1. The Art of Reconstruction

2.  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

4.  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.  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

7.  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

9.  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.  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

12.  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

14.  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

16.  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 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

21.  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

24.  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

26. CARIntegrins

27.  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

29.  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.  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

33.  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

35.  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

37.  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.  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

40.  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

42. Swine Flu HIV Smallpox Avian Flu