Viral Infection: Virus Entry and Subsequent Steps
Transcript of Part 2: Endocytosis and Penetration
00:00:01.20 My name is Ari Helenius and I am from the Institute of Biochemistry 00:00:07.06 at ETH Zurich in Switzerland. In the second presentation, 00:00:14.00 we will follow the viruses into the cell. The voyage of the virus from the 00:00:20.24 cell surface continues now by endocytosis and eventually inside the cell 00:00:26.12 the penetration will take place where the virus moves its genome 00:00:30.10 and accessory proteins into the cytosol. All viruses so far tested 00:00:35.29 move first to the cytosol wherever their final replication site is. 00:00:42.09 Many viruses, as I mentioned in the first lecture, then continue to the nucleus. 00:00:48.26 So here is the pathway again, the overall pathway binding to receptors 00:00:54.03 on the cell surface is followed by lateral diffusion of the virus 00:00:58.00 along the plasma membrane. So the initiation of signaling events 00:01:02.16 that very often end with endocytic uptake of the viral particle. 00:01:08.01 The virus then continues to additional organelles inside 00:01:12.14 the cytoplasm to which I will come in just a moment, and somewhere 00:01:16.28 along the line the penetration happens, the viral genome moves 00:01:20.14 from this extra-cytoplasmic cytosolic compartment into the cytosol. 00:01:29.25 Eventually, many viruses continue to the nucleus. So it's again a pathway 00:01:37.20 with several steps and we'll now continue talking about the endocytic process. 00:01:43.12 Now endocytosis can be defined as a process in which fluid, solutes, 00:01:52.09 membrane, and particles are internalized by cells by forming 00:01:57.19 an invagination at the plasma membrane which then pinches off as a vesicle 00:02:04.07 and the cargo is then transported in this membrane 00:02:08.26 bounded vesicle or vacuole inside the cytoplasm. 00:02:14.01 Receptor-mediated endocytosis is a specialized form or specialized forms 00:02:19.18 of endocytosis in which the cargo, the material to be internalized, 00:02:27.25 first binds to receptors on the cell surface and that then endocytosis 00:02:34.13 occurs not just on the cargo itself but on the whole receptor cargo complex. 00:02:42.10 The receptor binding is very important because 00:02:45.12 it helps to concentrate molecules on the cell surface and obviously from 00:02:51.09 what you heard in the first lecture, viruses use different forms 00:02:54.29 of receptor-mediated endocytosis. They bind first and that 00:02:59.08 and they are internalized together with one or the other of the receptors. 00:03:03.19 Endocytosis of course is a very complex process and I will not go 00:03:11.27 into this in detail but what you have to realize is that there are 00:03:15.01 many different mechanisms of endocytic uptake. Best known are here 00:03:20.14 the clathrin mediated endocytosis, which I talked about for virus uptake, 00:03:24.16 many viruses use it. Probably the majority of viruses may use it. 00:03:29.08 And here you can see another pathway, which is the phagocytic uptake 00:03:34.19 where large particles are internalized in tight fitting large vacuoles. 00:03:39.25 This type of uptake process is often used by cells that internalize bacteria. 00:03:48.16 But if one wants to now look at all the others it becomes a problem 00:03:53.01 in classifying all the different forms of endocytosis. First of all, 00:03:57.13 the classical classification is phagocytic uptake, that's particle uptake like here, 00:04:03.01 an actin-dependent process and pinocytosis, which is the uptake 00:04:07.25 of fluid and solutes and small particles. But here you can see that 00:04:12.07 pinocytosis has a wide spectrum of mechanisms and some of them involve 00:04:18.14 caveolin, some of them contain other diagnostic features, 00:04:26.03 which I will not go into in detail. But as you can see, we're starting to know already which 00:04:31.06 of these pathways contain viral ligands, which viruses use which type 00:04:36.14 of endocytic processes. A word of caution of course here is that some cells 00:04:42.00 internalize a particular virus by one mechanism, and in another cell the virus 00:04:47.14 may enter by another one. That is for example shown here 00:04:50.10 that SV40 can go in by two different mechanisms. Here influenza virus 00:04:56.07 also seems to use more than one type of mechanism. Underneath this hole, 00:05:01.20 if we move from the cell surface and the formation of this 00:05:05.03 primary endocytic vesicles downwards into the cell, then there is a 00:05:09.27 maze of organelles involved of which the main ones are shown on this schematic. 00:05:16.09 The most important ones are the classical early endosome, 00:05:21.03 almost all of these pathways as you look here lead to transport of cargo 00:05:26.18 into the early endosome. Material then either can return to the cell surface through 00:05:33.00 a recycling endosome over there or continue to other places. 00:05:37.20 You have to realize also that this is rather simplified. 00:05:41.02 Typical cargo move to the late endosome, which is more acidic, 00:05:45.20 and then for degradation into the lysosomes, the pH drops all the time 00:05:51.00 from about 6 or 6.2 in early endosomes to 5.5 and even lower in lysosomes. 00:05:57.13 Some other pathways that are here on the right have different types 00:06:03.22 of mechanisms. Macropinocytosis gives rise to a poorly characterized 00:06:08.12 primary vacuole called a macropinosome, and phagocytosis leads 00:06:14.23 to the formation of large phagosomes. These also in many cases seem 00:06:19.17 to feed into this central pathway of endosomes. From the endosomes, 00:06:24.25 there are different arrows, all of them are not shown here, but one of them 00:06:28.03 seems to be from endosomes to the endoplasmic reticulum, 00:06:31.09 which some viruses are known to use. Okay, that is a little bit 00:06:35.28 of background, but you have to realize there is huge complexity 00:06:39.16 here and I prefer to show it this way. What you are looking at here 00:06:43.11 is fluorescent transferrin, one of the physiological ligands that are taken up 00:06:48.27 by most cells, and how it looks when its moving through the maze 00:06:53.14 of endocytic organelles. These are vesicles, vacuoles, tubular structures, 00:06:57.24 endosomes, and so on. They are processing the traffic of this 00:07:04.29 nutrient carrier protein and in the cell you can see how 00:07:09.09 complicated this all is. This is the pathway and this is 00:07:12.18 the membrane trafficking systems that many viruses have learned to 00:07:20.17 take advantage of during entry. Okay, let's go back now to our example, 00:07:26.25 the last one we talked about, this is the Human Papilloma virus 16. 00:07:31.08 It has moved around on the cell surface, moved down along filopodia 00:07:36.04 and then it is being endocytosed here. It looks like these vesicles 00:07:40.08 are not coated, they don't contain clathrin probably, but it is not entirely 00:07:47.14 clear at the moment. All the work on the Papilloma viruses that I'm talking 00:07:52.02 about is a collaboration between Mario Schelhaas in my lab and 00:07:55.13 Patricia Day and John Schiller at the NIH. Now if you went long enough 00:08:00.10 we can see the virus moving into endosomes. I'll just show one 00:08:04.14 picture here, this is an endosome, probably a late endosome which 00:08:08.22 has viral particles here and some other membranous material. 00:08:13.01 So the virus moves at least into late endosomes, we see it also probably 00:08:17.18 in lysosomes. The question is what type of endocytic pathway does this 00:08:22.00 virus use? One way to look at it is using all of those perturbations 00:08:26.15 that I looked at before, knowing that each pathway that I mentioned 00:08:30.27 before have different dependencies on endocytic machinery components. 00:08:36.25 They may use clathrin, dynamin, caveolin, many others, and one 00:08:40.23 can actually test it, which of those endocytic machinery factors 00:08:45.14 of the cell are important. They may or may not be dependent 00:08:49.04 on actin and microtubules or cytoskeletal elements. 00:08:52.16 The signaling molecules are maybe involved. There are many kinases 00:08:58.18 that may or may not be used. All of this can be tested. Regulatory factors, 00:09:03.19 GTPases of the Rho family, Rabs, Arfs, and so on may be playing a role here as well. 00:09:10.04 Channels and acidification machinery, the list goes on, 00:09:14.06 but these are all things which one can experimentally test and measure 00:09:18.19 whether an inhibitor, for example of acidification through 00:09:23.04 the vacuolar ATPase, perhaps it will block infection of a given virus, 00:09:28.00 that tells you that the virus requires a cue, perhaps the low pH in endosomes. 00:09:35.14 And so you can build up a picture of what a particular virus is using. 00:09:39.13 Some lipids are in some cases also essential. Now if we go back 00:09:44.15 now to the Papilloma virus, it is using as you already saw noncoated pits 00:09:49.26 in these cells that we are studying and it transports to late endosomes. 00:09:54.08 That is pretty clear from morphological and light microscopy studies. 00:09:58.29 What is unusual here is that this entry process is super slow. 00:10:04.00 So the halftime of endocytosis is 3 hours so before the average virus is 00:10:10.02 internalized by cell it takes 3 hours. We are not seeing 00:10:14.04 that for any other viruses. And also the exposure to acid that is required, 00:10:18.26 for that it has to wait 10 hours. So that is very unusual, super slow entry mechanism. 00:10:26.01 It doesn't need components that I mentioned of 00:10:30.13 clathrin associated proteins, dynamin-2, there is a list of things we know for 00:10:36.25 sure the virus doesn't care about. It doesn't require them, and 00:10:40.09 there is a whole other list that's growing in size for what it does need 00:10:44.21 to infect the cell, acidification, certain kinases, sodium-proton exchanger 00:10:51.09 and so on, and in this way we can start to build up the picture of what 00:10:55.22 exactly is the mechanism used by Papilloma virus. 00:11:00.05 I won't go through it in detail but just summarize to you what the current 00:11:04.12 situation is. The virus binds to cell surface, very often to filopodia, 00:11:10.03 it then surfs down the filopodia and then it is endocytosed by 00:11:15.28 a non-clathrin non-caveolin pathway which in its features looks new, 00:11:20.18 we haven't seen anything precisely like it before, transported then 00:11:24.14 to early but also perhaps directly to late endosomes, and then the low pH 00:11:29.14 in a very slow event induces somehow penetration into the cytosol, 00:11:35.00 this virus we know has to get to the nucleus, otherwise infection will not occur. 00:11:39.14 So we are starting to build here a picture of a new pathway 00:11:43.13 which probably is used by other viruses that we also have studied. 00:11:48.03 So the endocytic process, we'll leave it behind and now come 00:11:52.17 to the penetration. The event that allows the viral genome to move into the cytosol. 00:11:58.09 It's this event shown schematically here and it is one of the events 00:12:03.12 where the virus actually has to do something actively itself. 00:12:07.08 Most of these steps are mediated solely by the cellular machinery. 00:12:13.07 Here something has to happen where the virus actively participates. 00:12:19.16 Now the site at which the penetration occurs is variable from 00:12:27.20 one virus to the other. I mentioned that some viruses like HIV 00:12:32.00 can fuse directly with the plasma membrane, they don't 00:12:34.23 have to be endocytosed. But those that are endocytosed can then 00:12:38.22 be activated for penetration in early endosomes, late endosomes, 00:12:42.09 sometimes perhaps even in lysosomes and some viruses go all the way 00:12:47.29 to the endoplasmic reticulum and then penetrate there. 00:12:52.02 The mechanism that allows the capsid to move from one side of 00:12:57.25 the membrane to the other is membrane fusion. I mentioned earlier 00:13:01.22 that enveloped viruses invariably used this mechanism. They simply fuse 00:13:06.15 their envelope with, in this case, the limiting membrane of the vacuole. 00:13:12.00 You can also induce escape of the virus like adenoviruses do. 00:13:17.09 Some adenoviruses, they lyse the vacuole of the endosome by bursting 00:13:23.03 the membrane, they can escape into the cytosol. Other viruses form 00:13:28.08 some sort of pores that allow their genome to pass through 00:13:32.16 the membrane and perhaps these endoplasmic reticulum viruses 00:13:37.10 using the mechanism called ER-associated degradation. There is some 00:13:41.21 indication that, for example, SV40 virus does that. So there are different ways 00:13:46.19 of doing this. If we now look a little closer at what might be 00:13:51.02 happening and the evidence particularly for the non-enveloped viruses 00:13:55.01 is not overwhelming at this point but what happens for adenovirus, 00:13:59.29 this is quite clear. The viral particle is binding to the cell surface, 00:14:03.13 this is the one that has these long fibers and the virus is then endocytosed 00:14:10.03 and mainly by clathrin coated pits, it depends on on which viral strain 00:14:14.17 we are looking, and then it enters the endosome here. 00:14:17.19 Now it's exposed to low pH, that causes a change in the particle, 00:14:23.15 which makes some components of the particle lytic, that means 00:14:27.05 they can now break up the membrane and the virus can escape through 00:14:33.06 the broken membrane into the cytosol, then move to the nuclear pore complex. 00:14:37.15 So there is a mechanism of lysis, which will not kill the cell 00:14:41.22 because it is only this vesicle that lyses, the rest are all intact. 00:14:46.14 Polio virus may be the best example now of viruses which use some sort of 00:14:53.02 pore strategy to enter. So the main point here is that these 00:14:58.18 non-enveloped virus particles binds to its well-characterized receptors, 00:15:02.12 is probably endocytosed, in most cells, into cells and then some cues 00:15:10.22 lead, for example receptor binding, lead to a conformational change 00:15:14.13 in the particle that allows the particle to insert into the membrane 00:15:20.14 in part and then simply release the RNA into the cytosol. The viral particle 00:15:26.03 itself is not entering the cytosol, simply the genome, in this case the viral particle 00:15:31.15 stays inside or outside the cytoplasm. So that's very different, 00:15:36.24 the overall idea and then you have here. So here the viral particle 00:15:43.27 is able to make a transient channel or pore through which 00:15:48.04 the nucleic acid is transported to the cytoplasm, cytosol. Fusion is the way in 00:15:56.23 which enveloped viruses enter and you've already seen some of this. 00:16:02.17 The viral particle for example, HIV in this case has spike glycoproteins 00:16:07.06 on its surface, which are activated in this case by receptor interactions 00:16:12.11 to become fusion active and the envelope of the HIV then simply fuses 00:16:17.06 with the plasma membrane. This then releases the capsid into the cytosol 00:16:22.15 and many further events later, the capsid is in that case, 00:16:28.08 the DNA made up from this capsid synthesized from the RNA, 00:16:34.10 will then enter the nucleus. There is also evidence that this HIV 00:16:39.09 can enter by endocytic mechanisms like the influenza here. 00:16:43.02 This virus is an influenza virus and the viral spike glycoproteins, 00:16:49.10 they influence the hemagglutinin allows it to bind to the cell surface 00:16:53.01 to get endocytosed with clathrin coated pits and with some 00:16:57.03 other mechanisms and then in the early endosome or late endosome 00:17:01.26 where the pH approaches 5.5, the hemagglutinin conformation changes 00:17:08.26 and it becomes a membrane fusion factor. The virus now fuses 00:17:12.15 its membrane with a limiting membrane releasing the viral genome 00:17:17.16 into the cytosol. Okay, let's look at this. It sounds very easy when you say 00:17:24.19 the virus simply fuses with a limiting membrane, but what it takes 00:17:31.07 is actually a very sophisticated fusion machine. In many cases now 00:17:38.15 we know in some detail how these fusion proteins on the surface 00:17:43.26 of the viral envelope link. The first study, and one of the best studie 00:17:49.08 s today is the hemagglutinin of influenza shown here. 00:17:52.03 It is as you remember the major protein that covers the envelope 00:17:57.28 of the influenza virus as this visible 135 angstrom long spikes. 00:18:04.12 It has two functions, one is to bind the virus to the cell surface, 00:18:08.25 and at low pH to induce the fusion. Now other viruses have 00:18:15.14 quite different looking fusion machines, remember this is the 00:18:20.03 Semliki Forest virus, an alpha virus, where the surface is covered by these 00:18:24.19 propeller-like trimers here, three winged structures, which are composed 00:18:30.23 of this complex, where the major components are a glycoprotein 00:18:36.02 called E2, the gray one here that forms the outer part and then 00:18:41.02 the colored one, yellow, red and blue here, which is 00:18:46.05 the fusion protein E1. At neutral pH, this is how it looks. 00:18:51.21 Each of the propellers are present on the cell surface of the virus 00:18:56.17 in that form. The third virus is an example taken from the flavivirus from 00:19:04.04 the family of Dengue virus, yellow fever, and so on down here, 00:19:08.10 where the surface looks completely different, but also covered by glycoproteins 00:19:12.21 in this case, glycoprotein dimers, here shown, which lie flat along 00:19:18.24 the membrane, you can see the gray and the blue forming a pair. 00:19:23.04 All these are acid activated fusion proteins, which when exposed 00:19:29.03 to the correct pH, convert like a transformer, like a toy that children have, 00:19:40.18 to totally different conformation, and in that new conformation 00:19:44.05 they are fusion proteins. Let's look at an example of what happens here. 00:19:49.23 Similar studies have shown for the other ones, but we want to focus on 00:19:55.24 what happens to this complex propeller at acid pH. It's called fusion 00:20:03.19 protein of class II and if you look at this schematic picture from 00:20:10.00 the work of Kielian and Rey, first you have the neutral pH structure 00:20:14.18 where you have the grey protein and then the colored protein forming 00:20:18.06 a complex together, each one of these here is one of those trimers 00:20:25.09 that I showed before. Now in the endosome, this structure is exposed 00:20:30.16 to low pH and first the whole structure opens up the E2, the grey one, 00:20:37.26 and the color one separate from each other and the grey one 00:20:43.04 has no longer any function, it has been involved in bringing the virus 00:20:48.19 in by receptor interaction, now it is disposable. The fusion proteins, 00:20:53.25 the E1s from these spikes come together here and form homo-trimers, 00:20:59.03 they form the new structure which is no longer flat along the membrane 00:21:03.25 but forms these elongated spikes, and at the same time what happens 00:21:09.22 is that the fusion peptide, which is shown as a little red dot is exposed. 00:21:15.09 Let me go back and look at the fusion peptide here. One thing that 00:21:20.12 combines all these different mechanisms, is that they have 00:21:23.13 a fusion peptide, and they are shown here by these different arrows 00:21:27.21 They are in the neutral pH structure located hidden away somewhere, 00:21:31.24 it’s a hydrophobic peptide sequence, which in the acid pH will be exposed. 00:21:37.11 In different places they are there waiting to be exposed. So when they then 00:21:41.26 eventually are exposed they insert themselves into 00:21:48.05 the target membrane, in this case the limiting membrane of the endosome. 00:21:52.02 So what happens is that an intermediate in this fusion process, 00:21:56.09 the spike glycoprotein is hydrophobically anchored 00:22:01.01 by its own cytoplasmic tail in the viral membrane and then by this 00:22:05.21 fusion peptide into the target membrane. That alone will not 00:22:11.00 bring about fusion, because the distance here is too long. The membranes 00:22:15.10 have to be brought together, but the fusion comes in the next stage 00:22:18.26 when the conformational change of these trimers changes. 00:22:22.16 You can see it here, schematically shown, they sort of buckle over 00:22:26.08 each one of them and bring the membranes together, in a focal point 00:22:30.28 between them and that forces the lipids into contact with each other and 00:22:36.18 fusion is then thought to happen first with the outer leaflet fusing, 00:22:40.04 it's called hemifusion, and then as a final step the inner, 00:22:46.01 the closest membranes, and the outer leaflets also fuse. And now you 00:22:49.22 have formed a fusion channel. So in this way viruses have developed 00:22:54.15 or evolved to have sophisticated fusion proteins, the function of which 00:23:01.09 is to bring about this fusion without any external energy in the form of ATP. 00:23:06.16 The energy for the fusion is built into the conformation 00:23:12.01 of the spike glycoprotein. So the penetration event is very important 00:23:19.16 and also as we now start to understand quite a sophisticated 00:23:23.24 step in the whole interaction of the incoming virus with the cell. 00:23:29.07 Work from Lakadamyali and others have allowed us to look at 00:23:35.00 the series of event in real cell context. What you see here, down here, 00:23:41.26 and this is a movie actually in a moment. This is an influenza virus particle 00:23:46.13 in which the membrane, the envelope, has been doped with 00:23:50.07 fluorescent lipid at such high concentrations that it is almost 00:23:55.05 completely quenched. The fluorescence is not coming out of that particle, 00:23:59.24 but when the membrane fuses with the larger membrane 00:24:03.07 of a late endosome, which will happen up here in a moment, 00:24:06.29 then the fluorescence increases and you can see actually a flash 00:24:12.19 of fluorescence forming. You can see this is the cell, 00:24:16.01 the nucleus is here, and you are looking at the cytoplasm here. 00:24:20.27 The viral particle will first move around on the cell surface a bit 00:24:24.02 then its endocytosed and in a microtubule mediated transport step 00:24:28.10 it moves out, enters the late endosome here and then you can see 00:24:32.01 the fusion event happening. Yes, now it moved up there, and it’s still now 00:24:45.02 moving deeper into the cell, and then the fusion event happens right there. 00:24:51.20 So that is the course of events, binding, endocytosis, acid exposure, 00:25:00.11 and fusion. And that seals the fate, probably, of this cell. 00:25:06.10 Now we come to what happens after the fusion event has happened. 00:25:11.27 The capsids or the capsid is in the cytosol here, and now it has to move 00:25:18.13 to wherever replication is happening and then uncoated. 00:25:23.03 Many viruses replicate in the nucleus, practically all the DNA viruses 00:25:28.08 and many of them use the intracellular transport machinery 00:25:33.08 to move wherever they are moving. That is they use microtubules, 00:25:36.24 microtubule motors, dynein in this case, and they move them as a particle, 00:25:42.23 they take advantage of this system to move eventually 00:25:46.14 to nuclear pore complexes where the genome is released into the nucleus. 00:25:50.28 I will not go through this step in detail, except that the viruses 00:25:56.03 are skillfully using existing transport machinery in the cell. 00:26:01.00 Look at this though, this is a schematic of different viruses. 00:26:07.16 I won't go through all of them in detail. All of these replicate in the nucleus, 00:26:12.17 they have to get their genome in here somehow. One of them 00:26:16.11 is retroviruses like HIV-1, or lentiviruses which transport 00:26:22.14 the DNA after reverse transcription to the nucleus, this step 00:26:27.14 is dependent on microtubules. The uptake of adenovirus is shown here, 00:26:34.21 remember it lyses the membrane and it's then transported 00:26:37.25 to the nuclear pore by microtubules. The same is true for herpes complex 00:26:42.19 herpes simplex virus capsids and so on. So in many cases this transport 00:26:48.14 is similar. Here I'll show you one video sequence of herpes simplex virus 00:26:55.22 entering cells. The fusion here happens at the plasma membrane. 00:26:59.22 The capsid is released into the cytosol and it’s then moved to the nuclear 00:27:04.20 pore complex. And we have in the following movie labeled capsid, 00:27:09.02 or it has a GFP labeled protein in it and this is where you can see it coming 00:27:14.22 from 11 o'clock, then downwards along microtubules to 00:27:18.24 the microtubule organizing center. Perhaps this was another particle that 00:27:22.25 was released and then this particle then moves on, we don't see in this case 00:27:28.05 the final docking of the viral capsid on the nuclear pore 00:27:33.23 and that is eventually what happens. So that's an illustration of how 00:27:39.07 the viruses take advantage of microtubules. Now to release the genome 00:27:45.20 into the nucleus happens in most cases through the nuclear pore complex. 00:27:49.24 And here different viruses have evolved different strategies. 00:27:54.07 I'll just go through a few of them. Influenza virus, one of the few RNA viruses 00:27:59.10 that enters the nucleus, has a sub-genomic genome. 00:28:03.12 That is, it has eight individual RNAs packaged into individual 00:28:08.11 ribonuclear protein particles, each of which are small enough to go 00:28:13.10 through the nuclear pore using normal import and export machinery. 00:28:17.21 So import and export machinery in this case. So they use importins 00:28:23.13 and carrier ferrins and they enter as intact particles through the pore. 00:28:30.26 The herpes simplex virus, which herpes viruses which you just saw 00:28:36.03 in the previous movie they dock onto the nuclear pore complex 00:28:39.29 and then simply release their DNA through the nuclear pore. 00:28:45.22 It's not so simple actually, but the result of it is that there’s an 00:28:50.05 empty capsid left behind on the pore and the DNA is internalized. 00:28:54.19 Adenoviruses also bind in intact form on the surface of the nuclear pore, 00:29:02.16 and then they dissociate. They are pulled apart and the DNA goes 00:29:08.10 through into the nucleus and the structural proteins of the virus 00:29:12.29 dissociated from each other. And then there are some viruses such 00:29:18.04 as parvoviruses, which themselves are small enough to penetrate 00:29:22.00 without the associations with the pore. This is obviously a critical step 00:29:28.03 and we must learn much more about the molecular details of this later. 00:29:32.26 So I'll finish off this second seminar by a very obvious thought 00:29:39.25 which is clear in this field and it has two parts. One is that to 00:29:45.11 understand how viruses enter cells and to understand viruses in general 00:29:50.27 one must understand the cell. The host cell is a key to the 00:29:54.13 understanding of virology. But on the other hand, one can learn 00:29:59.10 about the cell, amazing things, simply by following and studying viruses. 00:30:05.13 So the viruses are telling us things about cells and cell biology which would 00:30:09.23 otherwise be very difficult to study. I'll finish there with this second part. 00:30:15.26 Thank you very much.
