Ari Helenius (ETH Zurich) Part 2: Endocytosis and Penetration

my name is Ari helena's and I'm from the Institute of biochemistry at ETH Zurich in Switzerland in the second presentation we will follow the viruses into the cell the voyage of the virus from the cell surface continues now into by endocytosis and eventually inside the cell the penetration will take place where the virus moves its genome and accessory proteins into the cytosol all viruses so far tested moved first to the cytosol wherever their final replication scientists many viruses as I mentioned in the first lecture then continue to the nucleus so here is the pathway again the overall pathway binding to receptors on the cell surface is followed by lateral diffusion of the virus along the plasma membrane the initiation of signaling events that very often then end with anesthetic uptake of the viral particle and virus then continues to additional organelles inside the cytoplasm to which I will come in just a moment and somewhere on the line the penetration happens the viral genome moves from this extra cytoplasmic cytosolic compartment into the sides eventually many viruses continue to the nucleus so it's a again a pathway with several steps and will now continue talking about the endocytic process now endocytosis can be defined as a process in which fluid solutes membrane and particles are internalized by cells by forming an invagination at the plasma membrane which then pinches off as a vesicle and the cargo is then transported in this membrane bounded a vesicle or vacuole in inside the cytoplasm receptor-mediated endocytosis is a specialized form or specialized forms of endocytosis in which the cargo compound the material to be internalized first binds to receptors on the cell surface and that then endocytosis occurred not just of the on the cargo itself but of the whole receptor cargo complex the receptor binding is very important because it helps to concentrate molecules on the cell surface and obviously from what you heard in the first lecture viruses use different forms of receptor mediated endocytosis they bind first and that they are internalized together with one or the other of their receptors endocytosis of course is a very complex process and I will not go into this in detail but what you have to realize is that there are def many different mechanisms of endo static uptake best-known are here the clathrin-mediated endocytosis which i talked about for virus uptake many viruses use it probably the majority of viruses may use it and here you can see another pathway which is the phagocytic uptake where large particle are internalized in tight-fitting large vacuoles this type of object process is often used by cells that internalized bacteria but if one wants to now look at all the others it becomes a problem in classifying all the different forms of endocytosis first of all the classification classification is phagocytic uptake that's particle uptake like here and acting at dependent process and pinocytosis which is the uptake of fluid and solutes and small particles but here you can see the pinocytosis has a wide spectrum of mechanisms and some of them involve caviola some of them in other diagnostic features which I will not go into in detail but as you can see we're starting to know already which of these pathways contain viral ligands which viruses use which type of endocytic processes a word of caution of course here is that some cells internalize a particular virus by one mechanism and in another cell the virus may enter by another one that is for example shown here that sv40 can go in by two different mechanisms here influenza virus also seems to use more than one type of mechanism underneath this whole if we move from the cell surface and the formation of these primary endosit equesticles downwards into the cell then there is a maze of organelles involved of which the main ones are shown on this schematic the the most important ones are the classical early endosome almost all of these pathways as you look here lead to transport of cargo into the early endosome material then either can return to the cell surface through recycling and the song over there or continue to other places this is you have to realize also Strothers simplified typical cargo moved too late endosome which is more acidic and then for degradation into the lysosomes the pH drops all the way all the time from about six six point two in early endosomes to five five and even lower in lysosomes some of the pathways that are here on the right have different type of mechanisms macropinocytosis gives rise to a poorly characterized primary I said back you are called the macro – ohm and phagocytosis leads to the formation of large phagosome these also in many cases seem to feed into this central pathway of endosomes from the end ourselves there are different arrows all of them are not shown here but one of them seemed to be from endosomes to the plasmic reticulum with some viruses are known to use okay that's a little bit of background but you have to realize there is a huge complexity here and I prefer to show it this way what you're looking at here is fluorescent transferring one of the physiological ligands that are taken up by ourselves and how it looks when it's moving through the maze of energetic organelles these are vesicles vacuole tubular structures and the psalms and so on we are processing the traffic of of this nutrient carrier protein and in the cell you can see how complicated this all is this is the pathways and this is the the membrane trafficking systems that viruses have learned too many viruses have learned to take advantage of during entry ok let's go back now to our example the last one we talked about this is the human papilloma virus 16 it has moved around on the cell surface move down up along Phillip Odia and then it is being endocytosed here it looks like these vesicles are not coated they don't probably contain clattering probably but it is not entirely clear at the moment all the work on the papilloma viruses and I'm talking about is a collaboration between Mario she has in my lab and Patricia de and John Schiller at the NIH now if we wait long enough we can see the virus moving into endosomes I just show one picture here this is an endosome probably a late endosome which has viral particles here and some other membranous material so the virus moves at least into late endosomes we see it also probably in lysosomes question is what type of endocytic pathway does this virus use one way to look at it is using all those perturbations that I looked at before knowing that each pathway that I mentioned before have a different dependencies on endocytic machinery components they may use clattering dynam in Kabul in many others and one can actually test it which of those andesitic machinery factors of the cell are important they may or may not be dependent on acting and microtubules so cytoskeletal elements the signal signaling molecules may be involved there many kindnesses that may or may not be used all these can be tested regulatory factors gtp Isis of the ro family revs laughs and so on and playing role here as well ion channels and acidification machiner the list goes on but these are all things which one can experimentally test and measure whether an inhibitor for example of acidification through the vacuole or a TPS perhaps it will block infection of a given virus that tells you that the virus that requires a cue perhaps the low pH in endosomes and so and you can build up a picture of what a particular virus is using some lipids are in some cases also essential now if we go back now to the popular virus it is using as you already saw non coated pits in these cells that we're studying and it's transport to late endosomes that is pretty clear from morphological and light microscopy studies what is unusual here is that this entry process is super slow so the half time of endocytosis is three hours so before the average virus is internalized by the cell it takes three hours that we're not seeing that for any other viruses and also the exposure to acid that is required for that it has to wait 10 hours so that's very unusual super slow entry mechanism it doesn't need components that I mentioned or clattering associated proteins done I mean – there's a list of things we know for sure the virus doesn't care about it doesn't require them and there's a whole other list that's growing in size for what it does need to infect acidification certain kindnesses the sodium proton exchanger and so on and within this way we can start to build up the picture of what exactly is the mechanism used by a balloon of ours I won't go through it in detail but just summarize to you what the current situation is the virus binds to cell surface very often to fill up olia you heard it then then surfs down the philip Odia and then it's endocytosed by a non clattering non-covalent pathway which in its features looks new if we haven't seen anything precisely like it before transported then too early but also perhaps directly too late in the songs and then the low pH in a very slow event induces somehow penetration into the cytosol these virus we know has to get to the nucleus otherwise infection will not occur so we are starting to build here a picture of a new pathway which probably is used by other viruses what we also have studied so all the endocytic process we leave it behind now come to the penetration the event that allows the viral genome of to move into the cytosol it's this event shown schematically here and it is one of the events where the virus actually has to do something actively itself most of these steps are mediated solely by the cellular machinery here something has to happen while the virus actively participates now the site at which the penetration occurs is variable from one virus to the other I mentioned that some viruses like HIV can fuse directly with the plasma membrane they don't have to be endocytosed but those that are endocytosis can then be activated for penetration in early endosomes late endosomes sometimes perhaps even in lysosomes and some viruses go all the way to the endoplasmic reticulum and then penetrate there the mechanism that allows the capsid to move from one side of the membrane to the other is membrane fusion I mentioned earlier that enveloped viruses invariably use this mechanism they simply fuse their envelope with a in this case the limiting membrane of the vacuole you can also induce escape of the virus like adenoviruses do some other no viruses there lies the vacuole never endosome by bursting the membrane they can escape into the cytosol other viruses formed some sort of pores that allow their genome to pass through the membrane and perhaps these endoplasmic reticulum viruses are using a mechanism called ear associative degradation there is some indication that for example sv40 virus does that so there are different ways of doing this if we now look a little closer at what might be happening in the evidence particularly for the non-enveloped virus is not overwhelming at this point but what happens for adenovirus this is quite clear the viral particle is binding to the cell surface this is the one that has these long fibers and the virus is then endocytosed and mainly by clathrin-coated pits depends on which viral strain we are looking and then it enters the endosome here now it's exposed to low pH that causes a change in the particle which makes some components of the particle lytic that means they can now break up the membrane and the virus can escape through the broken membrane into the cytosol then move to the nuclear pore complex so there is a mechanism of lysis which will not kill the cell because it's only this vesicle that that lysis the rest are all intact poliovirus may be the best example now of viruses which use some sort of poor strategy to enter so the main point here is that this non-enveloped virus particle binds to its well-characterized receptors is probably endocytosed in your cells into cells and then some cues lead for example receptor binding lead to a conformational change in the particle that allows the particle to insert into the membrane in part and then simply release the RNA into the cytosol the viral particle itself is not entering the cytosol simply the genome in this case the viral particle stays in the inside or outside the side of the cytoplasm so that's very different overall idea than you have here so here the bio particle is may able to make a transient channel or pore through which the nucleic acid is transported into the cytoplasm cytosol fusion is the way in which enveloped viruses enter and you've already seen some of these the viral particle for example HIV in this case has spiked like a proteins on its surface which are activated in this case by receptor interactions to become fusion active and the envelope of the HIV then simply fuses with the plasma membrane this then releases the capsid into the cytosol and many further events later the capsid is and in that case the DNA made up of a from this capsid synthesized from the RNA will then enter the nucleus there's also evidence that this HIV can enter by endocytic mechanisms like the influenza here this virus is an influenza virus and the viral spike lack of proteins the influenza hemagglutinin allows it to bind to the cell surface to get endocytosed with clathrin-coated pits and with some other mechanisms and then in the early endosome or late endosome where the ph approaches 5.5 the hemagglutinin is conformation changes and it becomes a membrane fusion factor and the virus now fuses its membrane with a limiting membrane releasing davara do you know I'm into the cytosol okay let's look at this it sounds very easy when you say the virus simply fuses with the with a limiting membrane but what it takes is actually a very sophisticated fusion machine in many cases now we know in some detail how this fusion proteins on the surface of the viral envelope look the first studied and and one of the best study today is the hemagglutinin of influenza shown here it is as you remember the major protein that covers the envelope of influenza virus as this visible 135 long short long spikes has two functions one is to bind the virus to the cell surface and at low pH to induce the fusion now other viruses have quite different looking fusion machines remember this is the same leaky forest virus and Alpha virus where the surface is covered by this propeller like trimers here 3m wing structures which are composed of this complex where the major components are a glycoprotein called e to the grey one here that forms the outer part and then the colored one yellow reading and blue here which is the fusion protein a one at neutral pH this is how it looks each of the propellers are present on the surface of the virus in that form the third virus as an example taken is a fluffy virus from the family of dengue virus yellow fever and so on down here where the surface looks completely different but also covered by glycoproteins in this case glycoprotein dimers here shown which life lie flat along the membrane you can see the the gray and the blue forming a pair all these are acid activated fusion proteins which when exposed to the correct pH confer con convert like a con transformer like a toy that children have two totally different conformation and in that new conformation they are fusion proteins let's look at an example of this this what happens here similar studies have shown for the other ones but we also focus on what happens to these complex propeller at acid pH it's a it's called a fusion protein of class 2 and if you look at this schematic picture from the work of Killian and Rhea first you have the neutral pH structure where you have a grey protein and then the color protein forming a complex together each one of these here is a is one of those primers that I showed before now in the endosome this structure is exposed to low pH and first the whole structure opens up the e to the grey one and the colored ones separates from each other and the the grey one has no longer any function it is it has been involved in bringing the virus in by receptor interaction now it is disposable the fusion proteins the e ones from these spikes come together here and form homo trimers they form a new structure which is no longer flat along the membrane that forms these elongated spikes and at the same time what happens is that the fusion peptide which is shown as a little red dot is exposed let me go back and look at the fusion peptide here one thing that combines all these different mechanisms is that they have a fusion peptide and they are shown here by these different arrows they are in the neutral pH structure located hidden away somewhere it's a hydrophobic sequence which in the acid ph will be exposed in different places they are there waiting to be exposed so when they then eventually are exposed they insert themselves into the target membrane in this case the limiting membrane of the endosome so what happens is as an intermediate in this fusion process the spike lipoprotein is hydrophobic ly anchored by its own cytoplasmic tail in the viral membrane and then by this fusion peptide into the target membrane that alone will not bring about fusion because the distance here is too long the membranes have to be brought together but the fusion comes in the next stage when the conformational change of these tremors changes you can see it here schematically shown they sort of buckle over each one of them and bring the membranes together in a focal point between them and that forces the lipids into contact with each other and fusion is then thought to happen first with the outer leaflet fusing is called Hemi fusion and then as a final step the the inner the closest membranes and the outer leave that's also fused and now you have formed a fusion channel so in this way viruses have developed or evolved to have sophisticated fusion proteins the functional which is to bring about this fusion without any external energy in the form of ATP the energy for diffusion is built into the conformation of the spike like a protein so the penetration event is very important and also as we now start to understand quite sophisticated step in the whole interaction of the incoming virus with the cell work from Locka meolli and others have have allowed us to look at to see this event in the real real cell context what you see here down here and this is a movie actually in a moment this is an influenza virus particle in which the membrane the envelope has been doped with fluorescent lipid at such high concentrations that it's it is almost completely quenched to the fluorescence is not coming out of that particle but one of the membrane fuses with the larger membrane of a late endosome which will happen up here in a moment then then the fluorescence increases and you can see actually a flash of fluorescence for me you can see this is the cell the nucleus is here and you are looking at the peripheral cytoplasm here the vile particle will first move around on the cell surface a bit then it's endocytosed and you know microtubule immediately transport step it moves out enters the late endosome here and then you can see the fusion event happening so is anything moving yes now it moved up there and I'm still now moving deeper into the cell and then now I think the fusion even happened right there so that is the time the the course of events binding endocytosis as it exposed the fusion and that that seals the fate probably of this cell now we come to what happens after the fusion event has happened to set the cap seeds or the capsule is in the cytosol here and now it has to move to wherever replication is happening and then uncoated many viruses replicate in the nucleus practically all the DNA viruses and many of them use the intracellular transport machinery to move wherever they are moving that is they use microtubules microtubule motors dining in this case and they move them as a particle they take advantage of this system to move eventually nuclear pore complexes where the genome is released into the nucleus I will not go through this time step in detail except that the viruses are skillful using existing transport machinery in the cell look at this though this is schematic of different viruses I won't go through all of them in detail all of these replicate in the nucleus they have to get the genome in here somehow one of them is retro viruses like HIV one or an anti viruses which transport the DNA after reverse transcription to the nucleus this step is dependent on microtubules the uptake of adenovirus is shown here remember it lies is the membrane and it's then transported to the nuclear pore why I could use the same is true for herpes Catholics herpes simplex virus capsid and so on so in many cases this transport is similar here I'll show you one video sequence of herpes simplex virus entering cells the fusion here happens at the plasma membrane the capsid is released into the cytosol and it's then moved to the nuclear pore complex and we have in the following movie labeled the capsid or it has a GFP labeled protein in it and this is where you can see it coming from 11 o'clock then downwards along microtubules to the microtubule organizing Center and perhaps this was another particle that was released and then this particle then moves on we don't see in this case the final docking of the viral capsid on the nuclear pore and that is eventually what happens so that's an illustration of how the viruses take advantage of microtubules now to release the genome into the nucleus happens in most cases through the nuclear pore con and here different viruses have evolved different strategies I'll just go through a few of them influenza virus one of the few RNA viruses that enters the nucleus has a sub genomic genome that is it has eight individual RNAs packaged into individual ribonucleoprotein particles each of which are small enough to go through the nuclear pore using normal import and export machinery sorry import X in this case so they use importance and reference and they enter as intact particles through the port the herpes simplex virus which and herpes viruses which you just saw in the previous movie they dock onto the nuclear pore complex and then simply release the DNA through the nuclear pore it's not so simple actually but the result of it is that there's an empty capsid left behind on the poor and the DNA is internalized adenovirus is also bind in intact form on the surface of the nuclear pore and then they dissociates they are pulled apart and the DNA goes through into the nucleus and the the structural proteins of the virus dissociated from each other and then there are some viruses such as para viruses which themselves are small enough to penetrate without dissociation through the pore this is obviously a critical step and we must learn much more about the molecular details of this later so I'll finish off this second seminar by very obvious thought which is clear in this field one and and and it has two parts one is that to understand how viruses enter cells and to understand viruses in general one must understand the cell the host cell is a key to the understanding of a viral but on the other hand one can learn about the cell amazing things simply by following and studying viruses so the viruses are telling us things about cells and cell biology which would otherwise be very difficult to to study I'll finish there on this second part thank you very much

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