Virus Life Cycles in 3D

The Art of Reconstruction

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

Virus Life Cycle

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

Virus Life Cycle

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

Enveloped Virus

SFV as an example

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

Non enveloped Viruses

Non enveloped virus

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

Cell Attachment

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

The Receptor binding region of HRV14

The Receptor binding region of HRV14

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

HRV16 complexed with ICAM-1

HRV16 complexed with ICAM-1

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

Simian Rotavirus

Simian Rotavirus

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

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

SFV Spikes

SFV with envelope and capsid

SFV spike structure

SFV spike structure

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

SFV spike conformational changes

SFV spike conformational changes

SFV process of fusion

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

Adenovirus

Adenovirus 2 and hexons

Adenovirus uses 2 receptors

CAR Integrins

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

Structural changes in penton

Structural changes in penton

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

Genome Release

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

Flock House Virus

Flock House Virus

FHV γ helices

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

FHV entry into cell

FHV entry into cell

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

CCMV particle expansion

CCMV particle expansion

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

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

Transcribing DLP of Rotavirus

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

L-A Virus

L-A virus transcription

The End!

Swine FluSwine Flu

HIV

SmallpoxAvian Flu