1. Quasispecies Theory and the Behavior of RNA Viruses Sumeeta Singh, Steve Bowers, Greg Rice, Tom McCarty BINF 704 02/19/13

3. What is a virus? Viruses are obligate intracellular parasites. Small infectious agents bearing nucleic acid instructions. Classified based on the form of nucleic acid, DNA or RNA.

4. Virus types Plus strand RNA virus Minus strand RNA virus Double strand RNA virus Retrovirus Single strand DNA virus Double strand DNA virus

5. RNA Virus types

6. DNA Virus types

7. “Quasispecies Theory” Mathematical framework describing evolution of macromolecules (Eigen, 1971) Extends the classic population genetics ideas of mutation-selection into quasispecies (Eigen, Schuster, 1977) Eventually borrowed to describe RNA virus evolution dynamics

8. Virus Replication Error Plus strand RNA virus Minus strand RNA virus Double strand RNA virus Retrovirus Single strand DNA virus Double strand DNA virus RNA dependent- RNA polymerase Error rate =10 -3 to 10 -5 DNA polymerase Error rate =10 -7 to 10 -9 Reverse Transcriptase Error rate =10 -4 to 10 -5

9. Requirements and Consequences Polymerase responsible for high error rates. Estimated that each single and some double nucleotide sequence changes occur. Resulting in a collection of related sequences around a “master” sequence. Variation is related to ability to survive (population genetics) AND probability of occurring based on sequence neighbors (quasispecies).

10. Virus Replication Swarm

12. “Quasispecies Theory” 1971 1977

13. Quasispecies and RNA viruses Survival of the “flattest” Error Catastrophe Fidelity and Fitness

14. Survival of the Fittest or Survival of the Flattest A flat species is a species which exists in a genetically diverse group. Not dominated by one variant. A quasispecies must be a flat species. A fit species (in this part of the presentation) is a species which reproduces very fast.

15. Flat Species

16. Properties of a flat species A flat species will have high mutation rates. A flat species is able to mutate without a major effect on fitness.

17. Advantages of a flat species Different mutants in a flat quasispecies can help each other. Flat species are better able to adapt.

18. Dengue-1 Virus Lives as a quasispecies One variant of the virus which is found in high concentrations cannot survive on its own. It can only survive because other viruses (infecting the same cell) create the protein which it lacks.

19. Viroid Experiment Theory: More genetically diverse (flatter) species are better able to adapt to mutations. Viroid 1 - CSVd – very fit Viroid 2- CChVMd – more flat, but less fit Procedure: Infect plants with each viroid Subject the plants to two environment either normal, or UVC light

20. Results UVC light will cause mutations Results: Under normal conditions the fitter viroid did better, then the flatter viroid. Under the UVC light the two were about equal Under the UVC light the flatter became more diverse. The fitter did not become more diverse.

21. Fitter or Flatter, which is better able to survive? The fit species will grow faster in an ideal environment. The flat species will be able to adapt more quickly.

22. Error Threshold RNA Viruses have a high mutation rate The point at which accumulated mutations reduce fitness: - Too much mutation can lead to loss of vital information - Too little mutation can lead to host defenses overcoming the virus Error Threshold: position in informational space where a phase transition occurs such that the genomic sequence information can no longer be perpetuated. The greatest fitness is when mutation rates approach the error threshold

23. Model of Error Catastrophe http://www.pnas.org/content/98/12/6895.full

24. Error Catastrophe Extinction of a virus as a result of excessive RNA mutations – lethal mutagenesis Decrease viral fitness by increasing the rate at which new mutations appear. There is an intrinsic limit to the maximum variability of viral genetic information before it loses meaning. If an RNA virus quasispecies goes beyond that mutation limit, the population will no longer be viable.

25. Increasing Mutation Rate Ionizing radiation (eg X-rays) – cause mutations by damaging DNA. Base Analogs – chemicals that replace one of the usual nucleotides in the DNA. These mutagens cause copying errors.

26. APOBEC3G Humans have the ability to induce lethal mutagenesis Protein found inside cells that has a very specific antiviral role Cytidine deaminase enzyme Unfortunately, HIV has the ability to bind to APOBEC3G proteins and cause their degradation

27. APOBEC3G It de-aminates the cytosine base, thus mutating it to a uracil base

28. APOBEC3G

29. 29 Fidelity and Fitness: Mutation Rate Evolutionary Theory: viral error rates RNA Virus: low fidelity generates diverse population of variants Homogenous population vs dynamic environment

30. 30 Fidelity and Fitness: Mutation Rate Ribavirin and Lethal Mutagenesis Hypothesis: mutant with low mutation rate less sensitive to LM and resistant to ribavirin Poliovirus experimental groups: G64S polymerase mutation

31. 31 Fidelity and Fitness: Mutation Rate/Pathogenicity 3D-G64S Virus Is Less Pathogenic than Wild-Type Virus in Mice Competition between 3D-G64S and Wild-Type Virus in Mice

32. 32 Fidelity and Fitness: Virulence G64S species attenuated in transgenic mouse model for poliovirus infection Virulence determined by diversity of coinfecting population Quasispecies diversity, rather than the selection of individual variants, correlates with enhanced virulence

33. 33 Fidelity and Fitness: Attenuation Vaccine Design Vignuzzi(2008): G64 engineered mutants stimulated high titers of neutralizing antibodies in mice Fidelity modulation as therapeutic strategy

34. 34 Future Perspectives How do models apply to infected hosts? What is best measure of viral fitness in dynamic population? How does population diversity influence pathogenesis (subpopulation cooperation)? Improved assays for characterizing viral populations Modern techniques: Deep sequencing, Molecular barcoding 34