Virus Life Cycles in 3D - PowerPoint Presentation

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Virus Life Cycles in 3D

The Art of Reconstruction

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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 cell3. Enter the cell4. Release its genome in order to replicate5. Replicate its genome6. Transcribe and translate its viral proteins7. Package its genome and proteins8. Escape from the cell

Virus Life Cycle

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Virus Life Cycle

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All these processes can be visualized by

cryo

These visualizations allows for a better understanding of viruses and may lead to vaccination developmentFor each virus, there is a unique life cycle but all viruses accomplish the same steps in order to survive Virus Life Cycle

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Semliki Forest Virus is an enveloped

Alphavirus

It has 2 transmembrane proteins (E1 and E2) in its envelopeThe virus binds to the cellular receptor, endocytosed, and fuses with the endosome membrane to release its nucleocapsid for replication

Enveloped Virus

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SFV as an example

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Poliovirus is a non enveloped virus in the

Picornavirus

familyIt 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 replicationAlso releases from the cell by lysis instead of budding

Non enveloped Viruses

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Non enveloped virus

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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 survivalCell Attachment

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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 cryoWas believed that the binding site for ICAM-1 was located in the canyon region of HRV16

The Receptor binding region

of HRV14

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The Receptor binding region

of HRV14

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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 receptorHRV16 complexed with ICAM-1

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HRV16 complexed

with ICAM-1

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VP4 of rotavirus is important to the viral life cycle

It is a determinant of virulence, has

hemagglutination activity and is also a neutralization siteThe reconstruction showed that VP4 extends from the surface of the virus, which may then be able to bind to the cellular receptor more easilySimian Rotavirus

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Simian Rotavirus

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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.

Activation

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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 spikePreferential extraction and reconstruction comparison determined that E1made up the outside of the spike while E2 extended from the centerSFV Spikes

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SFV with envelope and capsid

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SFV spike structure

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SFV spike structure

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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 proteinE2 moves outward while E1 moves inward to form a trimmer and trigger fusionSFV spike conformational changes

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SFV spike conformational changes

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SFV process of fusion

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Adenovirus is made up of hexons

and two proteins at the five fold

vertice: penton base and fiberIt binds two receptors: CAR and an integrinCAR binds to the fiber while the penton base binds the integrin and causes activationAdenovirus

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Adenovirus 2 and hexons

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Adenovirus uses 2 receptors

CAR

Integrins

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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 orientationStructural changes in penton

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Structural changes in penton

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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: ReovirusesGenome Release

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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 onγa lies in pentamers under the five foldγb interacts with the bulk RNA and γc

γc

also

interactes

with the ordered RNA

Flock House Virus

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Flock House Virus

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FHV γ

helices

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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 membraneThe RNA is then dragged into the cell by its contacts by the other γ peptide contacts

FHV entry into cell

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FHV entry into cell

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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 RNAHowever, the three fold vertices open up which allow for flow of moleculesCCMV particle expansion

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CCMV particle expansion

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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.Transcription and Translation

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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

Transcribing DLP of Rotavirus

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Transcribing DLP of Rotavirus

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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 metabolitesL-A Virus

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L-A virus transcription

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The End!

Swine Flu

Swine Flu

HIV

Smallpox

Avian Flu