Lecture 2: "Coronavirus biology"

Hi, I'm Richard Young, your host with Fukundo Batista and Lena Feyen for the MIT course on Covet 19 Sars Kobe 2 and the pandemic. There are more than 200 species of viruses known to infect humans, and several new species, at least two, are discovered each year. -thirds of these also infect non-human hosts and some of them have the ability to jump from one host species to another. This transmission between species from animals to humans depends on many factors. Exposure to the virus. The presence of a suitable receptor in the. surface of our cells the ability of the virus to exploit our own cellular machinery to replicate and escape our many defenses and the ability to transmit from one person to another well enough to cause epidemic outbreaks the purpose of this course is to learn what we know today on covet 19 of the world's best scientists last week dr. bruce walker introduced us to the general scientific issues surrounding the pandemic today i have the pleasure of introducing you to a talented virologist, dr.

Britt Glonsinger, who has been at the forefront of educating those scientists in the public about the new

coronavirus

. Dr. Glonsinger is a professor at the University of California at Berkeley and a researcher at the Howard Hughes Medical Institute. Her award-winning research focuses on the ways in which that RNA viruses hijack the machinery of host cells professor glonsinger thank you for bringing our current understanding of this virus to our students and viewers, thank you very much for the invitation, I just wanted to make sure you can see my screen now before we start, yes Okay, so, I'm really delighted to be able to participate in what you're looking for.

It's going to be a pretty exciting class this semester, as Rich mentioned for most of my degree. I have studied how viruses manipulate postgene expression and cooperate with the cellular gene expression machinery to benefit the virus and its replication cycle. In fact, I have generally studied. This during the DNA virus infection, but since March of this year, of course, it has been expanded to include

coronavirus

es and, as Dr. Walker pointed out in his seminar last week, it is really part of a global change without precedents in the focus of scientists from all disciplines toward a singular objective. of understanding this new virus to combat the pandemic and I just want to take a second to highlight that, in fact, many of these efforts are volunteer-based and need to be recognized, so I just want to mention that on this slide, for example.

These are two of my lab's postdoctoral fellows, Azra Lari, Divi, and Ananda Kumar, who over the past few months have donated many hours of their time to help set up and manage a coveted 19-test diagnostic lab that was organized at the University of California at Berkeley. through the innovative genomics institute both to do on-campus testing but also to test underserved components of the community where this has been limiting and although they are grassroots scientists, they have entered the clinical arena just to contribute to these pandemic efforts and really I liked Azera's statement here, which was that at first I was lukewarm about how much I could offer, but there really is no insignificant part of the process, each seemingly small task contributes to the larger goal and for me this is really a reminder of the can. of working in a coordinated and collaborative way, something we are seeing a lot during this pandemic, could be a gray of light in what would otherwise be a lot of darkness, so the part of this larger story that I am going to tell you today it's how the virus itself operates and that's because the better we can understand how the virus works, the better prepared we will be to design truly effective interventions and I'm going to divide the seminar into three general topics, today we're going to start discussing. in some detail how cov2, which is the name of the virus that causes Skillvid19 of course, enters your cells, then once there how it activates the viral machinery that is encoded in your genetic instruction set, it's Chinese and finally, how to use. that machinery to dramatically reorganize the cell and turn it into a virus factory.

I know we have a pretty diverse audience today in terms of academic background, so what I'm going to do is certainly provide some molecular details about each one. of these topics, but in general I'm going to try to keep most of the

lecture

at a level that I think a first-year science student could understand, so if you start to get lost, don't worry, I'll try to backtrack. let's look at the bigger picture pretty regularly, okay, let's start by just going over what constitutes a coronavirus particle, actually, what constitutes any virus, the definition of a virus is basically something that has a set of genomic instructions, genetic instructions, that It may be in the DNA. or in the RNA for coronaviruses it is in the RNA and that set of genetic instructions is protected in a house that is made of proteins or a mixture of proteins and lipids or fats that is called envelope and the proteins come from the virus, but in At any time there is a Lipid involved in a viral particle that is always stolen from the host.

No virus can produce or generate its own liquid molecules, so let's take an image of the coronavirus and cut it in half so this diagram shows what the components of the coronavirus particle are. You see, an orange is the RNA genome. It is wrapped around the inside of the particle. It's a surprisingly large genome. We will talk about this in more detail later. It is a genome that is what we would call positive sense RNA. means it is ribosome ready, once it enters the cell it can be read directly by the protein machinery and the genome itself is encoded in a viral protein called nucleic acid.

This is something that helps protect the RNA but we also think is important to help them. New viral particles are formed after the RNA has been replicated in the process of morphogenesis and that nucleocapsid RNA is then surrounded by a lipid envelope which, as I said, is taken from the host cell and that lipid envelope is studded with three different viral proteins, two of which are the matrix. The protein shown in purple, the envelope protein shown in yellow here, is again involved in the formation of the virus particles, helping the viral RNA to bud into them.

The third one, the spike protein, is what gives the virus its name, and that comes from looking at this protein, this virus. Under electron micrographs, the spike protein, which is the most prominent feature of the viral particle, is something that stands out, it looks like a corona, which means corona, a crown of spikes or a halo in an eclipse and, despite of protein, we are going to spend some time talking about it. Because it plays two critical roles in allowing the virus to enter cells, it is also the primary target of neutralizing antibodies to inactivate the virus, and therefore the focus of almost all vaccine efforts is on generating antibodies. neutralizers against the spike protein, so let's take a closer look at Spike Spike is actually a group of three of the same protein, it's a homotrimer that binds together and forms each of these structures on the surface.

Actually, we know this thanks to several cryo-electron microscopy studies. We know a lot about the Spike protein and the atomic. level of detail so I'm just showing you an image from one of those posts here and to highlight that the spike has a couple of different regions or domains that are important first is this upper globular domain here that is involved in engaging the receptor to which you can think of as a key looking for a cellular lock to enter, it can only be inserted into a lock that fits the key, so the virus can only access cells that have the correct lock and ace2 is the name of that law.

Because it's the protein or the receptor on the surface of the cells that allows the virus, so this upper globular domain is the part of the spike that binds to that receptor, but that's not the only thing that the spike does, in fact, it has this bottom domain that houses a The second critical activity essentially has what's called a fusion machine or a fusion activity buried in this bottom region of the protein, similar to a reservoir. You can think of the fusion machinery as something that runs on a spring, like a jack-in-the-box, where when you have the lid closed on the box, that spring is in a condensed, inactive form and that lid is this globular receptor binding domain. , so what needs to happen for this fusion machinery to turn on and allow the virus membrane to mix with the cell's plasma membrane so that the virus can penetrate that external cellular defense of its plasma membrane is that you have to remove this lid to allow this fusion peptide to expand and access the cell, so that's the second really critical function of the spike i' I'm going to show you a movie about this in just a second, but first you'll notice that it's color coded and is color-coded to show the degree of sameness or conservation between closely related spike proteins from members of the same subgenus.

The caveat is that the areas in light blue or teal are the most variable region and the darker areas in purple are the regions that are least variable and therefore it is not surprising that for all viruses, it is true that the receptor binding domain or almost all is a region of the virus is prone to intense evolutionary pressure, usually coming from the immune system due to neutralizing antibodies, so it is part of the viruses that will generally change much more rapidly than many others regions of the virus and that also applies to coronaviruses, however, don't. the opinion that these are changing like crazy because, in fact, coronaviruses at the peak generally have quite low genetic diversity, meaning that few new mutations tend to arise during multiple rounds of viral replication and keep that thought in mind because we're going to touch Later in this talk we'll explain why that is, but the fact that it is is important because it means it's probably good news for a vaccine strategy.

We are concerned that vaccines that could be made well may mutate the virus very quickly and move away from the neutralizing antibodies to escape the ones that are generated, and I think that is less likely to happen with this coronavirus than with other viruses that have mutation rates. much higher levels, such as HIV and influenza, does not mean that changes are not occurring in the virus. Of course there are, and some of you may have heard of this d614g mutation that has been in the news a lot. lately and this refers to the fact that an aspartic acid at position 614, which is sort of at the boundary between the receptor binding domain and the fusion machinery quite early in the pandemic, I think in March or April it changed with quite quickly from being an aspartic acid to being a glycy and a recent report shown here has shown that this change was perhaps something that was positively selected, meaning that this change may have benefited the virus by creating a spike variant that was more transmissible or could access the inside of cells more easily, so these changes are occurring and are being documented through these widespread sequencing efforts, but the virus overall is not mutating at the rapid rate of other viruses, okay so let's summarize what the spike is doing for the virus to enter the cell first, like I said it must act like a key to attach to the lock of that h2 receptor protein so that only the cells that have this receptor have the ability to allow the virus to come in through that interaction, but as I said, that's not enough.

Also, the second critical activity of the spike is that it has to activate that fusion machinery in the lower region of the bridge and the removal or opening of that cap is done by cutting it using a kind of what can be thought of as molecular scissors called a protease enzyme. The protease that has been shown to play an important role in cutting and activating that spike fusion machinery is called temporis ii, although it is thought that others could be involved such as furin as well, so that the protease cuts the tip of the h2 receiver head and what this does is allow the spring loaded internal fusion machine to turn on and insert.

Upon entering the cell plasma membrane, the viral membrane comes very close to the cell membrane and fuses them. This creates a portal between the inside of the virus and the inside of the cell so that the virus can now deposit its payload, which is the RNA genome. If the cytoplasm starts to replicate, then it's already in, what the virus needs to do is convert that genome into a large number of proteins that will do the job of remodeling the host cell, copying that viral genome and assembling new progeny virions like does that? Let's first take a look at theviral genome itself.

What is it made of? Like I said, it's a long genome made of RNA. It encodes about 13 different genes, so they are described in this diagram here or it means open reading frame. so it's a gene that is produced in a shorter or longer form and has things like envelope matrix proteins or spikes, other types of things from these 13 genes, at least 27 proteins are produced, actually , suggested a recent report on ribosome profiling. that there may be 20 additional protein products made through things like upstream open reading frames or other things like that, so how do you get 27 or more proteins made from just 13 genes in the first place?

Well, the answer is that the virus has to use some tricks and it has to use some tricks for two different reasons: the first is that, although the virus can make use of the cellular machinery, it basically must comply with the rules that that cellular machinery It normally operates in and within a eukaryotic cell like ours. In our own cells, we generally get one gene converted to a protein for each messenger RNA, so our messenger RNAs, which are the ones read by the ribosomes, generally have a five-prime cap that helps recruit the protein synthesis machinery. proteins in the correct area, which then scans through what is called the untranslated region until it locates a start codon, this tells the protein synthesis machinery to start making proteins, start copying this gene from its from RNA to protein form and it will read it until it hits a molecular stop signal and then the ribosome will exit.

Everything that happens after that stop signal is not seen by the correct cell, so it is shown here. The ribosome that will scan will produce proteins that are within that blue region that I have shown here and then in a dissociated form, this is a problem for a virus like the coronavirus. true, because I just told you that it has a whole series of genes, it doesn't have one gene in its RNA, it has 13 genes in its RNA, but according to eukaryotic translation rules, these genes will mostly not be seen by the ribosome. the ribosome is going to show up, it's going to see gene one, it's going to find the star codon, it's going to translate it into protein until it gets to the stop codon of gene one and then it's going to dissociate and it's going to drop off and then it's going to restart it.

Gene one starts over. All of these genes downstream or to the right of gene one are basically going to be invisible to the ribosome, so all of this RNA is not going to make protein one, two, three, four, and five, it's just going to make protein one, so how do you avoid the coronavirus that potential? barrier to expressing its genes well, the first thing it does is use a strategy where it encodes many different proteins in a single gene, so it does this for this first giant gene that occupies a large part of the viral genome actually and the The way it does this is by inserting these genes just into this open reading frame removing those start and stop signals that normally sit on the barrier and tell the ribosome when to start and when to finish so that you only have one start signal. at the end of the first protein coding sequence and then it has another direction at the end after the ribosome has gone through 11 in this case or if there are frame shifts here, up to 16 different genes can give me, this is called polyprotein, it is essentially a giant fusion of between 11 and 16 different proteins made from a single open reading frame, a single gene.

Now, how these proteins are separated from each other is by using those molecular scissors again, but this time not the cellular molecular scissors that the virus encodes. its own protease enzymes that can come and cut this long polyprotein into individual protein subunits that will then have different activities. You can imagine that this protease is a really critical activity for the virus and is therefore a target for antiviral drugs. safe development, okay, so that's a strategy that the virus uses to get up to 16 or more proteins made from one gene, but that doesn't solve the problem of all these other genes that are now on the far right of the genome, how do they cure me?

The virus uses a second strategy to convert these sets of genes into proteins and the way it does this is by generating new copies of RNA and those new copies of RNA are subgenomic in length, they are less than the full length of the genome, so the We call subgenomic RNAs and they are a nested set, which means they all have this, they all have the same three primes and the same right end here which is basically the end of the viral genome and Sometimes, however, it will be long and will have all the genes present and sometimes it will be shorter and only have some of the genes present, so the way this happens is that as the viral copying machine creates these RNAs at the boundary between each of these.

The genes found on the right side of the genome are a signal called the transcriptional regulatory sequence or trs and that signal basically gives the RNA copying machine the option to stop or continue and maybe like a traffic light, we don't know exactly how. It works, but still allows the copier to sometimes stop at that signal, in which case it would have a short RNA if it stops at the first signal, or sometimes it reads that first signal all the way to the second signal and then does so. It's kind of crazy to jump to the other end of the genome and add this special sequence of five top leaders, so everyone has the same five cousins.

That leading sequence can help with translation, it can help with other related functions. to these RNAs, okay, so why does this solve the virus problem? It solves the problem because by creating these subgenomic RNAs, each of these subsequent genes has the opportunity to be represented as the first gene that the ribosomal protein machine makes or sees. in this case, gene two, which is similar to the spike protein, here it is the first gene that the ribosome sees, so it will be translated into protein and you will have spikes, all these other genes downstream of course, not they will be seen, but that is fine because there is another RNA where gene 3 has the opportunity to be the first gene detected by the ribosome and so on and in this way each of the proteins located on the right end of the genome can be synthesized from of these subgenomic RNAs, so it's actually a combination of this giant polyprotein fusion and the creation of subgenomic length messages from the other end of the RNA that allows the virus to express all of its proteins that will do the job of copying the RNA virus and remodel the host's interior. cell, so let's talk for a minute about this RNA copying machine.

It's quite fascinating. In fact, all RNA viruses have to encode their own RNA copying enzyme. This is called RNA-dependent RNA polymerase or rdrp and that's because our own cells don't have this. machine in them for viruses to steal, we have machinery to make proteins that they can steal, but we don't have machinery to copy RNA, so each RNA virus has to bring its own RNA copying machine for coronaviruses. This RNA copying machine is remarkably sophisticated. I have the core polymerase shown here in pink encoded by a non-structural protein or nsp12. This is the core RNA-dependent RNA polymerase, but you'll notice that there are eight or nine different additional viral proteins that form what's called this replicase complex.

These are things. that help the polymerase stick to the RNA and be more processive that they process the RNA as it is made that they can unwind the RNA to potentially traverse problematic structured regions, but the complexity of this RNA copying machine is very high for the right viruses , it actually rivals those DNA ones that are much more sophisticated overall than these RNA photocopiers, why might that be? Well, actually, this is a pretty fascinating facet of coronavirus

biology

, but just so you understand I have to remind you that, first of all, RNA copiers generally make a lot of unintentional mistakes, but on average they will insert one wrong nucleotide every one thousand or ten thousand uh bases that are copying well, so this is what we call an error prone polymerase, an air prone nature of the polymerase and it is what is driving the high mutation rates that are seen in many viruses of RNA, things like HIV have an astronomically high mutation rate because their RNA copying machine is inherently sloppy.

The influenza Ebola virus, all these RNA copying machines are sloppy and can't correct their mistakes and that's what leads to high mutation rates. Remember I said that the coronavirus genome does not appear to be mutating at these high rates that we see in many other RNA viruses. The reason is this sophisticated RNA copying machine that the coronavirus uses. has an ace up its sleeve, it has its own built-in review mechanism, so it's basically a text editor that travels along with the polymerase and if the polymerase makes a mistake, it puts the wrong nucleotide in the sequence, this text editor will kick him. eliminate the error and allow it to be replaced with the correct nucleotide for the sequence, so when comparing this type of corrected genome you could see that there would be many fewer errors compared to a viral genome that does not have this correction activity.

This bottom graph here has data. of the sars coronavirus shows in fact that if you remove this proofreading enzyme called xon, the number of errors that occur randomly in the genome increases about 20 times, so this is probably the underlying reason why coronaviruses tend to have a lower error rate and also likely turns out to be an important component, at least in why coronavirus genomes are so large. In fact, the coronavirus genome, which has about 30,000 different nucleotide bases, is two to three times larger than an average RNA virus; most RNA viruses are in the range of eight to eighteen thousand pairs.

Of bases, things like rhinovirus, which is the cause of most common colds, have less than 10,000, as does hiv influenza, it's a bit more than that, but you'll notice that sars cov2 and other coronaviruses have its genome. is huge, in fact, they are approaching the theoretical limit of how large the genome of an RNA virus can be. What sets that limit is this error rate, so the problem of having an RNA genome is a problem for the virus, and actually it usually is. But the thing about genome size is that the longer the genome, the more likely it is that, as the RNA copying machine goes by, it will make a random error, so if you make a mistake once every 10,000 bases and if you have a genome that's 10,000 bases long, well, you'll have a random error somewhere every time you copy that RNA longer, but if you have a longer genome, you'll have more of one error every time you copy that RNA.

And it turns out that viruses can tolerate a certain amount of random errors, but if there are too many of them, this decreases what we would call the fitness of the viral population and they suffer from what is called error catastrophe. Many bugs make the virus too weak. Too many problems. and the entire population can collapse, it is this that helps determine the lifespan limit of a virus, so the fact that the coronavirus can correct its mistakes allows it to have a much larger genome before it can reach this threshold of just catastrophe and that means that the coronavirus genome can encode more genes, more proteins to remodel and perhaps have a more sophisticated interaction with the host.

Now that the virus has created its set of viral machinery, what is it going to do with that machinery? How do you use each of them? those viral proteins to reorganize the host cell and begin producing many new progeny baryons. Well, it turns out that it remodels in a really surprising way the inside of an infected cell and it does so by essentially creating new rooms where the virus can replicate and new rooms that I mean is that viral proteins are made that act on cell membranes derived from the endoplasmic reticulum, the rough endoplasmic reticulum that takes this cell membrane, moves it, stretches it, and uses it to form these circular double-membrane vesicles that form throughout the cytoplasm. of a cell infected with coronavirus, these are called transcription and replication complexes or rtcs, but essentially what they are, as you can imagine, are separate rooms within the cell that allow the virus to copy its genome efficiently, so There are probably two reasons, at least two.

One of the reasons the virus creates these rooms is that it helps the virus do what it's doing, which is copy its genome to a more secret location. The cytoplasm of the cell is full of sensory proteins that look for evidence that the cell has been infected. It is looking for things that look strange because if it detects those strange things it will alert the broader immune system to try to launch the adaptive immune response that produces antibodies. and killer T cells and also to warn all neighboring cells to melttheir immune defenses to protect against infection and a main signal that these sensor proteins look for is double-stranded RNAs because they are generally not formed within our own cells very effectively, but if you think about how an RNA genome is copied, we are starting with an incoming strand of RNA and the RNA copying machine has to make a copy of that and for that copy it can form double stranded RNA intermediates which are a key signal to the immune system that they are infected so if you create specialized In environments where these RNA copying reactions occur, there may be less chance that the immune system can detect them.

A second hypothesis for why they are beneficial is that they allow the virus to concentrate basically all the things it needs to copy its genome and transcribe its genome in one place and therefore the whole process can be more efficient. These are the rough endoplasmic reticulum, which is riddled with ribosomes around the periphery of these, allowing new messenger RNAs that are made from the viral genome to perhaps be translated into proteins in the same general location as well, so this is an image taken of a cell infected with the sars coronavirus just to give you an idea that these double membrane vesicles are actually filling the cytoplasm of these cells infected with the coronavirus, they are practically everywhere these circles are these vesicles that the virus is forming and a zoom in here this is a cryo-electron tomography experiment and this is a 3D representation that basically allows you to build a three-dimensional structure to see what the inside of a cell looks like allows you to see the pseudo color of these vesicles, the inside of these vesicles would be this area here that is surrounded by the purple color and then because they are double membraned, you have this second membrane that is yellow, which is contiguous but surrounds the vesicles and therefore within each of these circular areas would be where the copying of the viral RNA takes place.

In addition to remodeling the cytoplasm membrane environment, the virus is also quite dramatically changing the gene expression environment of the host's genes and one of the ways it does this is through a protein. That is the first protein formed from the viral genome and is called a non-structural protein. One is a viral protein that is capable of powerfully restricting the expression of cellular genes and does so through a double strategy: inducing the cleavage of RNAs messengers through a mechanism that is not yet well understood and repress the translation of messenger RNAs. this is something that for the sars cov2 nsp1 protein there have been a number of recent studies in preprints that have really helped elucidate the mechanism of how this protein restricts translation, so the 40 subunit of the ribosome is one of the main components of the ribosome Of course, that helps translate messages into proteins and the way it does that is that it has an entry channel that allows the messenger RNA to be fed to the ribosome so that it can be read by the ribosome and turned into a protein.

It turns out that what nsp1 does is I got this unstructured domain that's highlighted in pink here that inserts into that 4ds ribosome mRNA entry channel like a cork, which essentially prevents messenger RNA from accessing the ribosome and so Therefore, it reduces the translation of these messages. So why might reducing the translation of cellular messages be beneficial? One hypothesis is that it could divert available ribosomes preferentially toward viral messages, for example, if they were translated more efficiently. Another hypothesis that has been widely discussed is that this could be a very good general immune evasion strategy for the virus, so remember those sensor proteins that I was talking about that in cells are constantly scanning the cellular environment for evidence of infection and when they detect that evidence of infection, they will use that information to transmit a signal to the nucleus that tells the cell to turn on. genes that might be involved in fighting infections, genes like those that code for interferon, which is a kind of important emergency signal, or genes stimulated by interferon, or a whole set of things that cells can use to activate the response innate immune and the issue with the need to activate these genes is that the cell has to transcribe them into its own messenger RNA, those messages have to be read by the ribosome to produce things like interferon, so if nsp1 shows up and blocks this message so that it is not read by the rhizome, then the cell will be able to produce less signal that activates the innate immune response and that is shown here in this preprint from Tom's, which shows that in conditions where the innate immune response is strongly activated , this is actually due to infection with another RNA virus called sendai. virus, these cells will secrete a large amount of interferon beta, which is one of these molecules that activates the immune response;

However, if the cells express the nsp1 protein, much less interferon beta will be produced because the interferon beta messenger RNA cannot be translated efficiently by the ribosome, so this fits with some observations from transcriptomic studies that were published by the group of antenna burst showing that in the sars code moi infected cells under these viruses tend to induce or infected cells tend to have a fairly limited innate. antiviral state low expression of interferon stimulated gene products like interferon beta and interferon lambda um, so this activity of nsp1 could be contributing to that there are probably a set of other proteins that also contribute to that.

I will note that in the coronavirus genome in addition to the enzymes that copy RNA and the structural proteins that make up the viral particle, there is a complete set of what are called accessory genes. These are genes that are generally unique to each virus and are thought to fine-tune the interaction between them. that virus and the environment of the host and vipo, so while we do not know the function of many of the accessory genes for copie2, the hypothesis is that, based on what we know about related coronaviruses, they are likely involved in modulating . the immune response as well and probably contributing to this phenotype, so now the last thing the virus needs to do is basically take those copied genomes that it has created and assemble them into new progeny variants that can then be released from the cell. a process that again is largely mysterious for coronaviruses and partly because I told you that the RNAs themselves are produced in these protective vesicles, they somehow need to be transported out of those vesicles and coated with the nuclear protein and directed to a new set. of vesicles that are derived from the er golgi intermediate compartment or the urgent compartment, these vesicles have been previously studded with the spike protein, the matrix protein, the envelope protein that will form the exterior of that new coronavirus particle and Therefore, interactions between the core protein-encoded genome and probably the matrix protein drive a budding reaction that causes the viral genome to become enveloped within these vesicles to form these new particles, so you see something like this where the genome would be absorbed, wrapped and then pinched so that these vesicles would now have a series of new virus particles inside them which would then be transported via an exocytosis-like mechanism to the surface of the cell where they would be transported. released to continue on and infect neighboring cells or be exhaled into the environment to infect a New Coast, a recent paper just published shows that Bureau e6 cells infected with Kobe 2.

In this scanning electron microscopy image, you can see that the viral particles that are these little dots here or here seem to be being released from these big bumps. of the infected cell, these are called filipodia and are filled with a molecule called actin. They are rich in actin. Actin is a component of the cellular cytoskeleton. It helps form the structure of the cell, but is also important for cell movement in things. and we know that other viruses like oxinia, which is a member of the poxvirus family, or ebola or others, are known to dramatically remodel actin or use actin to help them escape from cells, so one hypothesis here is that cov2 is also using actin to help create or um sequester these filopodia to push the viral particles away from a cell in which they are produced towards the extracellular environment or towards the neighboring cell and that will be an exciting area of ​​future research.

Well, let me summarize what I have covered today. which is basically the entire life cycle of the coronavirus inside a cell, we discussed how the first thing that happens after the virus is inside the body is that it has to locate cells that have the correct h2 receptor and that spice spike protein is join to that. receptor in a mechanism similar to a lock and key, but then it uses a second activity, it is a spring-loaded fusion machine to enter beyond the plasma membrane of the cell, uh, defense to enter the door, open the door fusing the viral membrane with the host plasma membrane by depositing its RNA genome into the cell, it then uses some viral tricks to translate 27 or more different viral proteins from the information of this single incoming RNA and that is through a combination of a giant polyprotein fusion that is processed and produces smaller subgenomic RNAs like these viral proteins. then it does the work of rearranging the inside of the cytoplasm of the infected cell, stretching the cell membranes to form these new spaces in the cytoplasm, these double-membrane vesicles where the RNA copying machine, which has proofreading activity, can then do many copies of the virus. genome and produce subgenomic RNAs to produce more viral proteins.

These proteins, the structural proteins, are then embedded in new vesicles in the cytoplasm and the newly copied RNA is transferred to those nuclear protein-coated vesicles and buds into them to form progeny variants before they are released from the cell, so that in this way the virus can start with a single incoming baryon, a single RNA, and release 100 to hundreds of progeny variants from that single infected cell in a remarkably short period of time, eight to ten hours for the average coronavirus . infection, okay, I'll end there and just acknowledge that I had a lot of help with image preparation while preparing this and other coronavirus-related talks from my lab ella hartinian divi ananda kumar jessica tucker michael lee and azra lari everyone helped We often use biorender to make these images.

I also had a wonderful collaboration with Scientific American to create several of the images from the movies you saw with editor Mark Foshetti and illustrator Veronica Hayes, so you can see those movies on this interactive website here and I also want to acknowledge that the data that was showing today are not really from my group, but from a whole collection of coronavirus researchers, scientists and doctors who have become coronavirologists to generate this type of information. We can learn how this virus works in an attempt to combat penta, so I hope I've left enough time to answer some questions.

Thank you very much, Professor Glenn Singer. There are a lot of questions Charles wants to know. The matrix and spike proteins, the only proteins that are found in the coronavirus envelope, yes, as far as I know, those are the only viral proteins that are there, it is very possible that some cellular proteins come with us, but if there are, there are cellular proteins there, we don't know any function they might have and it could just be that they are on that membrane when the virus is fighting, so normally you will identify the proteins that are there by collecting viruses and subduing them. to things like mass spectrometry, so there may be others, but these are the only functional ones and Jarek's question: can the virus selectively repress the translation of cellular genes without repressing the translation of its own RNAs?

Yeah, this is a great question and it kind of is, um, there's some conflicting information on that, I would say in the literature conceptually it would make more sense for viral RNAs to escape this translational repression and it's not clear that they do that at all, but let me tell you the thinking behind this, so one hypothesis is that um even If viral RNAs could be subject to this repression, it turns out that from coronavirus-related studies where you look at the composition of these membrane vesicles where RNA is produced, the nsp1 protein is not there, so it is possible that it is localized. during infection in other parts of the cytoplasm in areas where cellular RNAs are typically translated, but not very close to where viral RNAs are translated, so there might be some sort of location separation that helps youselectively dampen host gene expression there.

There is also some data to suggest that the viral leader sequence added to that end of viral RNAs may confer a translational advantage to those RNAs. This has been shown primarily in in vitro studies and a recent ribosome profiling study did not detect such translation. advantage, so I think the jury is still out to what extent this happens and then there is the idea that, along with maybe whether viral RNAs have a translational advantage, by selectively depleting some of the ribosomes or inactivating some of the ribosomes, you could bypass the rest of them anyway, they would be preferentially occupied by viral RNAs, so it's a complicated answer but a great question.

One of the students wants to know if the virus encodes its own protease and not the viral protease. be part of the polyprotein and, if so, how can it be spliced? Yes, another excellent question, is it part of the polyproteins, the viral proteases, there are two of them, they are actually part of the polyprotein and they function in both cis and trans, etc. what that means is that the protease as soon as it is translated by the ribosome and that domain of the protein folds, it can become active and self-cleave from the polyprotein so that it can be cleaved and then act on the rest of the polyprotein. junctions to cleave those polyproteins into individual unitary proteins, so you are right, it is in the polyprotein, but it can be cleaved from the polyprotein and act on the other junctions and Arbory ​​asks if the h2 receptor varies in abundance among the human population and does is that so? it is also found in bats and pangolins it is not restricted to the human population so ace2 can be found elsewhere if it varies in abundance in the human population it is an interesting question and I don't know the answer but I saw a good study done by ralph barrick's group a few weeks ago that studied the abundance of the ace2 receptor in lung tissue and what they found was that the highest concentration of ace2 was in the upper respiratory tract where most people get this coronavirus infection and the amount where the concentration of these two decreased in a gradient the lower the lung was and the deep lung had less, which could explain why fortunately not everyone is getting this fulminant deep lung infection, which is the most common form. dangerous and it is most often restricted to the upper respiratory tract, so the concentration of h2 in different tissues or different locations within the body can certainly vary and I would like to know how we know how viral genomes are successfully encoded in the nucleocapsid , why not the nucleocapsid? encapsulate host cell genomes or cellular RNAs, yes, another response we don't know why, probably a feature of the location of the nuclear capsid.

First, there may be fewer cellular RNAs because they have been used up in the process. of the posterior closure and the second nucleocapsid is within those compartments, as well as those replication compartments, so it is very possible that it is encoding the viral RNA there and together they are escaping and going to the place where viral morphogenesis or budding is located .happening, I think it is entirely possible that nucleic acid could bind to cellular RNA. The nucleocapsid is the most abundant viral protein produced in these cells and it is not surprising because it takes a lot of it to cover all the viral RNAs that are produced and played.

Additional regulatory roles that I didn't talk about beyond the morphogenesis component, but my hypothesis would be that because it is produced perhaps in the vicinity or is located in the vicinity of where those viral RNAs are amplified, it will preferentially encode those others. The student wants to know if there is a mutation in the proofreading enzyme that inactivates it, wouldn't it be beneficial for Kovi to allow it to evolve more quickly? Yeah, that's a fascinating concept, and to some extent, you know, the answer might be yes, but it turns out that most RNA viruses are thought to have hit the sweet spot for the maximum number of mutations they can. tolerate without exaggerating, since, since you are alluding to some mutation, it benefits the virus, right? so that the virus finds an inherent flexibility, if it finds a bottleneck somewhere, it counteracts an immune pressure somewhere, well, there could be some variant within the viral population that has arisen through this random mutational event that allows the virus avoids the air and so there are some beautiful early studies, actually with the polio virus, from Carly Kierkegaard's lab and Raul Medina's lab, that show that, in fact, you know it's really It is beneficial for the virus to have some level of mutation in a living environment, but it is a double-edged sword because there are too many mutants. the virus cannot tolerate and many of the antiviral drugs used against viruses or the best studied ones target the polymerase and several of them are of the class where they increase the error rate of the RNA extraction machine, maybe only two or three times, but that's enough to push that virus into what I described as a bug catastrophe.

They can't tolerate any more mutations and still have a fit population, so it's a very tight error rate, probably for each virus. Thank you so much. Much on behalf of the students, it has been very enlightening to hear you talk to us about this virus. Thank you for spending time with us. It's a pleasure. Thanks for inviting me.