1. The Art of Reconstruction
Virus Life Cycles in 3D
The Art of Reconstruction

2. Virus Life Cycle
In order to survive, viruses must be able to do the following:
1. Find a host cell it can replicate in
2. Bind to that cell
3. Enter the cell
4. Release its genome in order to replicate
5. Replicate its genome
6. Transcribe and translate its viral proteins
7. Package its genome and proteins
8. Escape from the cell

3. Virus Life Cycle

4. Virus Life Cycle All these processes can be visualized by cryo
These visualizations allows for a better understanding of viruses and may lead to vaccination development
For each virus, there is a unique life cycle but all viruses accomplish the same steps in order to survive

5. Enveloped Virus Semliki Forest Virus is an enveloped Alphavirus
It has 2 transmembrane proteins (E1 and E2) in its envelope
The virus binds to the cellular receptor, endocytosed, and fuses with the endosome membrane to release its nucleocapsid for replication

6. SFV as an example

7. Non enveloped Viruses
Poliovirus is a non enveloped virus in the Picornavirus family
It differs from SFV in that when it binds to its cellular receptor, it goes through a conformational change.
This conformational change may facilitate the release of genome into the cell for replication
Also releases from the cell by lysis instead of budding

8. Non enveloped virus

9. Cell Attachment
The first step in viral replication is to be able to bind to the correct host cell.
Virus recognize host cells by certain receptors.
Bind to these receptors through specific interactions.
Binding sites on viruses are typically conserved to ensure survival

10. The Receptor binding region of HRV14
Picornaviruses shield their receptor binding site in a region called the canyon in order to protect it from antibodies.
Must be conserved so that the virus can bind to the correct cell in order to replicate.
HRV16 + ICAM-1 interaction was one of the first to be studied through cryo
Was believed that the binding site for ICAM-1 was located in the canyon region of HRV16

11. The Receptor binding region of HRV14

12. HRV16 complexed with ICAM-1
HRV16 complexed with the 2 N terminal domains of ICAM-1
The footprint of ICAM-1 was centered over the canyon as predicted showing that the canyon was in fact the binding site of the receptor

13. HRV16 complexed with ICAM-1

14. Simian Rotavirus VP4 of rotavirus is important to the viral life cycle
It is a determinant of virulence, has hemagglutination activity and is also a neutralization site
The reconstruction showed that VP4 extends from the surface of the virus, which may then be able to bind to the cellular receptor more easily

15. Simian Rotavirus

16. Activation
Viruses must be stable enough to survive the extracellular environment but must also be unstable enough to release their genome when they reach susceptible cells.
Certain conformational changes must occur in the virus when it reaches the proper environment in order to release its genome in the correct place and at the correct time.

17. SFV Spikes
SFV has a spike protruding from its envelope comprised of E1, E2, and E3
Reconstruction showed that the spike has a hole in its center
From previous studies, E3 was determined to be on the outside of the spike
Preferential extraction and reconstruction comparison determined that E1made up the outside of the spike while E2 extended from the center

18. SFV with envelope and capsid

19. SFV spike structure

20. SFV spike structure

21. SFV spike conformational changes
In order to determine the conformational changes needed for activation, the particles were treated with low pH and vitrified within milliseconds
Comparison between treated and untreated particle reconstructions showed that E1 and E2 move around each other
E2 is the receptor binding portion while E1 is the membrane fusion protein
E2 moves outward while E1 moves inward to form a trimmer and trigger fusion

22. SFV spike conformational changes

23. SFV process of fusion

24. Adenovirus
Adenovirus is made up of hexons and two proteins at the five fold vertice: penton base and fiber
It binds two receptors: CAR and an integrin
CAR binds to the fiber while the penton base binds the integrin and causes activation

25. Adenovirus 2 and hexons

26. Adenovirus uses 2 receptors
CAR
Integrins

27. Structural changes in penton
The conformational changes needed for activation were determined by comparing particles which had the fiber attached and which did not
A small region which was determined to contain the RGD sequence by MAb binding changed orientation

28. Structural changes in penton

29. Genome Release
The genome of the virus is released in order to make viral proteins and reproduce the genome.
Viruses can employ several strategies to do this: injection, release into the cytoplasm, release into the nucleus
Exception: Reoviruses

30. Flock House Virus
FHV is comprised of 180 copies of a single protein which undergoes a post assembly cleavage
The cleavage produces γ peptides which lie in different orientations according to the subunit it is located on
γa lies in pentamers under the five fold
γb interacts with the bulk RNA and γc
γc also interactes with the ordered RNA

31. Flock House Virus

32. FHV γ helices

33. FHV entry into cell
This data suggested a method of FHV entry and release of genome
The virus binds and contacts the membrane at the five fold vertex
The contact releases a pocket factor which then allows the γa pentamer to insert into the membrane
The RNA is then dragged into the cell by its contacts by the other γ peptide contacts

34. FHV entry into cell

35. CCMV particle expansion
CCMV releases its genome by expansion
At low metal ion concentration and high pH, the particle swells
The particle does not fall apart due to interactions between subunits and RNA
However, the three fold vertices open up which allow for flow of molecules

36. CCMV particle expansion

37. Transcription and Translation
In order to multiply, the virus must be able to produce viral proteins and replicate its genome.
Process is intrinsically asymmetric which leads to difficulties in icosahedral reconstructions.
Reovirus have provided many clues to the process due to its unusual replication.

38. Transcribing DLP of Rotavirus
Acridine orange was used to visualize RNA in the reconstruction
Channels throughout the rotavirus capsid in which allow the newly synthesized RNA to be exported

39. Transcribing DLP of Rotavirus

40. L-A Virus
L-A virus is a fungal virus which contains 2 RNA dependent RNA polymerases on the inside of two of its capsid proteins
The RNA moves past the polymerases as it is synthesized and is exported through pores in the capsid
The capsid protects the RNA from degradation while allowing for the import of important metabolites

41. L-A virus transcription

42. The End!
HIV
Avian Flu
Smallpox
Swine Flu
Swine Flu