Structure of Viruses “Theoretical” considerations Molecular data Evolutionary pressures

Some important methods Transmission electron microscopy Cryo electron microscopy and image reconstruction X-ray crystallography-atomic resolution Sequence data Reverse genetics Modeling and graphics

Electron Microscopy Permits High Resolution Visualization of Virus Particles Negatively Stained Plant Rhabdovirus Negative staining using uranylacetate or phosphotungstic acid is very useful to visualize partly purified viruses of various shapes. Cryoreconstruction of Reovirus Particles Virion Subviral Particle Core Cryoelectron microscopy and image reconstruction can provide high resolution images of virus structure

X-ray Crystallography Can Provide Atomic Resolution of Viruses that can be crystallized Highly purified symmetrical viruses that can be crystallized in solution can be studied by X-ray Crystallography. X rays are diffracted by the crystals and diffraction patterns are recorded on film. The diffraction patterns are analyzed using light wave and physical principles to determine structure at high resolution. These analyses involve complex physical calculations. Molecular genetic analyses to modify proteins can help refine structure.

Comparisons of Some Spherical Virus Capsids The capsids of some viruses shown at the same scale. (A) Tomato bushy stunt virus; (B) poliovirus; (C) simian virus 40 (SV40); (D) satellite tobacco necrosis virus. The structures of all of these capsids have been determined by x-ray crystallography and are known in atomic detail.

Terminology Capsid protein Structural subunit Morphological subunit Capsomere Shape vs symmetry Protein domain (e.g. beta barrel)

The Two “Laws” of Virus Structure (1) Genetic Economy (2) Equivalence

Genetic Economy Watson and Crick (1956) A genetic argument: viruses are small so their genes must be small. They cannot “waste” coding capacity on large proteins. Capsid must be built of small subunits.

Equivalence Caspar and Klug (1956-1962) Viruses must be stable in order to survive-capsid must be low free energy structure (not inert-metastable). Free energy is reduced when all subunits are in an identical environment (i.e. “equivalent”).

Limitations on particle design Maximizes genetic economy Small proteins of only a few types as structural subunits Minimizes free energy Symmetrical even if subunits are not Encloses space (for genome)

The concept of of symmetry- two basic types observed Helical (translational symmetry) shape: rod, cylinder, filament Icosahedral (rotational or cubic symmetry) shape: spherical, quasi-spherical, isometric Mixed or binal: tadpole SHAPE DOES NOT EQUAL SYMMETRY!!!

Two Forms of Symmetry Helix (rod) e.g., TMV Icosahedron (sphere) e.g., TBSV Helical and Icosahedral forms predominate in both Unenveloped and Enveloped viruses!!!!!!!

Viruses Shapes are Based on Helical and Icosahedral Forms

TMV leads the way (again) First virus to be crystallized Well-studied structurally since the 1950s TMV has one capsid protein of about 17kDa, 2100 copies per particle 300 x 15 nm 6.4 kb (+) ssRNA What do we know about the virion?

“Helical” symmetry virions How do you make a symmetric particle out of an asymmetric building block? (a problem for all viruses-not just TMV!) Make it 3-D by stacking the rings????

TMV capsid features-show more sophisticated approach NOT a stack of discs! Helical shape 3 nucts/subunit 49 nucts/helical turn Rise = 1.4 Angstrom per subunit 16-1/3 subunit per turn 23 Angstrom pitch Central channel

Cross section of two rings TMV does not merely enclose RNA…..

Advantages of helical symmetry Simple Easy to expand- up to a point…. potyviruses and closteroviruses (flexuous)

How do the helical viruses eg TMV address the limitations on particle design? (Review) Maximizes genetic economy Minimizes free energy Symmetrical even if subunits are not Encloses cargo space (for genome)

Icosahedral symmetry virions Another way to enclose a space Tetrahedron, octahedron, icosahedron all are symmetrical But the icosahedron is the only one actually found Used by diverse groups of viruses Why is the icosahedron favored in nature? Why is it special?

A preview of the answers Low free energy Encloses space efficiently with small subunits Expandable

Symmetry axes Football has 2 Axes of Symmetry (no laces)

Properties of icosahedra Vertex: 5-fold Face: 3-fold Edge: 2-fold Has three axes of symmetry

Building an icosahedral capsid Minimum of 3 asymmetric subunits per face Icosahedron has 20 faces 60 structural subunits minimum Called T = 1 (T = triangulation number)

An example of an extremely simple capsid T = 1, 60 subunits Very rare Parvoviruses Best example is Satellite Tobacco Necrosis Virus 21 kDa capsid protein 195 AAs 1239 nuct ss RNA Small capsids

T=1 Satellites have Perfect Icosahedral Organization 3-Fold Axis 18 nm 2-Fold Axis 5-Fold Axis Satellite Panicum Mosaic Virus

T=1 Capsids are Very Small How do you build a larger capsid to make a”real” virus? And address the limitations… Caspar and Klug look for an answer

Enlarging the capsid By Genetic Economy-must add more copies By Equivalence-must maintain equivalent contacts Sub-triangulation is the solution

Quasi-equivalence T >1 capsids cannot have full equivalence Caspar and Klug hypothesize: close is good enough Quasi-equivalence Capsid proteins flexible enough to accommodate

Caspar and Klug Show How to Calculate T May be calculated- T = Pf2 where P = h2 + hk + k2 ( h and k are any two integers with no common factors) and f = 1, 2, 3, 4, etc.

Some T classes T = 1: rare- Microviridae, Geminiviridae T = 3: most common- Comoviridae, Picornaviridae T = 4: also frequent T > 7: skew capsids, larger viruses 60T = # of structural subunits T can be considered the number of small triangles (facets) on each face

A T = 3 particle-some features Complete equivalence not possible Shows quasi-equivalence Note vertices Note deformation of face Icosadeltahedron is quasi-spherical

Variation of T = 3 Pseudotriangulation number P Picornaviruses have P = 3 capsids or pseudoT = 3 or pT =3

Another variation- cowpea mosaic virus and related viruses of plants Comoviruses have P = 3 capsids

Common fold in capsid proteins

TBSV capsid protein-cartoon

Domain Comparison

Beta barrels in picornaviruses VP1, VP2, VP3 are major capsid proteins VP4 not shown

Beta barrels in comoviruses L protein has 2; S has 1

T = 1 capsomeres

Canine parvovirus and STNV T = 1 capsids

T = 3 image reconstruction

Human rhinovirus 14 (Picornaviridae)

Poliovirus