My name is Ari Helenius and I work in the Institute of Biochemistry at the ETH Zurich,
the Swiss Federal Institute of Technology. What I will be talking about today is
the cell biology of virus entry and in this first part will concentrate on the general
aspects of virus-cell interactions. It only concerns viruses of animal systems,
animal viruses. I've chosen this title for the first part because it shows that virus particles,
when they enter a cell, require assistance by the cell itself.
Many cellular processes and cellular factors are involved in bringing the viral particle
into the cell so inadvertently the cell helps the virus to infect it.
We'll start first by talking about the viral particle itself. What is a virus particle?
The most important component of the virus particle is the genome,
which is made out of RNA or DNA and the genome encodes the genes required
for building a virus particle in the infected cell. These viral genes may be only
a handful in number for some viruses, and in others a few more, but in principle
it is a very small gene with very few proteins genes in it. The genome
in the virus particle is present in a highly condensed state in order to
take as little space as possible and this can happen in two ways either it is
coiled up together with proteins in so called helical capsid, or as shown here,
it may be in an icosahedral protein shell that surrounds it. In some viruses
both structures exist, the virus is coiled up with proteins inside then covered
by this protein shell that forms the particle. What you have there is a so-called
non-enveloped virus, which means there is nothing more, only nucleic acid and protein.
A large number of viruses and virus families have in addition to this type of
central capsid structure, a lipid bi-layer membrane, and this membrane surrounds
the capsid and provides, in this case, the outermost layer of the virus.
The envelope contains additional virally encoded proteins, so-called viral envelope proteins.
So these are the two forms of viruses in animal systems,
a non-enveloped and an enveloped virus system.
The function of the viral particle itself is actually very simple.
It's a carrier particle to carry the viral genome, and sometimes
accessory proteins from the infected cell to a non-infected cell. It can be
a cell transfer that happens between cells in an organism, or it can be
from one organism to another, for example, from one human being
to another human being. Anyway it is the viral particle that transmits
the infection between cells. Important in this process is that the particle
has to also help to bring the viral genome into the cell, uncoat it,
and that it is then delivered in this way in the replication competent form.
So it all starts with the viral particle in the extracellular space, entering a host cell,
an un-infected host cell, and then inside the host cell the virus has to uncoat
its genome, and then the cell can use this genome as the information
needed to produce new virus particles. These are formed inside the cell,
and eventually released into the extracellular space again
and the whole cycle begins again. It means that the virus
is an obligate intracellular parasite. It cannot replicate by itself,
it always needs the help and machinery of a host cell. There are many
different types of viruses, and this schematic picture shows on the top part
here some DNA viruses, some of them have a lipid bilayer envelope,
like these two here, that is they are envelope viruses, and on this side
a few non-enveloped viruses. This is a herpes virus, this is a pox virus,
and up here we have a virus that causes warts. It's called papilloma virus.
We'll talk about that later in this lecture. Down here are so-called envelope viruses
that I mentioned. They are all RNA viruses. They contain a lipid bilayer envelope,
some of them have an icosahedral capsid like shown here. Others have
a helical capsid as I already mentioned before.
Up here is the influenza virus, down here is the SARS virus, or a related virus,
so-called coronavirus, and I will in a moment talk about a virus from this family here,
which is so-called alphavirus that causes encephalitis.
Now viral particles look very different in the electron micrograph microscope,
this is how the influenza virus looks. The virus particles are not identical in shape,
but they all have this envelope and in the envelope you see the projections
which form the envelope glycoproteins, which are very important during
the virus entry into cells. This is an alphavirus, the Semliki Forest virus,
which I also will talk about in a moment. It has almost all of its surface covered
by this spike glycoproteins. The envelope is only visible as blue spots
in the background of this protein shell. The next one is electron micrographs
again from SARS virus, a coronavirus. It's enveloped and it has spike glycoproteins
on its surface. The final virus is a non-enveloped virus, the papillomavirus,
which has this protein shell and the DNA of this virus is inside this central cavity of the particle.
Before looking at this entry in more detail, it is important to point out that viruses
are a very important health risk in the world. Infectious diseases in general
are the second most common cause of death in humans and half of those
are thought to be caused by viruses. There are many established human
and animal diseases such as polio, measles, and so on, and there are re-emerging
viral diseases, which are known to be human viruses before
which are now extending and expanding again in the world. In addition,
which is a very big concern is that there are emerging new viral diseases.
SARS virus is a good example of that, HIV another one, which were not always
in the human population but now appear from different sources. It is also important
to realize that some viruses are potential agents for terrorists
and that is a major concern that one has to take seriously. Now, I'm only going
to mention a few viruses so you get an idea of how large the numbers
are of people affected. The AIDS disease caused by HIV1 is widely spread.
40 million people are thought to be infected today, and about 25 million
have already died from this disease. Hepatitis B virus is probably
the most widely spread human virus-caused disease. About 400 million people
are infected chronically today, and some 25% of those are probably going
to succumb from liver disease or liver cancer caused by this virus.
Rotavirus is perhaps not so well know, but it causes big problems in children
in particular in Latin America with almost 1 million children dying from it yearly.
Influenza virus is a major threat. It's known that in the Spanish disease
of 1918-1919, about 40 million people around the world died.
And of course the avian influenza virus is a potential pandemic threat right, H5N1.
The final one on the list is the SARS virus where the number of people affected
was not very big, but it is also clear that there were huge financial losses caused
by this relatively limited infection. Now the transmission of viruses
from one person to another, from one organism to another, occurs in many different ways.
One of them is direct contact, and another important one is in the form of
aerosols, for example influenza virus is transmitted that way. But one shouldn't
forget insect bites for insect carried viruses, and contaminants in food and water,
and contaminated syringes and so on. We have been studying virus entry
for many years now, and we are using many different techniques. One of the
problems of course is that viruses are extremely small. If you take a typical virus
particle, the size of the particle if you magnify it 1 million times is the size of an orange.
That's 1 million times enlarged. If you take a host cell and do the same,
enlarge that, magnified 1 million times it would be the size of a big circus tent.
So the fascination that has always been there for me has been how can this tiny
little particle, relatively speaking to its host, enter a huge cell and then
within hours in many cases transform it completely so that it is now basically a virus factory,
it produces viral particles in thousands of thousands of numbers.
What we have been using is a series of approaches that come partly from
virology, obviously, but we also use cell and molecular biology
as important techniques. In addition, biochemistry and biophysics
are needed and more recently we have tried to apply also techniques
of systems biology and computer science. You'll see some examples
of that in a moment. More specifically, what we do,
and what have done in this field is the sophisticated use of light and electron microscopy.
Light microscopy usually now in live cell experiments. We also take advantage
of in vitro systems, you'll see an example in just a moment of lipid bilayers
without cells used in virus entry studies. Where biologists
and molecular biologists today are particularly skillful are perturbations,
one can perturb the cell and the virus in many different ways and then find out
how that affects infection by using chemical inhibitors,
by using mutant viruses and mutant cells, and also then modify the cells
using dominant inactive and active constructs. In addition,
one can modify cells using siRNA and as you will hear later, you can then use this
siRNA silencing technology to apply to automated high throughput screens
to find out cellular proteins involved in infection. Okay, here you see
where the study started many years ago. We were trying to understand how
Semliki Forest virus, a small enveloped RNA virus enters cells in tissue culture.
The surface of the cell is shown here by electron microscopy and first the features
include the filopodia, these are long actin containing extensions
of the plasma membrane, and what is here is probably a lamellapodium,
another common structure present on cell surfaces. But most importantly,
these small spots that you see are viral particles attached to the cell surface,
and some of them are being internalized in invaginations such as here
and there are other ones up here. Here's one where the viral particle is disappearing
into the surface of the cell in a deep invagination. What happens
is that the virus is being internalized by endocytosis, the bound particle
first is taken up into clathrin coated pits, and these invaginate forming
clathrin coated vesicles, and using this standard pathway of endocytosis,
the virus is delivered to an organelle called the endosome. And in this endosome,
the virus is exposed to a reduced pH, around 6, and that induces a
conformational change in the spike glycoproteins
resulting in the activation of membrane fusion between
the envelope of the virus and the limiting membrane of the endosome.
As a result, this icosahedral capsid, with its RNA genome is released now into
the cytosolic compartment and almost immediately uncoated. That means that
the capsid falls apart, the viral RNA is released, and then it is used here,
as the messenger RNA for the synthesis of the first viral proteins.
This is how it looks in an electron microscope. You see the first step virus being
internalized in a clathrin coated pit, which has this electron dense material
on the cytosolic side, these are clathrin coated vesicles. In some cases
one can actually see the capsid down here being released from
an endosomal structure before it has had time to uncoat. Many studies
like this with different viruses have shown what the general program
of virus entry looks like. The virus
entry and infection always starts with the virus binding to the cell surface.
It binds to receptors, that is cell surface components, which serve
as binding sites for the virus, and after binding, typically the viral particle
starts to move around on the surface, laterally along the membrane.
During this time, already, the virus induces signals by activating the cells
own signaling pathways and in this way the virus prepares the cell for the invasion.
One of the things that then typically happens is that the viral particle is internalized
by different mechanisms of endocytosis. There are some virus families
which are able to penetrate and go straight through the plasma membrane
without endocytosis, but the majority are endocytosed first. The endocytic
vesicles that are formed carry the virus into a secondary organelle inside the cell.
In the case of Semliki Forest virus, this would have been an early endosome,
and here then the penetration of the capsid into the cytosol is triggered
by the conditions in this compartment. The next step once the virus has made
it all the way to the cytosol is movement into the location where uncoating and
replication of the virus can take place. For most DNA viruses that involves
transport along microtubules to the nucleus and to the nuclear pore complex.
Then through different mechanisms the genome can be transported through the
nuclear pore and into the nucleus, and then uncoated in the process.
Viruses that replicate in the cytosol have different other locations where
they are moved. So as you look at this whole pathway, you can see that there
is a whole program of steps, one consecutive to the other, resulting finally
in the transport of the genome into a specific location that's also then
where mostly the final uncoating of the genome takes place.
Many viruses have been analyzed by us and others, and the general picture
is starting to emerge and I'll summarize basically what the main points are.
First of all, the entry process occurs in multiple steps. It's not a very simple process.
You have to go through each step otherwise infection does not occur.
As the virus moves from the plasma membrane inwards into the cell deeper and deeper,
that program is connected to an uncoating program at the end of which
the viral genome is then released and in a form that it can be replicated.
So entry and uncoating go hand in hand. The virus particle itself
is constructed in such a way that it has the uncoating program
already built into it, and what it means in practice is that the proteins,
all the virus particles itself, is metastable structurally. That these proteins
and the capsid can undergo major changes in response to biochemical cues,
and the biochemical cues in this case are provided by the cell. I already gave one example,
that is the low pH in the early endosome triggers a change
in the spike glycoprotein of Semliki Forest Virus,
and makes it a fusion protein. That type of cue is important, low pH in this case,
is a cue given by the cell. But there are many other cues, I'll come to that,
many types of cues. The main point is that the cell is providing information to the virus.
Do this, do that. Basically the virus is a blind man, and the cell takes it
by the hand and brings it through into the cell, and through its entry program.
So what is very important from the very moment of first contact is
the presence of cellular factors and processes. The virus depends on them
at every stage of its entry program. They are very critical components.
Now in the dialogue between the incoming particle and the cell, it's not only the cell
that provides information to the virus, but also the virus engages the cell in a dialogue
where it triggers this activation of these signaling pathways, and in that way
the information is given both by the pathogen and the host. Very important in a
sort of very general sense is that the virus particle must speak
the language of the cell. It must know the pin codes and all the passwords and it
has to know exactly how to activate the cells processes and functions that it needs.
So that is probably the most important realization that has come through the study
of many different viruses and their host cells. Now if we look at the type of cues
that I mentioned that different viruses require to go through the orderly process
of their entry program, at low pH, as you see here, exposure to low pH is
a very common one, but it's not the only one. Very often viruses require cues
by binding to specific cell surface molecules, so called virus receptors
, that induces changes. The low pH is another one. Also sometimes the cell has
to induce cleavages in specific viral proteins in order to activate them
and in some cases the re-entry of the virus from the extracellular space, which is oxidizing,
into the reducing environment of the cytosol serves as a cue.
All sorts of different things build up and help the virus do the thing that they need to do.
In some cases its exposure to specific enzymes such as thiol oxidoreductases.
So the virus is exposed to these changes and is modified by the cell
in order to be active in its entry. One final, very important general point
is that there is a basic difference in the strategy used by enveloped viruses,
those that have a lipid bilayer, and those that do not, the non-enveloped viruses.
The enveloped viruses do their transfer of the genome in a very smart
and intelligent way. They use the same principle by which the cells themselves
transfer macromolecules from one membrane bound compartment
to the other that is a vesicle transfer mechanism in which the cargo, in this case it's the capsid,
a large macromolecular complex, is built into a vesicle, here,
and this vesicle by membrane fission pinches off, in this case
the plasma membrane with the capsid inside. This capsid then is transferred
to a new cell and then through a membrane fusion reaction, either at the plasma membrane
or in an endocytic compartment here releases the capsid into the cytosol.
As you see here, the plasma membrane may not always be the case
where the virus is formed, it can also happen in intracellular organelles.
But the main point is that no macromolecular structure of the virus
needs to pass through the hydrophobic barrier of a bilayer, it's all taken care of
by membrane fission, fusion, coupled fusion reactions. Non-enveloped viruses
have a much bigger problem. They have no membrane, they cannot do this.
Typically they exit from the infected cell by a lytic event. They break open
the membrane and the virus is released and then as they enter the new cell
they have to either lyse these vesicles and I'll come back later into
what type of mechanisms they use. Typically these mechanisms are not as well
characterized as the ones used by enveloped viruses. So now I want to go through
some early events that happen on the plasma membrane and then in
the second lecture I'll talk about the intracellular events. So let's go back
to the beginning. Now the virus has to bind to the cell surface. That step is
very important for many reasons. The virus cannot infect the cell
which it cannot bind to, so there has to be a first contact and binding otherwise
nothing will happen, the cell will not get infected.
The viral receptors that I have been alluding to are typical, normal,
everyday plasma membrane proteins of the cell. Either proteins,
lipids, or carbohydrates. Viruses have evolved to use some of these
for binding to and to mediate their entry into cells. Now-a-days we distinguish
between two types of attachments. One is the so-called attachment factors. These factors
simply bind the virus and help to concentrate the viral particles on the surface of the cell.
Then the real receptors come into play. The receptors in addition
to binding the virus help to give the virus information for example
by inducing conformation changes. They may be helpful in generating signals
that I mentioned before, or they may be involved in endocytosing the particles.
So they do than just bind. Many viruses can use more than one type of receptors.
Some use two or more receptors consecutively. You may know that HIV uses two.
And also it's important to realize the binding is typically multivalent so the virus binds
to more than one receptor at a time. So there are many contacts with the cell surface.
Now the type of molecules that serve as receptors are variable. They depend
on which virus we are talking about. So this picture shows some molecules
and the viruses that use them, and as you can see in this case these cell surface proteins
are quite different. And the choice of receptor for a virus is very important
because that determines which cell types in the body and which species
can be infected by the virus. So virus can obviously only infect cells which have
that particular receptor on its surface that it needs. Eventually the choice of receptor
is very important in determining what cells are infected and what type of disease
results from the particle invasion. We won't go through this in detail.
These are glycoproteins and proteins and they come from many different families
for different viruses. One the side of the virus, there must be of course something
that binds to the receptor, and that also varies. For example, in enveloped animal viruses,
the glycoproteins that cover the surface of the bilayer membrane
are the ones that bind to receptors. For example in this case the influenza virus,
the blue structures here are influenza hemagglutinin molecules and they
are responsible for binding to sialic acid containing receptors.
In non-enveloped viruses, as in the adenovirus that you see down here,
the first contact with the first receptor is through the fibers and the little knob at the end of the fibers.
Here is a rhinovirus, which binds to this yellow receptor molecule
shown in this crystal structure which in fact binds to small indentations
present on the surface of the virus. So they can be surface protrusions or
surface indentations. So viruses have developed specific sites which can bind
multiple receptors like this. Now let's look at one specific virus as an example.
In this case it is the Simian virus 40, polyomavirus family member.
It's a non-enveloped virus and it's structure is extremely well characterized
as you see here by X-ray crystallography. The particle is composed of
a surface protein called VP-1, which is present in these donut shaped structures,
which contain five VP-1 molecules each.
There are seventy-two of these pentamers, five-mers, organized
in an icosahedral structure with symmetry of T=7.
The VP-1 molecule is the one that binds to the receptor, and the receptor
in this case is a lipid molecule, the ganglioside called GM1.
Here is a picture of that ganglioside. It is a sphingolipid.
It has a carbohydrate moiety and a VP-1 molecule binds to some of the sugars
at this moiety. Here is a crystal structure recently published that shows
how exactly this interaction works. You have the pentamer here seen from the side,
and the sugar moieties are shown in the binding sites on the surface of this pentamer.
So here the interaction with the receptor is extremely well characterized.
The surface has multiple sites, each pentamer can bind five receptor molecules.
What you see here is the surface of CV-1 cells, a host cell for SV-40,
and viral particles in this case SV-40 particles have been labeled
fluorescently so they are visible on the surface of a live cell using
total internal reflection microscopy. Some of them are like this one,
fixed in place already, it does not move anymore. Others are really moving
in a random fashion around the surface of the cell. If one looks at virus particles
when they are binding initially they first go through a phase where they are mobile,
and then they stop, pretty much, or maybe drift a little bit,
but there is a free random motion followed by fixing the virus in place.
And then eventually the viral particles are endocytosed.
Now, it is possible in this case to study this interaction in a cell-free system,
in which one takes simply lipid vesicles, artificial lipid vesicles, liposomes,
containing the receptor GM1 and allows them to interact with
the coverslip or the glass surface and they will form a uniform bilayer on that surface,
which then will bind viruses and if you do that this is how it looks,
the particles bind nicely like they do on the cell surface and now they are all mobile.
All of the are moving and their movement is completely random.
Now in this case this lipid bilayer serves as a model system for the plasma membrane.
Now to find out a little more in detail how this motion works, is the virus sliding along
the membrane or is it rolling, we have collaborated with some terrific biophysics
at ETH Zurich, mainly Philipp Kukura and Vahid Sandoghdar,
who have been able to look at this question by following the viral particle by new technology
which is called interferometric scattering detection, iSCAT. It's a label free
detection system where they can follow the viral particle itself and we coupled
one quantum dot fluorescent probe, a single one to the viral particle,
and that could then be followed by its fluorescence.
The system allows nearly molecular spatial resolution and extremely high temporal
resolution. By combining in this case the following of the tracking of the viral particle
and this quantum dot, it is possible to get three-dimensional information
about the motion of the particle on the surface of these lipid bilayers.
This shows again the set-up a little more. We have a viral particle with a single quantum dot.
We can follow the viral particle not by fluorescence, but by this interferometrics,
and this is the type of spot you can see in the microscope.
Of course light microscopy allows you to go down to about 200-300 nanometers,
but since the viral particle is known in size exactly, we can define the center
of one of these from the point spread function with 2 to 3 nanometer resolution.
So the resolution of the system is extremely good, it's almost as good
also for finding exactly where this quantum dot is located.
So now when one combines both the interferometric analysis
and the fluorescence analysis, one can get the trajectory
of the virus moving on the cell surface where one can see that the quantum dot
and the particle are not exactly following the same trajectories.
They are moving a little bit differently, and on the whole of course they follow
each other and one can then through computers analyze
what that means in terms of 3D structure. And here you can see the outcome of that.
So this is the surface of the lipid bilayer that contains the receptor,
and the viral particle is moving randomly around. It is not exactly sliding
nor does it seem to be rolling, but it's sort of wobbling,
probably moving from one receptor to another.
This is what we expect also of something like this happening on the cell surface.
Now before finishing this section, I would like to talk a little bit about the
surface behavior of this particular virus. It's the Human papilloma virus 16,
the major cause of cervical cancer. It's a DNA virus,
a non-enveloped virus, 55 nanometers in diameter. It replicates in the nucleus
and receptors for this virus are not entirely clear expected that they do use as
an important component, proteoglycan heparan sulfates.
The virus is acid activated and it is entering by endocytosis. Electron microscopy
here shows that the virus on cell surfaces likes to bind to filopodia.
These are the actin containing extensions. You can see them
in a section here and the viral particles are attached. Many viruses bind
to filopodia as you'll see later. Here is just an enlargement
of a particle and the plasma membrane underneath it.
When Mario Schelhaas, who did most of these studies together with Patricia Day and John Schiller at NIH
looked at this, they found that the viral particles when they are sitting on this filopodia
are actually moving down the filopodia towards the cell body.
The filopodia is here stained with GFP-labeled actin and this surfing of viral particles
towards the cell body happens for many viruses, it was first observed by Walther Mothes at Yale,
and we see it now for many different viruses.
So the viral particles in this case are not moving randomly on the surface of the cell,
but they bind to specific structures and then they move in a very directed
motion down these actin containing filopodia. The movement is entirely
dependent on the retrograde actin flow inside the filopodia
the actin is also moving from the tip down to the cell body.
Now this same phenomenon can be seen, well part of it, by electron microscopy.
You have here the cell surface and here is the filopodia or the beginning of it
and even the actin filaments are visible and this may be a virus which is moving down
to the cell body. What then happens is the endocytosis of particles into the cell,
the cell now internalizes the particles by endocytosis. Here we see already a vesicle
which contains a viral particle probably emanating from the cell surface
and we can see this happens to many viruses. They are actively taken up by the cell.
I will finish here, but I want to stay as long as we are still on the plasma membrane
and then in the next seminar talk about later events, but I would like to summarize
a few of the points that happens here. What is happening here underneath
this picture is that first of all there is a multivalent association
of the virus with these receptors. Receptors that clustered underneath the virus.
Somehow this clustering and interaction of the viral particle with its receptor
triggers a transbilayer coupling from the outside surface to the inside of the cell,
and this then activates a signaling pathway or more, which informs the cell
about the viral particle. Basically the virus is sitting on the surface and saying,
ping, ping, ping, ping, I'm here, do something.
And in this case as we see here the activation of an endocytic reflex
in the cell occurs. One or the other endocytic mechanism
is activated to bring in the viral particle. These endocytic vesicles then
help to move the virus from the surface into the center of the cell, and that is then
for many viruses where the penetration happens into the cytosol.
So I will for the first part stop here and then in the next seminar
discuss events that viruses go through after they have been endocytosed by the cell.
Thank you very much.