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Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis.

Stockdale SR, Collins B, Spinelli S, Douillard FP, Mahony J, Cambillau C, van Sinderen D - PLoS ONE (2015)

Bottom Line: Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled.Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production.Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Western Road, Cork, Ireland.

ABSTRACT
Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

No MeSH data available.


Molecular model depicting TP901-1 phage assembly.See Discussion section for details. Protein names in red are putatively depicted as virion-comprising proteins; however, their localization and copy number, if indeed present, is currently unknown. Colours assigned to proteins correspond to Fig 1.
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pone.0131676.g004: Molecular model depicting TP901-1 phage assembly.See Discussion section for details. Protein names in red are putatively depicted as virion-comprising proteins; however, their localization and copy number, if indeed present, is currently unknown. Colours assigned to proteins correspond to Fig 1.

Mentions: Western blot detection of TP901-1 proteins demonstrated the strong functional relationships between virion-composing proteins, how these interactions are both complex and essential, and facilitated a model for the assembly of TP901-1 virion (Fig 4). In this model, the portal protein of TP901-1 is important as it is predicted to initiate capsid assembly through interactions with other crucial capsid components. MCP1TP901-1 is presumed to act as a functional analogue of phage SPP1 capsid protein gp7 (see Results section) and thus is predicted to associate with the internal face of the portal protein complex. SPP1 gp7, present at an estimated three copies per virion, is proposed to bind DNA in a non-specific manner to control genome ejection at the onset of infection [92–94]. The in vitro assembly and lysogeny assays of TP901-1 mutants suggests that mutant MCP1TP901-1 assembles into full virions, as the mutation could not be efficiently complemented by the addition of purified capsids or tails. However, achieving high-quality EM images of MCP1TP901-1::Ter to confirm this result was not possible, as ultracentrifugation preparations of this mutant generated only capsids which appear devoid of DNA (see Fig 3B). Therefore, more work is required to understand the role of MCP1TP901-1 in viral assembly and possibly its role in virion stability. MCP2TP901-1 is depicted as a capsid protein in our phage assembly model; however, also in this case further work is required to confirm that MCP2 is indeed part of mature TP901-1 virions.


Structure and Assembly of TP901-1 Virion Unveiled by Mutagenesis.

Stockdale SR, Collins B, Spinelli S, Douillard FP, Mahony J, Cambillau C, van Sinderen D - PLoS ONE (2015)

Molecular model depicting TP901-1 phage assembly.See Discussion section for details. Protein names in red are putatively depicted as virion-comprising proteins; however, their localization and copy number, if indeed present, is currently unknown. Colours assigned to proteins correspond to Fig 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493119&req=5

pone.0131676.g004: Molecular model depicting TP901-1 phage assembly.See Discussion section for details. Protein names in red are putatively depicted as virion-comprising proteins; however, their localization and copy number, if indeed present, is currently unknown. Colours assigned to proteins correspond to Fig 1.
Mentions: Western blot detection of TP901-1 proteins demonstrated the strong functional relationships between virion-composing proteins, how these interactions are both complex and essential, and facilitated a model for the assembly of TP901-1 virion (Fig 4). In this model, the portal protein of TP901-1 is important as it is predicted to initiate capsid assembly through interactions with other crucial capsid components. MCP1TP901-1 is presumed to act as a functional analogue of phage SPP1 capsid protein gp7 (see Results section) and thus is predicted to associate with the internal face of the portal protein complex. SPP1 gp7, present at an estimated three copies per virion, is proposed to bind DNA in a non-specific manner to control genome ejection at the onset of infection [92–94]. The in vitro assembly and lysogeny assays of TP901-1 mutants suggests that mutant MCP1TP901-1 assembles into full virions, as the mutation could not be efficiently complemented by the addition of purified capsids or tails. However, achieving high-quality EM images of MCP1TP901-1::Ter to confirm this result was not possible, as ultracentrifugation preparations of this mutant generated only capsids which appear devoid of DNA (see Fig 3B). Therefore, more work is required to understand the role of MCP1TP901-1 in viral assembly and possibly its role in virion stability. MCP2TP901-1 is depicted as a capsid protein in our phage assembly model; however, also in this case further work is required to confirm that MCP2 is indeed part of mature TP901-1 virions.

Bottom Line: Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled.Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production.Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

View Article: PubMed Central - PubMed

Affiliation: School of Microbiology, University College Cork, Western Road, Cork, Ireland.

ABSTRACT
Bacteriophages of the Siphoviridae family represent the most abundant viral morphology in the biosphere, yet many molecular aspects of their virion structure, assembly and associated functions remain to be unveiled. In this study, we present a comprehensive mutational and molecular analysis of the temperate Lactococcus lactis-infecting phage TP901-1. Fourteen mutations located within the structural module of TP901-1 were created; twelve mutations were designed to prevent full length translation of putative proteins by non-sense mutations, while two additional mutations caused aberrant protein production. Electron microscopy and Western blot analysis of mutant virion preparations, as well as in vitro assembly of phage mutant combinations, revealed the essential nature of many of the corresponding gene products and provided information on their biological function(s). Based on the information obtained, we propose a functional and assembly model of the TP901-1 Siphoviridae virion.

No MeSH data available.