Viral Infection: Virus Entry and Subsequent Steps
Transcript of Part 1: Virus Entry
00:00:01.01 My name is Ari Helenius and I work in the Institute of Biochemistry at the ETH Zurich, 00:00:12.08 the Swiss Federal Institute of Technology. What I will be talking about today is 00:00:19.08 the cell biology of virus entry and in this first part will concentrate on the general 00:00:26.24 aspects of virus-cell interactions. It only concerns viruses of animal systems, 00:00:33.22 animal viruses. I've chosen this title for the first part because it shows that virus particles, 00:00:41.27 when they enter a cell, require assistance by the cell itself. 00:00:47.05 Many cellular processes and cellular factors are involved in bringing the viral particle 00:00:52.06 into the cell so inadvertently the cell helps the virus to infect it. 00:01:00.19 We'll start first by talking about the viral particle itself. What is a virus particle? 00:01:09.26 The most important component of the virus particle is the genome, 00:01:14.15 which is made out of RNA or DNA and the genome encodes the genes required 00:01:23.08 for building a virus particle in the infected cell. These viral genes may be only 00:01:30.27 a handful in number for some viruses, and in others a few more, but in principle 00:01:37.13 it is a very small gene with very few proteins genes in it. The genome 00:01:46.23 in the virus particle is present in a highly condensed state in order to 00:01:51.29 take as little space as possible and this can happen in two ways either it is 00:01:57.23 coiled up together with proteins in so called helical capsid, or as shown here, 00:02:04.08 it may be in an icosahedral protein shell that surrounds it. In some viruses 00:02:10.20 both structures exist, the virus is coiled up with proteins inside then covered 00:02:16.23 by this protein shell that forms the particle. What you have there is a so-called 00:02:22.08 non-enveloped virus, which means there is nothing more, only nucleic acid and protein. 00:02:28.20 A large number of viruses and virus families have in addition to this type of 00:02:33.26 central capsid structure, a lipid bi-layer membrane, and this membrane surrounds 00:02:40.08 the capsid and provides, in this case, the outermost layer of the virus. 00:02:46.07 The envelope contains additional virally encoded proteins, so-called viral envelope proteins. 00:02:53.27 So these are the two forms of viruses in animal systems, 00:02:58.24 a non-enveloped and an enveloped virus system. 00:03:03.17 The function of the viral particle itself is actually very simple. 00:03:08.13 It's a carrier particle to carry the viral genome, and sometimes 00:03:14.07 accessory proteins from the infected cell to a non-infected cell. It can be 00:03:19.18 a cell transfer that happens between cells in an organism, or it can be 00:03:24.22 from one organism to another, for example, from one human being 00:03:28.05 to another human being. Anyway it is the viral particle that transmits 00:03:33.09 the infection between cells. Important in this process is that the particle 00:03:39.02 has to also help to bring the viral genome into the cell, uncoat it, 00:03:47.19 and that it is then delivered in this way in the replication competent form. 00:03:54.07 So it all starts with the viral particle in the extracellular space, entering a host cell, 00:04:03.29 an un-infected host cell, and then inside the host cell the virus has to uncoat 00:04:11.09 its genome, and then the cell can use this genome as the information 00:04:17.25 needed to produce new virus particles. These are formed inside the cell, 00:04:22.24 and eventually released into the extracellular space again 00:04:27.06 and the whole cycle begins again. It means that the virus 00:04:30.13 is an obligate intracellular parasite. It cannot replicate by itself, 00:04:37.04 it always needs the help and machinery of a host cell. There are many 00:04:46.22 different types of viruses, and this schematic picture shows on the top part 00:04:51.12 here some DNA viruses, some of them have a lipid bilayer envelope, 00:04:56.00 like these two here, that is they are envelope viruses, and on this side 00:05:00.07 a few non-enveloped viruses. This is a herpes virus, this is a pox virus, 00:05:06.19 and up here we have a virus that causes warts. It's called papilloma virus. 00:05:11.05 We'll talk about that later in this lecture. Down here are so-called envelope viruses 00:05:18.03 that I mentioned. They are all RNA viruses. They contain a lipid bilayer envelope, 00:05:23.01 some of them have an icosahedral capsid like shown here. Others have 00:05:30.01 a helical capsid as I already mentioned before. 00:05:33.26 Up here is the influenza virus, down here is the SARS virus, or a related virus, 00:05:39.20 so-called coronavirus, and I will in a moment talk about a virus from this family here, 00:05:45.22 which is so-called alphavirus that causes encephalitis. 00:05:51.18 Now viral particles look very different in the electron micrograph microscope, 00:05:59.16 this is how the influenza virus looks. The virus particles are not identical in shape, 00:06:05.01 but they all have this envelope and in the envelope you see the projections 00:06:10.00 which form the envelope glycoproteins, which are very important during 00:06:14.04 the virus entry into cells. This is an alphavirus, the Semliki Forest virus, 00:06:20.07 which I also will talk about in a moment. It has almost all of its surface covered 00:06:25.19 by this spike glycoproteins. The envelope is only visible as blue spots 00:06:29.17 in the background of this protein shell. The next one is electron micrographs 00:06:36.24 again from SARS virus, a coronavirus. It's enveloped and it has spike glycoproteins 00:06:44.17 on its surface. The final virus is a non-enveloped virus, the papillomavirus, 00:06:50.16 which has this protein shell and the DNA of this virus is inside this central cavity of the particle. 00:07:01.10 Before looking at this entry in more detail, it is important to point out that viruses 00:07:08.14 are a very important health risk in the world. Infectious diseases in general 00:07:14.17 are the second most common cause of death in humans and half of those 00:07:21.17 are thought to be caused by viruses. There are many established human 00:07:27.15 and animal diseases such as polio, measles, and so on, and there are re-emerging 00:07:34.17 viral diseases, which are known to be human viruses before 00:07:38.28 which are now extending and expanding again in the world. In addition, 00:07:45.26 which is a very big concern is that there are emerging new viral diseases. 00:07:51.24 SARS virus is a good example of that, HIV another one, which were not always 00:07:58.00 in the human population but now appear from different sources. It is also important 00:08:03.16 to realize that some viruses are potential agents for terrorists 00:08:11.04 and that is a major concern that one has to take seriously. Now, I'm only going 00:08:22.07 to mention a few viruses so you get an idea of how large the numbers 00:08:29.01 are of people affected. The AIDS disease caused by HIV1 is widely spread. 00:08:37.15 40 million people are thought to be infected today, and about 25 million 00:08:43.05 have already died from this disease. Hepatitis B virus is probably 00:08:48.29 the most widely spread human virus-caused disease. About 400 million people 00:08:55.28 are infected chronically today, and some 25% of those are probably going 00:09:04.20 to succumb from liver disease or liver cancer caused by this virus. 00:09:11.00 Rotavirus is perhaps not so well know, but it causes big problems in children 00:09:23.15 in particular in Latin America with almost 1 million children dying from it yearly. 00:09:28.25 Influenza virus is a major threat. It's known that in the Spanish disease 00:09:34.21 of 1918-1919, about 40 million people around the world died. 00:09:40.13 And of course the avian influenza virus is a potential pandemic threat right, H5N1. 00:09:49.21 The final one on the list is the SARS virus where the number of people affected 00:09:55.24 was not very big, but it is also clear that there were huge financial losses caused 00:10:01.12 by this relatively limited infection. Now the transmission of viruses 00:10:09.24 from one person to another, from one organism to another, occurs in many different ways. 00:10:15.08 One of them is direct contact, and another important one is in the form of 00:10:21.00 aerosols, for example influenza virus is transmitted that way. But one shouldn't 00:10:26.00 forget insect bites for insect carried viruses, and contaminants in food and water, 00:10:34.13 and contaminated syringes and so on. We have been studying virus entry 00:10:41.23 for many years now, and we are using many different techniques. One of the 00:10:46.12 problems of course is that viruses are extremely small. If you take a typical virus 00:10:51.03 particle, the size of the particle if you magnify it 1 million times is the size of an orange. 00:10:57.29 That's 1 million times enlarged. If you take a host cell and do the same, 00:11:04.17 enlarge that, magnified 1 million times it would be the size of a big circus tent. 00:11:10.14 So the fascination that has always been there for me has been how can this tiny 00:11:16.00 little particle, relatively speaking to its host, enter a huge cell and then 00:11:24.00 within hours in many cases transform it completely so that it is now basically a virus factory, 00:11:30.18 it produces viral particles in thousands of thousands of numbers. 00:11:37.10 What we have been using is a series of approaches that come partly from 00:11:43.23 virology, obviously, but we also use cell and molecular biology 00:11:47.29 as important techniques. In addition, biochemistry and biophysics 00:11:52.18 are needed and more recently we have tried to apply also techniques 00:11:57.22 of systems biology and computer science. You'll see some examples 00:12:02.02 of that in a moment. More specifically, what we do, 00:12:06.17 and what have done in this field is the sophisticated use of light and electron microscopy. 00:12:13.03 Light microscopy usually now in live cell experiments. We also take advantage 00:12:18.20 of in vitro systems, you'll see an example in just a moment of lipid bilayers 00:12:24.18 without cells used in virus entry studies. Where biologists 00:12:30.02 and molecular biologists today are particularly skillful are perturbations, 00:12:34.26 one can perturb the cell and the virus in many different ways and then find out 00:12:39.14 how that affects infection by using chemical inhibitors, 00:12:44.22 by using mutant viruses and mutant cells, and also then modify the cells 00:12:53.29 using dominant inactive and active constructs. In addition, 00:13:00.10 one can modify cells using siRNA and as you will hear later, you can then use this 00:13:06.15 siRNA silencing technology to apply to automated high throughput screens 00:13:14.01 to find out cellular proteins involved in infection. Okay, here you see 00:13:21.29 where the study started many years ago. We were trying to understand how 00:13:27.14 Semliki Forest virus, a small enveloped RNA virus enters cells in tissue culture. 00:13:34.28 The surface of the cell is shown here by electron microscopy and first the features 00:13:39.26 include the filopodia, these are long actin containing extensions 00:13:46.12 of the plasma membrane, and what is here is probably a lamellapodium, 00:13:51.21 another common structure present on cell surfaces. But most importantly, 00:13:56.27 these small spots that you see are viral particles attached to the cell surface, 00:14:01.23 and some of them are being internalized in invaginations such as here 00:14:05.29 and there are other ones up here. Here's one where the viral particle is disappearing 00:14:12.07 into the surface of the cell in a deep invagination. What happens 00:14:18.25 is that the virus is being internalized by endocytosis, the bound particle 00:14:25.13 first is taken up into clathrin coated pits, and these invaginate forming 00:14:31.25 clathrin coated vesicles, and using this standard pathway of endocytosis, 00:14:36.09 the virus is delivered to an organelle called the endosome. And in this endosome, 00:14:41.13 the virus is exposed to a reduced pH, around 6, and that induces a 00:14:48.00 conformational change in the spike glycoproteins 00:14:50.20 resulting in the activation of membrane fusion between 00:14:55.26 the envelope of the virus and the limiting membrane of the endosome. 00:15:03.06 As a result, this icosahedral capsid, with its RNA genome is released now into 00:15:10.21 the cytosolic compartment and almost immediately uncoated. That means that 00:15:16.09 the capsid falls apart, the viral RNA is released, and then it is used here, 00:15:23.01 as the messenger RNA for the synthesis of the first viral proteins. 00:15:31.11 This is how it looks in an electron microscope. You see the first step virus being 00:15:37.16 internalized in a clathrin coated pit, which has this electron dense material 00:15:43.14 on the cytosolic side, these are clathrin coated vesicles. In some cases 00:15:48.06 one can actually see the capsid down here being released from 00:15:52.09 an endosomal structure before it has had time to uncoat. Many studies 00:16:00.20 like this with different viruses have shown what the general program 00:16:06.20 of virus entry looks like. The virus 00:16:09.17 entry and infection always starts with the virus binding to the cell surface. 00:16:14.17 It binds to receptors, that is cell surface components, which serve 00:16:20.11 as binding sites for the virus, and after binding, typically the viral particle 00:16:25.11 starts to move around on the surface, laterally along the membrane. 00:16:29.05 During this time, already, the virus induces signals by activating the cells 00:16:38.09 own signaling pathways and in this way the virus prepares the cell for the invasion. 00:16:46.20 One of the things that then typically happens is that the viral particle is internalized 00:16:51.04 by different mechanisms of endocytosis. There are some virus families 00:16:56.16 which are able to penetrate and go straight through the plasma membrane 00:17:00.06 without endocytosis, but the majority are endocytosed first. The endocytic 00:17:06.10 vesicles that are formed carry the virus into a secondary organelle inside the cell. 00:17:12.02 In the case of Semliki Forest virus, this would have been an early endosome, 00:17:16.13 and here then the penetration of the capsid into the cytosol is triggered 00:17:22.09 by the conditions in this compartment. The next step once the virus has made 00:17:28.21 it all the way to the cytosol is movement into the location where uncoating and 00:17:35.21 replication of the virus can take place. For most DNA viruses that involves 00:17:40.03 transport along microtubules to the nucleus and to the nuclear pore complex. 00:17:45.27 Then through different mechanisms the genome can be transported through the 00:17:53.11 nuclear pore and into the nucleus, and then uncoated in the process. 00:17:59.06 Viruses that replicate in the cytosol have different other locations where 00:18:05.16 they are moved. So as you look at this whole pathway, you can see that there 00:18:10.04 is a whole program of steps, one consecutive to the other, resulting finally 00:18:15.18 in the transport of the genome into a specific location that's also then 00:18:21.01 where mostly the final uncoating of the genome takes place. 00:18:28.01 Many viruses have been analyzed by us and others, and the general picture 00:18:36.27 is starting to emerge and I'll summarize basically what the main points are. 00:18:43.15 First of all, the entry process occurs in multiple steps. It's not a very simple process. 00:18:49.26 You have to go through each step otherwise infection does not occur. 00:18:54.25 As the virus moves from the plasma membrane inwards into the cell deeper and deeper, 00:19:01.16 that program is connected to an uncoating program at the end of which 00:19:07.18 the viral genome is then released and in a form that it can be replicated. 00:19:13.10 So entry and uncoating go hand in hand. The virus particle itself 00:19:21.10 is constructed in such a way that it has the uncoating program 00:19:26.18 already built into it, and what it means in practice is that the proteins, 00:19:31.29 all the virus particles itself, is metastable structurally. That these proteins 00:19:39.03 and the capsid can undergo major changes in response to biochemical cues, 00:19:44.25 and the biochemical cues in this case are provided by the cell. I already gave one example, 00:19:51.25 that is the low pH in the early endosome triggers a change 00:19:55.14 in the spike glycoprotein of Semliki Forest Virus, 00:19:57.26 and makes it a fusion protein. That type of cue is important, low pH in this case, 00:20:05.04 is a cue given by the cell. But there are many other cues, I'll come to that, 00:20:11.10 many types of cues. The main point is that the cell is providing information to the virus. 00:20:16.18 Do this, do that. Basically the virus is a blind man, and the cell takes it 00:20:22.12 by the hand and brings it through into the cell, and through its entry program. 00:20:28.28 So what is very important from the very moment of first contact is 00:20:37.13 the presence of cellular factors and processes. The virus depends on them 00:20:45.23 at every stage of its entry program. They are very critical components. 00:20:51.01 Now in the dialogue between the incoming particle and the cell, it’s not only the cell 00:20:58.07 that provides information to the virus, but also the virus engages the cell in a dialogue 00:21:05.13 where it triggers this activation of these signaling pathways, and in that way 00:21:14.21 the information is given both by the pathogen and the host. Very important in a 00:21:21.29 sort of very general sense is that the virus particle must speak 00:21:26.04 the language of the cell. It must know the pin codes and all the passwords and it 00:21:32.02 has to know exactly how to activate the cells processes and functions that it needs. 00:21:40.17 So that is probably the most important realization that has come through the study 00:21:46.07 of many different viruses and their host cells. Now if we look at the type of cues 00:21:57.03 that I mentioned that different viruses require to go through the orderly process 00:22:04.10 of their entry program, at low pH, as you see here, exposure to low pH is 00:22:09.20 a very common one, but it's not the only one. Very often viruses require cues 00:22:14.27 by binding to specific cell surface molecules, so called virus receptors 00:22:20.07 , that induces changes. The low pH is another one. Also sometimes the cell has 00:22:26.04 to induce cleavages in specific viral proteins in order to activate them 00:22:31.00 and in some cases the re-entry of the virus from the extracellular space, which is oxidizing, 00:22:39.22 into the reducing environment of the cytosol serves as a cue. 00:22:45.21 All sorts of different things build up and help the virus do the thing that they need to do. 00:22:51.05 In some cases its exposure to specific enzymes such as thiol oxidoreductases. 00:22:57.25 So the virus is exposed to these changes and is modified by the cell 00:23:03.07 in order to be active in its entry. One final, very important general point 00:23:10.01 is that there is a basic difference in the strategy used by enveloped viruses, 00:23:16.14 those that have a lipid bilayer, and those that do not, the non-enveloped viruses. 00:23:20.28 The enveloped viruses do their transfer of the genome in a very smart 00:23:27.12 and intelligent way. They use the same principle by which the cells themselves 00:23:32.18 transfer macromolecules from one membrane bound compartment 00:23:36.04 to the other that is a vesicle transfer mechanism in which the cargo, in this case it’s the capsid, 00:23:43.16 a large macromolecular complex, is built into a vesicle, here, 00:23:50.02 and this vesicle by membrane fission pinches off, in this case 00:23:57.00 the plasma membrane with the capsid inside. This capsid then is transferred 00:24:02.05 to a new cell and then through a membrane fusion reaction, either at the plasma membrane 00:24:07.01 or in an endocytic compartment here releases the capsid into the cytosol. 00:24:11.27 As you see here, the plasma membrane may not always be the case 00:24:16.17 where the virus is formed, it can also happen in intracellular organelles. 00:24:20.07 But the main point is that no macromolecular structure of the virus 00:24:25.04 needs to pass through the hydrophobic barrier of a bilayer, it's all taken care of 00:24:30.14 by membrane fission, fusion, coupled fusion reactions. Non-enveloped viruses 00:24:37.14 have a much bigger problem. They have no membrane, they cannot do this. 00:24:41.10 Typically they exit from the infected cell by a lytic event. They break open 00:24:47.04 the membrane and the virus is released and then as they enter the new cell 00:24:52.10 they have to either lyse these vesicles and I'll come back later into 00:24:56.23 what type of mechanisms they use. Typically these mechanisms are not as well 00:25:01.14 characterized as the ones used by enveloped viruses. So now I want to go through 00:25:09.25 some early events that happen on the plasma membrane and then in 00:25:14.03 the second lecture I'll talk about the intracellular events. So let's go back 00:25:20.05 to the beginning. Now the virus has to bind to the cell surface. That step is 00:25:25.27 very important for many reasons. The virus cannot infect the cell 00:25:30.05 which it cannot bind to, so there has to be a first contact and binding otherwise 00:25:37.06 nothing will happen, the cell will not get infected. 00:25:40.16 The viral receptors that I have been alluding to are typical, normal, 00:25:47.02 everyday plasma membrane proteins of the cell. Either proteins, 00:25:51.21 lipids, or carbohydrates. Viruses have evolved to use some of these 00:25:57.14 for binding to and to mediate their entry into cells. Now-a-days we distinguish 00:26:05.11 between two types of attachments. One is the so-called attachment factors. These factors 00:26:11.23 simply bind the virus and help to concentrate the viral particles on the surface of the cell. 00:26:18.17 Then the real receptors come into play. The receptors in addition 00:26:24.07 to binding the virus help to give the virus information for example 00:26:29.09 by inducing conformation changes. They may be helpful in generating signals 00:26:35.01 that I mentioned before, or they may be involved in endocytosing the particles. 00:26:41.13 So they do than just bind. Many viruses can use more than one type of receptors. 00:26:48.12 Some use two or more receptors consecutively. You may know that HIV uses two. 00:26:55.04 And also it's important to realize the binding is typically multivalent so the virus binds 00:27:03.22 to more than one receptor at a time. So there are many contacts with the cell surface. 00:27:08.19 Now the type of molecules that serve as receptors are variable. They depend 00:27:17.09 on which virus we are talking about. So this picture shows some molecules 00:27:21.27 and the viruses that use them, and as you can see in this case these cell surface proteins 00:27:27.11 are quite different. And the choice of receptor for a virus is very important 00:27:34.05 because that determines which cell types in the body and which species 00:27:40.17 can be infected by the virus. So virus can obviously only infect cells which have 00:27:47.06 that particular receptor on its surface that it needs. Eventually the choice of receptor 00:27:54.10 is very important in determining what cells are infected and what type of disease 00:28:00.10 results from the particle invasion. We won't go through this in detail. 00:28:06.14 These are glycoproteins and proteins and they come from many different families 00:28:11.00 for different viruses. One the side of the virus, there must be of course something 00:28:15.24 that binds to the receptor, and that also varies. For example, in enveloped animal viruses, 00:28:22.24 the glycoproteins that cover the surface of the bilayer membrane 00:28:27.11 are the ones that bind to receptors. For example in this case the influenza virus, 00:28:31.29 the blue structures here are influenza hemagglutinin molecules and they 00:28:37.03 are responsible for binding to sialic acid containing receptors. 00:28:42.19 In non-enveloped viruses, as in the adenovirus that you see down here, 00:28:47.25 the first contact with the first receptor is through the fibers and the little knob at the end of the fibers. 00:28:55.22 Here is a rhinovirus, which binds to this yellow receptor molecule 00:29:01.04 shown in this crystal structure which in fact binds to small indentations 00:29:06.16 present on the surface of the virus. So they can be surface protrusions or 00:29:11.24 surface indentations. So viruses have developed specific sites which can bind 00:29:17.14 multiple receptors like this. Now let's look at one specific virus as an example. 00:29:24.17 In this case it is the Simian virus 40, polyomavirus family member. 00:29:31.01 It's a non-enveloped virus and it's structure is extremely well characterized 00:29:35.07 as you see here by X-ray crystallography. The particle is composed of 00:29:39.21 a surface protein called VP-1, which is present in these donut shaped structures, 00:29:45.05 which contain five VP-1 molecules each. 00:29:49.01 There are seventy-two of these pentamers, five-mers, organized 00:29:54.02 in an icosahedral structure with symmetry of T=7. 00:29:59.05 The VP-1 molecule is the one that binds to the receptor, and the receptor 00:30:04.11 in this case is a lipid molecule, the ganglioside called GM1. 00:30:10.04 Here is a picture of that ganglioside. It is a sphingolipid. 00:30:15.09 It has a carbohydrate moiety and a VP-1 molecule binds to some of the sugars 00:30:23.28 at this moiety. Here is a crystal structure recently published that shows 00:30:30.08 how exactly this interaction works. You have the pentamer here seen from the side, 00:30:36.03 and the sugar moieties are shown in the binding sites on the surface of this pentamer. 00:30:41.27 So here the interaction with the receptor is extremely well characterized. 00:30:46.03 The surface has multiple sites, each pentamer can bind five receptor molecules. 00:30:54.11 What you see here is the surface of CV-1 cells, a host cell for SV-40, 00:31:05.14 and viral particles in this case SV-40 particles have been labeled 00:31:11.08 fluorescently so they are visible on the surface of a live cell using 00:31:16.18 total internal reflection microscopy. Some of them are like this one, 00:31:22.28 fixed in place already, it does not move anymore. Others are really moving 00:31:27.26 in a random fashion around the surface of the cell. If one looks at virus particles 00:31:33.12 when they are binding initially they first go through a phase where they are mobile, 00:31:38.06 and then they stop, pretty much, or maybe drift a little bit, 00:31:41.21 but there is a free random motion followed by fixing the virus in place. 00:31:47.26 And then eventually the viral particles are endocytosed. 00:31:52.27 Now, it is possible in this case to study this interaction in a cell-free system, 00:32:00.01 in which one takes simply lipid vesicles, artificial lipid vesicles, liposomes, 00:32:05.24 containing the receptor GM1 and allows them to interact with 00:32:10.21 the coverslip or the glass surface and they will form a uniform bilayer on that surface, 00:32:15.24 which then will bind viruses and if you do that this is how it looks, 00:32:20.16 the particles bind nicely like they do on the cell surface and now they are all mobile. 00:32:25.18 All of the are moving and their movement is completely random. 00:32:31.12 Now in this case this lipid bilayer serves as a model system for the plasma membrane. 00:32:37.20 Now to find out a little more in detail how this motion works, is the virus sliding along 00:32:46.24 the membrane or is it rolling, we have collaborated with some terrific biophysics 00:32:52.15 at ETH Zurich, mainly Philipp Kukura and Vahid Sandoghdar, 00:32:59.05 who have been able to look at this question by following the viral particle by new technology 00:33:05.03 which is called interferometric scattering detection, iSCAT. It's a label free 00:33:12.27 detection system where they can follow the viral particle itself and we coupled 00:33:18.11 one quantum dot fluorescent probe, a single one to the viral particle, 00:33:23.09 and that could then be followed by its fluorescence. 00:33:26.14 The system allows nearly molecular spatial resolution and extremely high temporal 00:33:33.22 resolution. By combining in this case the following of the tracking of the viral particle 00:33:41.20 and this quantum dot, it is possible to get three-dimensional information 00:33:47.12 about the motion of the particle on the surface of these lipid bilayers. 00:33:53.04 This shows again the set-up a little more. We have a viral particle with a single quantum dot. 00:33:58.12 We can follow the viral particle not by fluorescence, but by this interferometrics, 00:34:07.06 and this is the type of spot you can see in the microscope. 00:34:11.17 Of course light microscopy allows you to go down to about 200-300 nanometers, 00:34:18.07 but since the viral particle is known in size exactly, we can define the center 00:34:24.02 of one of these from the point spread function with 2 to 3 nanometer resolution. 00:34:29.27 So the resolution of the system is extremely good, it's almost as good 00:34:34.10 also for finding exactly where this quantum dot is located. 00:34:39.08 So now when one combines both the interferometric analysis 00:34:44.11 and the fluorescence analysis, one can get the trajectory 00:34:48.02 of the virus moving on the cell surface where one can see that the quantum dot 00:34:53.09 and the particle are not exactly following the same trajectories. 00:34:57.16 They are moving a little bit differently, and on the whole of course they follow 00:35:01.25 each other and one can then through computers analyze 00:35:05.06 what that means in terms of 3D structure. And here you can see the outcome of that. 00:35:10.02 So this is the surface of the lipid bilayer that contains the receptor, 00:35:15.23 and the viral particle is moving randomly around. It is not exactly sliding 00:35:21.27 nor does it seem to be rolling, but it’s sort of wobbling, 00:35:25.06 probably moving from one receptor to another. 00:35:29.04 This is what we expect also of something like this happening on the cell surface. 00:35:35.09 Now before finishing this section, I would like to talk a little bit about the 00:35:39.29 surface behavior of this particular virus. It's the Human papilloma virus 16, 00:35:44.28 the major cause of cervical cancer. It's a DNA virus, 00:35:49.00 a non-enveloped virus, 55 nanometers in diameter. It replicates in the nucleus 00:35:55.06 and receptors for this virus are not entirely clear expected that they do use as 00:36:01.21 an important component, proteoglycan heparan sulfates. 00:36:06.08 The virus is acid activated and it is entering by endocytosis. Electron microscopy 00:36:15.27 here shows that the virus on cell surfaces likes to bind to filopodia. 00:36:20.23 These are the actin containing extensions. You can see them 00:36:25.15 in a section here and the viral particles are attached. Many viruses bind 00:36:30.23 to filopodia as you'll see later. Here is just an enlargement 00:36:34.02 of a particle and the plasma membrane underneath it. 00:36:39.09 When Mario Schelhaas, who did most of these studies together with Patricia Day and John Schiller at NIH 00:36:48.28 looked at this, they found that the viral particles when they are sitting on this filopodia 00:36:53.08 are actually moving down the filopodia towards the cell body. 00:37:01.10 The filopodia is here stained with GFP-labeled actin and this surfing of viral particles 00:37:09.00 towards the cell body happens for many viruses, it was first observed by Walther Mothes at Yale, 00:37:14.21 and we see it now for many different viruses. 00:37:17.28 So the viral particles in this case are not moving randomly on the surface of the cell, 00:37:22.27 but they bind to specific structures and then they move in a very directed 00:37:27.13 motion down these actin containing filopodia. The movement is entirely 00:37:33.23 dependent on the retrograde actin flow inside the filopodia 00:37:38.02 the actin is also moving from the tip down to the cell body. 00:37:44.00 Now this same phenomenon can be seen, well part of it, by electron microscopy. 00:37:51.15 You have here the cell surface and here is the filopodia or the beginning of it 00:37:56.08 and even the actin filaments are visible and this may be a virus which is moving down 00:38:01.25 to the cell body. What then happens is the endocytosis of particles into the cell, 00:38:11.00 the cell now internalizes the particles by endocytosis. Here we see already a vesicle 00:38:17.18 which contains a viral particle probably emanating from the cell surface 00:38:22.17 and we can see this happens to many viruses. They are actively taken up by the cell. 00:38:29.15 I will finish here, but I want to stay as long as we are still on the plasma membrane 00:38:36.25 and then in the next seminar talk about later events, but I would like to summarize 00:38:41.23 a few of the points that happens here. What is happening here underneath 00:38:47.01 this picture is that first of all there is a multivalent association 00:38:51.23 of the virus with these receptors. Receptors that clustered underneath the virus. 00:38:57.04 Somehow this clustering and interaction of the viral particle with its receptor 00:39:03.21 triggers a transbilayer coupling from the outside surface to the inside of the cell, 00:39:09.28 and this then activates a signaling pathway or more, which informs the cell 00:39:17.23 about the viral particle. Basically the virus is sitting on the surface and saying, 00:39:21.18 ping, ping, ping, ping, I'm here, do something. 00:39:25.04 And in this case as we see here the activation of an endocytic reflex 00:39:30.21 in the cell occurs. One or the other endocytic mechanism 00:39:34.11 is activated to bring in the viral particle. These endocytic vesicles then 00:39:41.13 help to move the virus from the surface into the center of the cell, and that is then 00:39:46.21 for many viruses where the penetration happens into the cytosol. 00:39:52.23 So I will for the first part stop here and then in the next seminar 00:39:59.01 discuss events that viruses go through after they have been endocytosed by the cell. 00:40:06.12 Thank you very much.