1. Viruses are parasites Viral genomes small -- contain few genes compared with living cells – HIV genome has 9749 nucleotides - Human cells have ~3.2 x 10 9 base pairs (~30,000 genes) Viruses must hijack host cells. They can only reproduce if they force the host cell to make viral copies. New viruses leave host cell and infect similar cells. Some viruses cause diseases in their hosts.

2. Clicker question Viruses can infect 1) Humans 2) Animals 3) Plants 4) Bacteria 5) Bi 1 students 6) All of the above

3. Raison d’etre of a virus Deliver nucleic acid (single or double stranded; RNA or DNA) to host cell. Take over host’s biosynthetic machinery for replication, transcription, and translation of viral genes. Assembly progeny viruses in infected cell. Release progeny to infect other cells. Avoid host immune system.

4. Clicker question Are you currently symptomatic of a rhinovirus infection? 1)Yes 2)No 3)Decline to reveal 4)Huh?

5. Baker et al., 1999 Adding the third dimension to virus life cycles: 3D reconstructions of icosahedral viruses from cryoelectron microscopy. Microbiol. Mol. Biol. Rev. 63: 862-922 Many viruses are icosahedral We will look at two icosahedral viruses: rhinovirus (causes common cold) and poliovirus. Both are much smaller than HIV (diameters ~300 Å or 30 nm for rhino- and polioviruses compared with ~1000 Å for HIV) and both are non- enveloped (no lipid bilayer) viruses.

6. Icosahedral viruses Foxtrot by Bill Amend Human rhinovirus (HRV) and poliovirus are both picorna viruses (pico = small; rna refers to RNA). Lots of people are interested in picorna viruses.

7. Poliovirus Inserts RNA Directly into Target Cell Poliovirus RNA is then translated in the cytoplasm of the host cell Principals of Virology. Flint et al. Fig. 5.13

8. Clicker question Poliovirus and rhinovirus are retroviruses. 1) True 2) False

9. Clicker question Poliovirus and rhinovirus are retroviruses. 1) True 2) False -- Picornaviruses are plus strand RNA viruses, so their genomic RNA can serve directly as messenger RNA to translate viral proteins. One such viral protein is an RNA-dependent RNA polymerase, which makes a negative strand copy of the viral RNA that then serves as a template to make more viral RNA for packaging into new viruses.

10. Viral nucleic acid must be protected Viruses have protein shells to enclose their nucleic acid. Can’t make a single protein that is big enough to enclose and protect nucleic acid. Protein shells of viruses built up from many copies of one or more polypeptide chains, often resulting in rod-like or spherical shapes.

11. Spherical viruses Genetic economy -- build shell from many copies of a few kinds of subunit. Specificity -- subunits recognize each other to form interface of non-covalent interactions. Leads to SYMMETRY because of specific repeated bonding patterns of identical building blocks. Problem: protein shell is symmetrical, nucleic acid is not. Usually can’t see nucleic acid in crystal structures of spherical viruses.

12. How do you make a spherical object from identical units? Answered >2000 years ago by Greek mathematicians: icosahedron and dodecahedron have highest possible symmetry and therefore allow a maximum number of identical objects to form a closed symmetrical shell. EM by Aaron Klug in 1950s and 60s showed that viruses are icosahedral.

13. Buckminster Fuller Montreal Biosphere by R. Buckminster Fuller

14. Properties of an icosahedron Made from 20 identical equilateral triangles Put asymmetric object on icosahedron -- it is repeated 60 times by combining symmetry operations of 5-fold, 3-fold and 2-fold symmetry axes. Branden & Tooze, Fig. 16. 3

15. Clicker question What is the minimum number of protein subunits in the shell of an icosahedral virus? 1)1 2)5 3)20 4)60 5)120 6)None -- the shell of an icosahedral viruses is formed by a lipid membrane

16. How many protein subunits in an icosahedral virus? Look at number of symmetry related units in an icosahedron. Tiles on surface have 3-fold symmetry -- therefore 3 identical objects required to form one tile. 20 (# of tiles) x 3 (# of objects in a tile) = 60. Icosahedron has 20 3-fold axes, 12 5-fold axes, and 30 2-fold axes. One triangular face (20 total) divided into 3 A.U.’s Three objects related by icosahedral 3-fold placed on face Icosahedron with 60 objects: 1 per A.U. Branden & Tooze, Fig. 16. 4

17. The simplest icosahedral virus: STNV, a T=1 virus STNV (satellite tobacco necrosis virus) Smallest known virus (diameter = 180 Å). STNV is a satellite virus. It needs helper virus (TNV) in order to replicate. Self-sufficient viruses have more nucleic acid than STNV, so they are larger. RNA inside STNV only encodes viral coat protein. STNV has 60 identical protein subunits: T (triangulation number) = 1 (More about triangulation numbers later)

18. How to make a larger shell to enclose larger genome of a self-sufficient virus? Not by increasing size of each subunit (viral shell becomes thicker as subunits are enlarged). Instead, increase the number of subunits by having more than one subunit in each asymmetric unit of the icosahedron. Icosahedron has 60 asymmetric units, so number of subunits is a multiple of 60. Subunits can be identical or different, but more genetically economical if identical.

19. Poliovirus

21. Proteolysis (protein cleavage) is an important step in the maturation of many viruses Poliovirus RNA is translated into a poly-protein that is cleaved into separate proteins by a viral protease. Proteolysis by HIV protease is critical for HIV maturation. HIV protease is one of the targets of HAART (highly active anti-retroviral therapy).

22. Sites where antibodies bind to poliovirus Antibodies are proteins that are produced by B lymphocytes (B cells) in response to infection by a pathogen. Antibodies can neutralize the virus so it can’t infect cells. However, if the virus changes by mutation, antibodies usually can’t bind it anymore (rapid mutation of HIV is one of the main reasons antibodies aren’t very effective against HIV). Antibody binding sites on poliovirus represent sites of rapid mutation.

23. How are antibody binding sites identified? Passage virus in tissue culture cells in presence of neutralizing antibody. Viruses that are neutralized by the antibody can’t infect cells; mutant viruses can infect cells. Sequence RNA of viruses that are produced in the presence of the neutralizing antibody. Changes pinpoint the binding site (epitope) of the antibody. This process (growth of mutant viruses that aren’t affected by antibodies) is an excellent example of natural selection.

24. 1) Viruses mutate rapidly to avoid host immune system 2) Viruses enter cells by binding to host receptors 3) Mutation of host receptors would be deleterious for the host. How to reconcile these three points?

25. A canyon containing conserved residues encircles 5-fold axes in picornaviruses Host cell receptors bind in the canyon.

26. The Canyon Hypothesis

27. Host receptors for some viruses are structurally-similar proteins containing antibody-like domains Loop structures represent protein domains of ~100 residues each. Although the receptor domains are related to antibodies, the receptor proteins are monomers, whereas antibody binding sites are dimers. Antibodies are therefore “fatter” than these viral receptors and are excluded from places where these receptors can fit. Note that these proteins all serve vital functions for the host (e.g., CD4 is a T cell coreceptor), so the host cannot easily evolve to mutate or eliminate the receptor. Rossmann, MG (1994) Viral Recognition and Entry. Protein Sci. 3:1712-1725

28. Sequencing and analyses of all known human rhinovirus (HRV) genomes reveal structure and evolution Palmenberg et al., 2009, Science 324: 55-59 99 strains HRV-A and HRV-C share a common ancestor HRV-B is a sister group to the -A and -C common ancestor Branch lengths in the tree are proportional to similarity (p-distance). An example of a phylogenetic tree -- see Freeman Chapter 27. Also see B-3 “Reading a Phylogenetic Tree” in your textbook.

29. Summary of concepts Genetic economy leads to one or a few capsid subunits, which leads to symmetry. Viruses mutate to escape the host immune system. Mutations are on surface-exposed regions of viral proteins that can be accessed by host antibodies. Binding sites for host receptors must remain constant. These are inaccessible to antibodies.

30. Extra slides (not included in lecture) More examples of virus structures; additional details about triangulation numbers and quasi-equivalent symmetry

31. Relative size and shape of RNA-containing viruses Granoff and Webster (1999, Vol. 1, p. 401) ~300 nm

32. Relative size and shape of DNA-containing viruses Granoff and Webster (1999, Vol. 1, p. 401) ~300 nm

33. Triangulation numbers For specific values of multiples of 60, can pack subunits with only slightly different environments in a quasi-equivalent way. Multiples of 60 are called “Triangulation numbers”. Values of T are restricted: T = (h 2 + hk + k 2 )f 2, where h,k,f are integers. Specific values of T: 1,3,4,7... Extra slides at end of lecture discuss triangulation numbers, quasi- equivalent symmetry, and other interesting aspects of icosahedral virus symmetry.

34. Organization of different types of icosahedral viruses 180 identical subunits; 3 different environments Examples: plant viruses such as TBSV, TCV 60 identical subunits; identical environments Example: STNV, a satellite virus that co-infects plants together with TNV Pseudo T= 3 60 copies each of 3 different subunits; 3 different environments Examples: rhinovirus, polio virus T = 1 T = 3 P = 3 Picorna viruses

35. Considerations about minimum size for a virus Self-sufficient RNA virus must contain genes for an RNA-directed RNA polymerase (an enzyme to copy the viral RNA) and a capsid protein. Minimum size RNA for these viruses is 1.5 x 10 6 Da -- won’t fit in a T=1 shell. T=3 capsids can package 3 x 10 6 Da. But see http://www.rpgroup.caltech.edu/~paul/scripts/virus_packing/ e.g., TBSV e.g., STNV Diameter = 330 Å Diameter = 180 Å T=3 can package ~4x more RNA than T=1

36. Quasi-equivalent symmetry (Casper and Klug, 1962) Need to pack subunits in the same (or nearly the same) environment Not a problem for T=1 viruses -- all subunits have exactly the same environment and the same packing interactions If >1 subunit in each icosahedral asymmetric unit, the subunits see different environments depending on where they are in the asymmetric unit For certain multiples of 60, can pack subunits into icosahedron so that they are packed with “quasi-equivalence” T=3 virus. 180 subunits total A subunits around 5-fold B and C alternate around 3-fold -- result is pseudo 6-fold Note 3-fold relating A, B, and C is not exact. Branden & Tooze, Fig. 16.6

37. Compare T=3 and T=4 T=3; 180 subunits total T=4; 240 subunits total T=3. A,B,C in asymmetric unit. 3 environments: A around 5-fold, C across 2-fold, B and C alternate around 3-fold T=4. A,B,C,D in asymmetric unit. 4 environments: A around 5-fold, D around 3-fold, B,C,D around 2-fold to form pseudo 6-fold. 20 real 3-folds, 60 pseudo 3-folds (formed by A,B,C), so 80 3-folds. Branden & Tooze, Figs. 16.6, 16.7

38. Canyon in rhinovirus

39. Direct demonstration of the Canyon Hypothesis: CryoEM reconstructions of receptors bound to poliovirus and rhinovirus show that host cell receptors bind in the “canyons”

41. Viruses that infect bacteria (bacteriophage or phage) look like syringes Levine, A. “Viruses” p. 34

42. Why are viruses constructed from similar or identical subunits? Genetic economy: Nucleic acid codes for only ~11% of its weight as a protein (MW of triplet codon ~1000 Da; MW of amino acid ~110 Da). Viruses are 50-90% by weight protein, so must construct virus from limited number of different kinds of subunits. Fidelity: There are errors in DNA, RNA and protein synthesis. Less chance of deleterious error if use smaller protein/gene. Same argument for advantages of oligomeric proteins, e.g., if there is 1 error per 1000 residues, then the number of correct copies is: Single chain (1000 residues)e -1000 x 1/1000 = 37% Four chains (250 residues each) e -250 x 1/1000 = 78% Stability and Complexity: The small number of contacts in, e.g., octahedron or tetrahedron insufficient for stability (holes between subunits too big, particle would be leaky). More subunits --> more stability Larger virus particle --> more complex genome

43. Clicker question Which of these are the most numerous on Earth? A)Bacteria B)Phage ~10 31 phage on earth; 10x more than the next most numerous group, bacteria C)Humans D)Cockroaches E)Bi 1 students