1. Structure of Viruses “Theoretical” considerations Molecular data
Evolutionary pressures

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

3. 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

4. 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.

5. 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.

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

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

8. 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.

9. 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”).

10. 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)

11. 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!!!

12. 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!!!!!!!

13. Viruses Shapes are Based on Helical and Icosahedral Forms

14. 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?

15. “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????

16. 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

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

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

19. 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)

20. 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?

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

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

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

24. 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)

25. 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

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

27. 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

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

29. 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

30. 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.

31. 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

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

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

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

35. Common fold in capsid proteins

36. TBSV capsid protein-cartoon

38. Domain Comparison

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

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

41. T = 1 capsomeres

42. Canine parvovirus and STNV T = 1 capsids

43. T = 3 image reconstruction

44. Human rhinovirus 14 (Picornaviridae)

45. Poliovirus