The Art of Reconstruction
In order to survive, viruses must be able to do
the following:
Find a host cell it can replicate in
Bind to that cell
Enter the cell
Release its genome in order to replicate
Replicate its genome
Transcribe and translate its viral proteins
Package its genome and proteins
Escape from the cell
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
Semliki Forest Virus is an enveloped
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
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
The first step in viral replication is to
be able to bind to the correct host
 Virus recognize host cells by certain
 Bind to these receptors through
specific interactions.
 Binding sites on viruses are typically
conserved to ensure survival
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
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
VP4 of rotavirus is important to the viral life
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
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.
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
In order to determine the conformational
changes needed for activation, the particles
were treated with low pH and vitrified within
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
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
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
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
FHV is comprised of 180 copies of a single
protein which undergoes a post assembly
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
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
The RNA is then dragged into the cell by its
contacts by the other γ peptide contacts
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
In order to multiply, the virus must be able to
produce viral proteins and replicate its
Process is intrinsically asymmetric which
leads to difficulties in icosahedral
Reovirus have provided many clues to the
process due to its unusual replication.
Acridine orange was used to visualize RNA in
the reconstruction
Channels throughout the rotavirus capsid in
which allow the newly synthesized RNA to be
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
Avian Flu
Swine Flu
Swine Flu

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