Emerging Topics in Physical Virology

The following sections are included:

• Contributors

• Preface

• Contents

Cryo-electron microscopy (cryo-EM) is a structural technique that images biological macromolecules in native-like conditions, and has been widely applied to the study of viruses. Virus structures have been determined by cryo-EM at resolutions ranging from molecular (~30–50 Å) to near-atomic (~4 Å). Here we introduce cryo-EM of virus particles for the non-expert reader and review how some of the key cryo-EM studies have advanced our understanding of virus biology. We also describe the latest advances in cryo-EM. These advances are on the one hand driving cryo-EM studies of symmetric viruses towards atomic resolution. On the other, they are developing structural methods that allow the study of individual, pleiomorphic virus particles and the interactions they make with cellular machinery.

Structural analyses now provide a framework to understand some aspects of virus evolution, where the three dimensional structures of virus capsids essentially substitutes for a fossil record. It has emerged that whilst many aspects of viruses are subject to horizontal gene transfer, for many viruses the core structures responsible for capsid assembly and genome packaging are highly conserved. We define this set of indispensable properties as the virus 'self'. A 'viral lineage' then comprises a set of viruses sharing a recognizably common 'self'. In this review we summarize the history and current status of the endeavour to develop a structure-based taxonomy based around this concept, highlighting not only some of the insights that this has brought, but also the problems that have emerged and the prospects for a systematic structure-based classification of the virosphere.

Since the seminal work of Caspar and Klug on the structure of the protein containers that encapsulate and hence provide protection for the viral genome, it has been recognised that icosahedral symmetry is crucial for the prediction of viral architecture. In particular, their theory of quasi-equivalence invokes icosahedral symmetry to pinpoint the surface structures of viral capsids in terms of triangulations that schematically encode the locations of the protein subunits in the capsids. Whilst this approach is capable of predicting the relative locations of the proteins in the capsids, information on their tertiary structures and the organisation of the viral genome within the capsid are inaccessible. We present here a mathematical framework based on affine extensions of the icosahedral group that has been developed to predict a wide spectrum of features of the three-dimensional structure of simple viruses. This approach implies that the predictions of Caspar and Klug's quasi-equivalence theory are the consequences of a deeper level of structural organisation in viruses that orchestrates the full three-dimensional structure of simple viruses.

If a virus releases its genomic content prematurely, it loses its infective capability. Yet, the viral shell does need to open at a specific place and time to ensure genome delivery into a new host. Hence, the chemical and mechanical properties of capsids are carefully tuned to fulfill these constraints. Knowledge of these properties will help to elucidate the viral infectious pathway, to develop virus based therapies and to facilitate the use of viruses in nanotechnology. Here we focus on the material properties of viruses mainly based on data obtained by mechanical manipulation of single viral particles. The main tool for such experiments is the atomic force microscope (AFM) and the experimental basis of these measurements will be explained. Next, aspects of the capsid shell structure, presence of encapsidated material, capsid failure, maturation and capsid protein mutations will be discussed in relation to the viral material properties. By comparing the data of various viruses, similarities and differences in the mechanical properties will be highlighted.

The development of soft ionization techniques has made it possible to examine biological macromolecules using mass spectrometry. Proteomic approaches have been developed which are capable of identifying and chemically characterizing a wide variety of biological molecules in an efficient manner. A variety of mass spectroscopic experimental approaches have been developed which allow the investigator to obtain information about protein structure, dynamics and protein/protein interactions. This review focuses on the application of these techniques to the study of viral replication.

The following sections are included:

• Introduction

• Modeling Assembly
  • Stepwise Assembly
  • Contributions of Alternative Pathways to Stepwise Assembly
  • Molecular Dynamics and Stepwise Assembly
  • Protein Condensation

• Virus Capsid Assembly in Solution
  • Experimental Systems
  • Assembly of Bacteriophage P22
  • HBV Assembly
  • HBV and Small Molecules that Interfere with Assembly
  • Bromovirus Assembly

• Concluding Remarks and Future Directions

• References

We examine the assembly and disassembly of T = 13 viral shells using a deformable version of the Caspar-Klug deltahedral model. As the cohesive energy is reduced, the shell releases elastic energy by rupturing. The path of rupture is determined by the intrinsic elastic stress pattern of icosahedral shells. Surprisingly, spherical shells are more prone to rupture than icosahedral shells. Next, assembly of a T = 13 deltahedral shell is shown to be impossible along the conventional pathway of compact partial shells. Instead, we propose a non-compact assembly pathway, with the elastic energy directing the assembly through 'Whiffle-Ball' type assembly intermediates.

The following sections are included:

• Introduction

• Effect of Charge

• Effect of Preferred Curvature

• Effect of Polyanion Size

• Dependence of RNA Size on Sequence
  • Theory
  • Experiment

• So…What Determines the Size of an RNA Virus?

• Acknowledgement

• References

In this work, we review recent advances in the field of physical virology, presenting both experimental and theoretical studies on the physical properties of viruses. We focus on the double-stranded DNA (dsDNA) bacteriophages as model systems for all of the dsDNA viruses both prokaryotic and eukaryotic. Recent studies demonstrate that the DNA packaged into many dsDNA viral capsids is highly pressurized, which provides a force for the first step of passive injection of viral DNA into either bacterial or eukaryotic cells. Moreover, specific studies on capsid strength show a strong correlation between genome length and capsid size and robustness. The implications of these newly appreciated physical properties of a viral particle with respect to the infection process are discussed.

The following sections are included:

• Introduction:The Organization of Double-Stranded DNA in Bacteriophage Capsids
  • Spooling Models
  • Toroidal Models
  • Spiral-Fold Model
  • Liquid Crystalline Model
  • Other Models

• Knot Theory and Its Applications to Molecular Biology
  • Mathematical Knots
  • Knots and DNA
  • Computer Models of DNA Knotting
  • Algorithms for Generating Ensembles of Random Knots

• DNA Knots in Bacteriophage P4
  • Bacteriophage P4
  • Experimental Methods: P4 Production and DNA Extraction
  • Experimental Results

• Models of Random Packing
  • Computational Methods
  • Results
    • Knotting probability, average crossing number and writhe in confined random polygons
  • Distributions of Knots for Low Crossing Numbers: Non-Random Packing of DNA in Bacteriophage P4
  • Knotting Probability and Complexity in Biased Models

• Conclusions and Future Challenges

• Acknowledgements

• References

There is a growing need for medical treatments on the nanoscale in the fields of imaging, vaccine development and targeted drug delivery. Viruses are an ideal platform for these technologies because of their size, structure and ease of genetic or chemical modification. This chapter will highlight the use of viruses to achieve these goals, and will focus on the major contributions of a single member, cowpea mosaic virus. In the pursuit of advancing biomedical nanotechnology, significant contributions have been made to both medicinal applications and the understanding of the viruses themselves.

The following sections are included:

• Index