, which have fixed relative angular locations with which to measure the locations of these features. This work adds a new element to our understanding of the geometric arrangement of spherical viral capsid proteins; chiefly that the locations of protruding features are not found stochastically distributed in an icosahedral manner across the viral surface, but instead these features are found only in specific locations along the 15 icosahedral great circles. We have found that this result holds true as the T number and viral capsids size increases, suggesting an underlying geometric constraint on their locations. This is in spite of the fact that the constraints on the pentamers and hexamer orientations change as a function of T-number, as you need to accommodate more hexamers in the same solid angle between pentamers. The existence of this angular constraint of viral capsids suggests that there is a fitness or energetic benefit to the virus placing its protrusions in this manner. This discovery may have profound impacts on identifying and eliminating viral pathogens, understanding evolutionary constraints as well as bioengineering for capsid drug delivery systems. This result also suggests that in addition to biochemical attachment restrictions, there are additional geometric constraints that should be adhered to when modifying protein capsids.

Project description:Giant tailed bacterial viruses, or phages, such as Pseudomonas aeruginosa phage ϕKZ, have long genomes packaged into large, atypical virions. Many aspects of ϕKZ and related phage biology are poorly understood, mostly due to the fact that the functions of the majority of their proteins are unknown. We hypothesized that the Salmonella enterica phage SPN3US could be a useful model phage to address this gap in knowledge. The 240-kb SPN3US genome shares a core set of 91 genes with ϕKZ and related phages, ∼61 of which are virion genes, consistent with the expectation that virion complexity is an ancient, conserved feature. Nucleotide sequencing of 18 mutants enabled assignment of 13 genes as essential, information which could not have been determined by sequence-based searches for 11 genes. Proteome analyses of two SPN3US virion protein mutants with knockouts in 64 and 241 provided new insight into the composition and assembly of giant phage heads. The 64 mutant analyses revealed all the genetic determinants required for assembly of the SPN3US head and a likely head-tail joining role for gp64, and its homologs in related phages, due to the tailless-particle phenotype produced. Analyses of the mutation in 241, which encodes an RNA polymerase β subunit, revealed that without this subunit, no other subunits are assembled into the head, and enabled identification of a "missing" β' subunit domain. These findings support SPN3US as an excellent model for giant phage research, laying the groundwork for future analyses of their highly unusual virions, host interactions, and evolution.ImportanceIn recent years, there has been a paradigm shift in virology with the realization that extremely large viruses infecting prokaryotes (giant phages) can be found in many environments. A group of phages related to the prototype giant phage ϕKZ are of great interest due to their virions being among the most complex of prokaryotic viruses and their potential for biocontrol and phage therapy applications. Our understanding of the biology of these phages is limited, as a large proportion of their proteins have not been characterized and/or have been deemed putative without any experimental verification. In this study, we analyzed Salmonella phage SPN3US using a combination of genomics, genetics, and proteomics and in doing so revealed new information regarding giant phage head structure and assembly and virion RNA polymerase composition. Our findings demonstrate the suitability of SPN3US as a model phage for the growing group of phages related to ϕKZ.

Dataset Information

Mass Spectral Analyses of Salmonella Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

Publications

Mass Spectral Analyses of <i>Salmonella</i> Myovirus SPN3US Reveal Conserved and Divergent Themes in Proteolytic Maturation of Large Icosahedral Capsids.

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