Detailed Study Notes on Bacteriophage P22 Assembly and Protein Interactions

Published Ahead of Print

Publication Date: 5 March 2014.
Journal: Journal of Virology
Volume: 88
Issue: 10
Pages: 5287
DOI: 10.1128/JVI.00036-14.

Authors and Institutions

Authors: Carolyn M. Teschke, Juliana R. Cortines, Tina Motwani, Aashay A. Vyas
Affiliations:

  • Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA

  • Department of Chemistry, University of Connecticut, Storrs, Connecticut, USA

Abstract

Icosahedral virus assembly requires specific protein-protein interactions to form proper capsids. In bacteriophage P22, coat protein (gp5) and scaffolding protein (gp8) are essential for assembling a procapsid-like particle both in vitro and in vivo. This study identifies critical residues involved in the interaction between the scaffolding protein and coat protein's acidic residues, specifically focusing on the N-arm of the coat protein.

Key Findings:

  • Residue R293 of scaffolding protein is required for procapsid assembly.

  • Residue K296 of scaffolding protein contributes to this process but is not essential.

  • Site-directed mutagenesis revealed that changing coat protein N-arm residue D14 to A results in lethality, indicating its critical role in interaction with the scaffolding protein.

  • Residue E18 also plays a role in size determination for capsids, as mutations here affect capsid morphology.

  • Residue E15 shows weak interaction with the scaffolding protein.

  • The findings support the hypothesis that surface charge density drives interactions vital for virus capsid assembly.

Importance of the Study

  • Bacteriophage P22 serves as a model for studying icosahedral viral capsid assembly and its interactions with supporting scaffolding proteins.

  • Understanding these interactions can provide targets for antiviral strategies due to the limited number of distinct proteins utilized by viruses for capsid construction.

  • The study sheds light on how coat proteins assemble into functional capsids through specific charged interactions, fundamentally influencing viral morphology and replication processes.

Introduction

Icosahedral viral capsids are assembled chiefly from many copies of a singular coat protein, organized into distinct capsomers. The assembly involves several steps, beginning with protein-protein interactions that form intermediate structures known as procapsids. Key contributions in this pathway are made by auxiliary proteins, known as scaffolding proteins (SPs), which direct the coating protein to the correct morphology and shape.

Background on Bacteriophage P22

  • In P22, lack of scaffolding protein results in aberrant structures, including spirals formed from improper assembly of coat proteins.

  • Scaffolding protein initiates assembly by binding to coat proteins, creating an intermediate that encapsulates viral DNA.

  • Maturation of the capsid involves structural expansion, signifying a critical transition in viral assembly.

Structural Considerations

The coat protein structure is classified into different domains, critical for maintaining stability and ensuring proper interaction during assembly. The interaction zones within these domains include:

  1. N-arm: Amino acids 1 to 56, involved in initial assembly.

  2. E-loop: Amino acids 57 to 82, contributing to conformational flexibility.

  3. P-domain: Amino acids 83 to 143 and 346 to 420, important for capsid architecture.

  4. A-domain: Amino acids 144 to 222, facilitating structural integrity during assembly.

  5. I-domain: Previously termed the telokin-like domain, aiding in final capsid assembly via charged interactions.

Maturation Dynamics

Upon maturation, significant conformational shifts occur in the N-arm, P-loop, and A-domain, enhancing the stability and assembly of the capsid while enabling the expulsion of scaffolding proteins through openings in the capsid structure.

Experimental Procedures

Materials and Methods

The study utilized specific bacterial strains and plasmids to observe the interactions between the coat protein and scaffolding protein.

Bacterial and Phage Stocks

  1. Plasmids: pHBW-1 (bacteriophage P22 coat protein, gene 5) and pET3a (gene 8 - scaffolding protein).

  2. Mutants: Plasmid modifications through site-directed mutagenesis to create specific amino acid substitutions.

  3. Bacterial Strains: Includes Salmonella enterica serovar Typhimurium and Escherichia coli, engineered for phage expression and protein interaction studies.

Results and Discussion

Key Residue Interactions

  • Aspartic Acid (D14) in coat protein is fundamental for binding to R293 in the scaffolding protein, crucial for both assembly andfunctionality of the procapsid.

  • Mutational analysis demonstrated that even subtle changes at D14 lead to pronounced effects on assembly efficiency and morphology.

  • Glutamic Acid (E15) and E18 also play roles but exhibit less significant influence than D14, with E18 mutations leading to smaller capsids.

Protein Interaction Studies

The study demonstrated strong correlations between the identified residues and their roles in facilitating protein assembly dynamics.

  • Analyses using dual cysteine mutants supported the close proximity of interacting amino acids and validated hypotheses about their structural importance in capsid assembly.

Assembly Experiments

Interactions were further validated through assembly assays and evaluations of protein folding, with mutations directing significant outcomes regarding capsid morphology, favoring the hypothesis that the coat protein N-arm forms a primary interface with the scaffolding protein to establish the architecture of the procapsid.

Conclusion

The interrelation of charge interactions between the coat and scaffolding proteins is critical for the assembly and morphology of bacteriophage P22. Specific salt bridges, especially involving residues D14 and R293, highlight the nature of protein interfaces that drive viral capsid assembly.

Key Takeaways

  • Surface Charge Importance: The overall charge distribution plays a crucial role in capsid assembly dynamics.

  • Potential Antiviral Targets: The identified protein interactions present potential avenues for antiviral drug development due to the essential specificity of their interactions.

Acknowledgments

This study was supported by NIH grant GM076661. Special thanks to collaborators and technical staff at the University of Connecticut for their contributions.

References

Citations pertinent to the study and foundational works mentioned throughout the text, highlighting key findings in virology and protein interaction dynamics.

End of Notes