Virology Lectures 2020 #3: Genomes and Genetics

Good afternoon, before we start talking about viral

genomes

. I want to go back to the last time. I want to show you an example of two of the assays we talked about last time used in the new corona virus in China, so a paper was published. This is actually in the bio file, which is a preprint server. Now we can put our manuscripts here, but before and during the submission process so that everyone can see them. This is fabulous, so it is downloaded from the biography file server. You can see the link down there. at the bottom right discovery of a new coronavirus associated with the recent outbreak of pneumonia in humans possible origin in bats, you know the authors were from a laboratory in Wuhan and what they did, remember the last time we talked about how they did lung lavages , Ronquillo alveolar lavage. you put a tube in the lung you put some liquid in and then you take it out so they took some of that from a patient and added it to the cells we have to turn off the lights because you need to see this, they add it to These are V Rose cells, a V ro cell is a green monkey kidney cell, right, they use them and they also use the human cell line, but this is the result of the V Rho cell, so they took monolayers of these cells, removed the medium and added. a little bit of this alveolar bronchial lavage from a patient, very valuable material, right, they let the viruses absorb the sample, they don't know if there is any virus in it and then they put medium in it and then, 24 hours later, they looked in the microscope, like this which on the left is the mock infected monolayer.

You always need a mock infected monolayer. You just put a little bit of PBS on it to make sure things don't die in your lab and then on the left is the 24 hour mock infected monolayer. It's lovely. beautiful and fiscal cells and on the right is the monolayer where they put the patient's fluid and we see cytopathic effects. You see what I'm telling you is real, it is not false because other people use the same terms that use the same methodology, so one day Then you look at the scope and can you imagine how excited they were when they saw this?

We have something that causes CPE, which is probably a virus and then they characterized it, so that's one use of CPE, that's what I said. you can use it to see if you have a new virus. Fortunately, this virus causes cpe, of course there are many that don't, but if you have a virus that doesn't cause cpe, you can do something else that they did. They had antibodies against other corona viruses because people have been studying them for many years. They took some of those antibodies that are directed against a protein that is widely conserved between different corona viruses and took a replica of this monolayer and added the antibodies to the cells.

Typically you have two permeable eyes, the cells with methanol to allow the antibodies to come in and then you let the antibody stick for a while and then you add a second antibody with some kind of color indicator and then that's what they see here on the left. So on the next slide, on the left is the monolayer without any antibodies, it's infected. You can see a little bit of CPE there and on the right they added an antibody using a C cross-reactive virus and a P antibody, NP, which is a virus. protein that has an antibody against it and you see red fluorescence, so they are using a second detector antibody that has a red that will fluoresce red under ultraviolet light and now you can see that viral protein is being produced in these cells, so Another one of those trials we talked about last time uses serology to study viruses, so it's a really good real-time example of using these techniques.

That's what they did first to figure out what was infecting these individuals and, of course, they pulled out the gene sequence. In that document they also have that because I said I'm going to try to find things that are happening with these new viruses that are related to what we talked about here in class so that it's more relevant to you. Okay, today I want. to talk about viral

genomes

I want to go over all the different types there are you are a different number than us we have one type of genome we have a double stranded DNA genome but viruses all have different types I want to talk about all the different types and what they do and how we study them how we use

genetics

to study them and there was a breakthrough in the 1950s in

virology

remember we identified viruses around 1900 people start studying them in the 1950s cell culture is finally developed and then in the 1950s, we discovered through experiments.

I'm going to show you now that the viral nucleic acid is the genetic code. I have to say that for many years people didn't think that nucleic acid did anything, they thought that proteins specified the genetic code for many reasons, one of them was that there are only four bases in DNA and they said how could four bases code for something and so slowly data emerged that said DNA was the genetic code a great experiment 1944 Avery MacLeod and McCarty working at Rockefeller demonstrated that you could take DNA from one bacteria put it in another and transfer the properties of that first bacteria which they called Transformation we still use that word today when we put DNA into cells, so that was the first evidence, of course, in 1953 the structure of DNA was solved. and that was important for understanding what a template for genetic information could look like, but in the 1950s experiments with a DNA virus and an RNA virus showed that in viruses the nucleic acid is the genetic information.

Tobacco mosaic virus had crystallized in 1935 and the person who made it Wendell Stanley thought viruses were infectious proteins because 95% of his purified virus was protein, 5% was phosphate, but he ignored it, he thought it was a contaminant, of course, phosphate is part of the RNA that we now know is important. he got the Nobel Prize for crystallizing tmv even though he was wrong about the protein being infectious, so that's tmv here on the right, an electron micrograph and I'll show you an experiment done by Frank Conrad that shows that T RNA MV is the gene material and on the left the Hershey-Chase experiment with a bacteriophage called T 4, which is a DNA virus showing that DNA is the genetic material.

The one on the left you probably heard in high school. I know high school teachers teach this. I've heard it before here in college, and if not, this will be the first time you've heard it. This is the famous kitchen blender experiment in which these two individuals she, she, she, she was the researcher, Martha Chase, her assistant in 1952, here she is. The blender can't be seen very well, I took a photo of it. It's in Cold Spring Harbor, which is where she worked on Long Island and there's a library with a display case and behind the glass, oh, there's a blender on top.

They have one of her blenders there, so if you're ever there, go to the library and check it out, she's great and used the blender in this experiment. What he did was he said, "Okay, what's the genetic material?", the protein or the DNA of this virus, so he took the phage and labeled the viral protein with radioactive sulfur by growing the phage in the presence of radioactive sulfur. and then added the phage to the bacteria for just five minutes to get the phage to adhere and then put the mixture in a blender to cut up the phages, so that's where the blender comes in and then it cuts up the phage and presumably the nucleic acid is going in and then he lets the bacteria incubate and says that in the progeny the phages do it.

He found radioactivity and he didn't find anything when he used radioactive sulfur to label the protein, so he said the protein must not be the genetic material. I'm referring to a separate experiment that you have here labeled a viral DNA with radioactive phosphorus which, of course, is part of it. of the nucleic acid in this case the DNA again experiment adsorbed the virus cuts it in the blender and the progeny phages you can find radioactive DNA proving that DNA is the genetic material two comments about this experiment first the idea of ​​putting radioactivity in a blender scares me a lot, but no one cared, people were pretty careless about security in the old days.

Well, nowadays we don't use much radioactivity, that's fine. The other is that we now know that there are not too many E. coli phages. but there are some where the phage actually enters the cell, the whole phage, which would have confused this experiment. You wouldn't have had a clear answer. He worked with t4, where the phage rests on the salad, expels the DNA. in the bacteria that it was a good choice he didn't know it at the time but in retrospect but often in retrospect decisions look good like this, okay, that's Hershey-Chase DNA, at least in the t4 genetic material, this reinforces the growing idea that DNA may be the genetic material and then we have a very interesting experiment on the effect of tobacco mosaic virus carried out by Franco and Konrad.

I had two different strains of tobacco mosaic virus which I represent here as green and red. He could distinguish the shell proteins in the same way as these. Viruses are built and we'll talk more about this on Monday, the RNA molecule is here, it's shown as a coil and there are individual proteins, these little guys outlined here, that bind to each other and to the RNA and form a helix to protect themselves. the RNA and covered it and that's the virus particle so he had a way to distinguish the proteins from his two different strains of tobacco mosaic wire so what he did was he disassembled these two different viruses to get the protein and RNA separately, purified them and then took the green RNA and mixed it with red proteins to get a hybrid particle, so he discovered early on that if you purified the RNA and the protein and then mixed them again, they would become assemble and form a virus particle.

A very useful finding, so he mixed the proteins with the RNA Green RNA with red protein Red RNA with green protein and then used them to infect tobacco leaves, you let the infection go and harvested what came out and discovered that when you use an RNA green you have green proteins even though you assembled the green RNA with a red protein what came out is a completely green green virus so the RNA specifies the protein The RNA is the genetic material and the same with the other the red RNA complex with the Green protein ultimately gives rise to red viruses, so two really important experiments show that nucleic acid is the genetic material.

Now, of course, we have no doubt about this, but it's funny to look back and see that people really weren't sure about this, so nucleic acid is the genetic material. However, as we have studied viruses, we find all kinds of viruses, billions and billions of them, yet even though there are so many, we can narrow them down like we can put them into seven different groups and that's what I mean. We'll talk a little bit more because I think this is a very powerful rubric for organizing the genome. Just remember train number seven. If I ever ask you how many different types of viral genome there are, the answer is seven, very simple, and let's review. this and why one of the reasons behind this is that all viruses or viral genomes have to produce messenger RNA that can be translated by the host's translation system, because viruses do not encode their own translation system, so They have to produce an mRNA. that is supported there, therefore this is a strict rule, no one has found an exception.

No virus encodes a complete translation system, so they all have to produce mRNA that the cell can translate, so this gives you a common theme between all the viruses you can use to create. the rubric that I want to show you to help you organize these seven different types of genomes so that it's a ribosome, by the way, it's not a turkey, some people think it's a turkey every year, it won't be next time the Day comes Thanksgiving and you won't eat turkeys. ribosomes, so this is what I call the Baltimore scheme in my admiration for things on earth, we have viruses, we have the following plaque assays and then we have the Baltimore scheme.

I think this is amazing and I'm going to convince you that this is done. by a fellow named David Baltimore who's here and I did my postdoc with him, so I have a lot of respect for him and he's a great scientist who came up with this in the 1970s as a way of thinking about virus genomes and what he said it was all viruses. it needs to make mRNA, so let's put this in the center, let's put mRNA in the center and then see how the different viruses arrive. When he did that, he was able to put them into seven different classes, so they're all here on the outside of the class numbers start with one, so one is double-stranded DNA from which mRNA can be formed, another number two It is single-stranded DNA from which double-stranded DNA and then mRNA are produced.

Here you must remember some things that are very important. DNA to produce RNA, mRNA from DNA has to be double-stranded, always single-stranded DNA is not atemplate for mRNA synthesis nor for transcription, the same thing, so if you have a single-stranded D, you first have to go to double-stranded DNA before. you can make mRNA, so class three is double-stranded RNA which is both a messenger RNA and an anti-messenger RNA strand and interestingly, that strand of mRNA in the duplex cannot be translated because it is blocked by the other strand, so you actually have to make mRNA from a double stranded RNA, then we have group four where we have a genome that we call RNA plus.

We will talk about some definitions in a moment and that in some cases they can be translated directly, but to expand it I would go through a - RNA we have class five, which is - RNA, it cannot be translated, it is the opposite sense of the mRNA, for what needs to be copied and Class six is ​​here an unusual class of virus where the virus particle contains mRNA or RNA + and it is I copied the DNA which has become double stranded and from the DNA the mRNA is produced, which is in the outside. Here all these classes are what is in the virus particle and finally class seven, which we did not know at the time they were both created.

Its scheme consists of double-stranded DNA with gaps. This is exemplified by the Hypatia viruses or the hepatitis B viruses, which we will talk about and this is different because a DNA with gaps cannot be transcribed, it must first be repaired into double-stranded DNA and then from there it is created mRNA, so that's the classification screen, the Baltimore scheme. Now, a couple of definitions before we talk about this, a few more. very important, so the mrna, of course, is what is translated by the ribosomes and we call it positive strand just by convention, it has nothing to do with electrical charge or anything, it's just that at one point years ago someone said, Let's say mrna is the positive strand and we're stuck with that, okay, mRNA is the positive strand, so DNA of the same polarity, it's a double strand of DNA, would be the positive strand of DNA, but, Of course, if you want to make mRNA from double-stranded DNA, you have to copy it. the negative strand to make the positive strand okay and the RNA and DNA complements of the positive strands are called negative strands, only the complement again has nothing to do with the negatively charged strands and that is what I will use to talk about these more and less.

When I say more it means the same polarity as the mRNA and less is the complement. Now when I say plus I don't always mean mRNA because, as you'll see, not all Plus RNA is actually translated, we'll talk about that in a moment, but plus simply means that it has the same polarity as mRNA, whether it's translate or not, it's another step, so that's the Baltimore scheme and that's the terminology to explain all the pros and cons here, so for example the reason for the positive polarity could, in principle, be mRNA less. it's the opposite strand and of course double stranded DNA is more minus double stranded RNA is plus minus so that's the bolt and we're skiing the beauty of this is if I tell you a viral genome you can trace the basic steps. that are needed to make mRNA, I can rule you out any of these seven classes, and if you understand this classification system, you will know how to get to the core, which is mRNA.

For example, if I say how you get mRNA from double stranded DNA, you know that or this is something you have to memorize. I guess you have to memorize that double stranded DNA can be transcribed to produce mRNA, that is the process of making mRNA transcribed correctly, you can make it from double stranded DNA, so if I say what about a single stranded DNA virus? And we have those. You must remember that single-stranded DNA is not a template for transcription. It must first be made double-stranded. Only double-stranded DNA among DNA viruses is a model for transcription.

Neither single-stranded DNA nor double-stranded DNA can be transcribed, they must be repaired. RNA is not a translation template because it has the wrong polarity. There is no open reading frame for translation alone, plus mRNA can be translated, but again, not all. RNA is mRNA and finally a double-stranded RNA, although there is a positive strand in the double-stranded RNA duplex, the ribosomes cannot access it, that is something you have to remember, so to produce mRNA from a virus of double-stranded RNA, you actually have to copy the negative sign. strand and making a separate positive strand seems like a waste, but that's how it is because ribosomes can't translate double-stranded RNA, so that's the Baltimore system, we have seven kinds of viral genomes hollow double-stranded DNA double-stranded DNA and single-stranded DNA.

Stranded DNA, those are the DNAs that we call DNA viruses, so when I say for the rest of this class that DNA virus it means that it has a genome of one of those three types and then among the RNA viruses there is a more diverse one , we have double. Single-stranded RNA and then three classes of single-stranded RNA plus less and then plus with a DNA in between, it's an additional version of the plus class and these are all different classes with different pathways to the mRNA, so it's very simple, you know, the only. Single-stranded DNA, as you'll see, is actually more or less, they're both packaged into the virus particles, it doesn't matter, so we don't have an extra class of single-stranded DNA viruses, so let's see how that resonates. . you, the first question is why the mRNA is placed in the center because all virus particles have mRNA.

There is no reason why all genomes are sir because mRNA must be made from ancient genomes or because Baltimore studied mRNA. Well, it looks like we've saturated what we have here. 92% of you got the correct answer because mRNA must be made from ancient viral genomes, that is why virus particles do not contain mRNA. That's what Baltimore's scheme tells us. What's on the outside of those classes. That's what's in the viral particle. Single-stranded DNA, single-stranded DNA, etc., so if a virus has double-stranded DNA and cannot have mRNA and all our genomes are mRNA for the same reasons, they are not now, even though there are only seven classes of structurally viral nucleic acids. quite diverse and here you can see just a list of the ones you will find and these are the icons that I use for them on all my slides so they can be linear or circular so DNA r and i could be linear or circular we have here at the top a linear double-stranded DNA we have a circular double-stranded DNA we even have circular RNAs as you will see genomes can be segmented into pieces, meaning that a virus particle would have more than one piece of nucleic acid so the virus genome of influenza consists of eight pieces of single-stranded RNA.

Trent's RNA could have spaces, as we said, single-stranded more or less it can be ambi sense, meaning it has both plus and minus components that we will see today, it can be double. -stranded cannot have proteins attached to it, the ends can be covalently linked, so if you have a double-stranded DNA, this one in the middle here says labeled double-stranded, there are five and three prime ends, it's a chemical designation to have three ends at the base, they can sometimes be linked covalently, so the five leading ends can be joined via a bond to the three leading end giving you this circular end, which happens with some viruses and sometimes Sometimes there is also, in addition to a protein attached to the genomes, the RNA, so all different configurations, but what that means we will see as we talk about how these viruses replicate, but the main point is that they all replicate.

They divide into seven classes, no matter what the configuration of this genome is, you can put them in one of those seven classes and as I said to this day I don't know of any other configuration, it may be out there somewhere and an interesting exercise It's thinking about what other genome configurations there might be, but we haven't found them yet, so first of all, let's look a little bit about these genomes and what they do. Why are there seven classes and even beyond that? Why do we have all these different configurations? As I said the other day, it's hard to answer the why questions, so I'd put it differently.

In some ways, what is the function of genome diversity and I can answer a little bit of that question, but not all of it, and the first part I can answer is why do we have viruses with both DNA and RNA based genomes, because you already know everything else on the planet. it is based on DNA, as far as we know there are no organisms with RNA genomes, why are there viruses or what is the function of that? Well, I can tell you that the way we think life evolved on this planet is that many billions of years ago there was probably an RNA world first where organisms were based on RNA, not DNA, and these were pretty organisms. simple, they initially started out as self-replicating RNA molecules in the waters of the earth, the initial earth and people now study this and can get it. certain types of RNA molecules copied themselves and reproduced, so we think the early Earth had these eventually became simple cells, but there's a lot you can do with RNA because it's not very long, as you'll see in one moment.

Genomes are not long, so our base, our genome, is 3.2 billion base pairs. There could never be an RNA that long, so we think that at some point these RNA genomes change to DNA and we think that happened by producing a protein called reverse transcriptase. We will talk about it later, which exists to this day. Once you have a DNA genome, you can have larger organisms because they can have very large DNA. DNA is more stable, etc., so we think that was the driving force for the evolution of DNA. Probably a vote arose randomly initially from DNA, but then it had an advantage and that's why it persisted, but today RNA viruses are a kind of relic of this RNA world and in them we can see signs of an RNA world, so our new world, for example.

In any case, it would initially have no protein, so the RNA would also have to have a catalytic function. The ribosome, for example, most of the catalysis is carried out by RNA. You can remove the protein from a ribosome, it will still make proteins and there are ribosomes that have discovered autoenzymes that are integrated into RNA, there are RNAs, for example, that can cut themselves, they can split in two, they can split intermediate sequences, so that there is evidence that there were enzymatic activities that are compatible with an RNA world, so RNA viruses are relics. Actually, than they used to be, they are still evolutionarily competitive, so they exist later in the course, we will talk about a very interesting virus called viroid, which are small highly folded RNA molecules shown here is an example to the right and these.

They are mainly plant pathogens, there is one exception, there is a human pathogen that we will talk about, but they are interesting because they do not encode any proteins and they can enter a plant and reproduce, produce more of themselves, it does not have a protein coat. and they can spread from plant to plant and cause disease in plants without encoding any proteins, so they are probably a mineral version of what existed in an RNA world before cells existed and I think my view of evolution is that as ROS cells these self-replicating RNAs entered the cells and were selected to be able to multiply in one cell instead of that side.

I think it was more efficient to replicate in the cell, but when we talk about evolution, we'll get into that a little more. I can tell you what the reason or function of RNA and DNA genomes is because RNA viruses are relics of what used to exist and are still evolutionarily competitive; However, why do we have linear, circular, segmented, double-stranded, single-stranded? Genomes of different polarities. I can not tell you. I think the only thing we can say is that they have ecological niches where they all function and survive from my human point of view. All viruses should be more stranded RNA viruses because that makes more sense.

The virus has an RNA genome that is highly mutable and adaptable, as you will see, it is more RNA, as soon as it enters the cell, the protein is made, it has to do anything else and you will see, as we go through the cycles of replication, than the other types of genomes, the other seven kinds after all kinds of gymnastics to get to the mRNA, so I think that, from my human-centered point of view, more stranded RNA viruses should dominate, but they don't, there are all kinds of other configurations, so obviously their niches for each. of them, so now I can't tell you the function of many of these genome configurations.

I suggest you memorize them, although it is not necessary because when you have an exam you can have a sheet of paper with things written on it and you can draw the baltimore diagram but that's how it is, it takes up a lot of space and it is very easy to remember, but every year almost everyone has the baltimore scheme on their cheat sheets, they're not cheating because i let you bring it, but that's what everyone says right and you. I'm always amazed at how artistic people get with these andThey have the Baltimore scheme sandwich, but you have to remember that because, like I said, if you know this, you'll be able to tell me.

If I give you a genome, you know how mRNA is produced and how it is copied to produce more genomes, so here, for example, if you start with a positive RNA, you know that to produce more positive RNA you have to go through a negative intermediate , so whatever the virus enters a cell, it is not enough to produce many virus particles, you have to amplify the genome and from this you can also find out how that happens. Now, in this course, we will mainly talk about us, a handful of viruses. This is not a course focused on the type of virus.

We will have examples of each of these classes and they are written here and I am not asking you to memorize them, but sometimes you will see the name of a virus and if you are familiar with it, it will make more sense to you, so, for example, the DNA plus viruses, the parvoviruses, are one of the families that we will talk about in a moment, the adenoviruses, the herpes viruses, the hepatitis B for the gap viruses, the reowww for the doubles. stranded and this one will learn because these are some of these gastroenteritis agents, then we have the influenza viruses as prototypes for the RNA viruses for the RNA poliovirus plus, but of course the corona virus that we are dealing with now Worldwide. like a positive strand RNA virus, so we'll talk about that as well and finally, retroviruses are examples of positive RNA viruses that go through a DNA intermediate, so I would try to familiarize myself with some of these right here.

Slide just a handful of different viruses. Yes, does the nature of the genome make any virus more dangerous than others? So I would say that there are many DNA viruses that are pathogenic, but there are many more pathogens that are RNA viruses, so they are RNA viruses. Eukaryotic organisms dominate. I think that's because they're more mutable and can vary and occupy more niches, so I think having an RNA genome is probably better and you'll get more pathways when you look at pathogens later in this course, you'll see that most of them are virus, so whenever a new virus emerges, it is usually an RNA virus.

Let's look at the new emerging viruses in recent years, so we have corona virus several times, we have Ebola. virus which is an RNA virus, we have nepa and Hendra, which are RNA viruses, we had, of course, HIV, which is an RNA virus, and now if you go back far enough, thousands of years ago, smallpox appears, which is a DNA virus. viruses, but you get the idea, I think RNA viruses rule well, so what do genomes encode? I always keep saying they don't have a translation system but what do they encode and what don't they encode so they have gene products and signals in them for protein synthesis so every virus produces an mRNA that can be translated and most viruses don't encode components of the translation system.

In fact, when I started teaching this course it was before we knew about giant viruses. I always used to say that no virus encodes any part. of the protein synthetic machinery now that these giant viruses with huge genomes have been discovered and it turns out that they can encode components of the translation system such as T RNA and initiation factors, etc., but never the complete translation system, they encode proteins to replicate The viral genome to assemble the genome into a capsid and package it, they encode proteins that time the replication cycle, as you can see, when we talk about the larger DNA viruses, replication is divided into phases to coordinate the activities and their proteins, which makes it very interesting.

What is encoded in each viral genome is at least one protein to modulate the host's defenses, as I told you on the first day, we have a great immune system, if viruses did not encode antagonists of that immune system, they would all be eliminated because our system immune does not work. It's surprising, but every virus has at least one protein that it antagonizes: it's innate or adaptive immunity and we'll talk about some of them and they're encoded in the genomes and then their gene products that allow viruses to leave cells and spread to other cells. . In the south there is a giant virus that was discovered a couple of years ago and I want to point out that now giant viruses are a thing in themselves, they have a separate taxonomic classification because it turns out that they have several genes that they share and these are viruses. with huge genomes, I'll show you some examples in a minute and now there are dozens and dozens of examples of these affecting many different types of organisms and when they were first discovered they were unusual because they had really big genomes at first. and large particles.

I showed them a photo of my virus on the first day, much larger than anything we'd ever had, and secondly, they encoded parts of the translation system that we'd never seen before, except for their tRNA genes and some bacteria phages. They are unusual, this is a pan virus, which is magnificent, this is an electron micrograph of the particles and they are just beautiful, they are not blue of course, but they look really unusual, they have a round capsid on a sort of tail that stands out. I don't know what purpose the genome of this virus encodes everything except the ribosome for protein synthesis.

If you know anything about protein synthesis, you need aminoacyl trna synthetases to put the amino acids into the trna. Right, you need T-RNA. This virus genome encodes 70 of them. You need initiation and elongation proteins. This virus encodes all the genes for the maturation of T-RNA and mRNA, everything except the ribosome, and in fact I say it's just missing the ribosome because that is the virus that was discovered. Some of the authors were French and that's how they put it because if you know how you would say this in French, it's different from English, right, it's just missing the ribosome, so it's a little bit French, so it's true that this virus did it.

It has everything and that is notable and why. It does, it's a great question, why does the virus, you know, have to encode everything except the ribosome when the cell provides all of those components anyway? True, many hypotheses, maybe the virus needs to have more translation than the cell can provide, so it produces additional components maybe it has a specific amino acid triplet code usage, so we don't know the answers, but it is very interesting that it has so many things encoded that that is what is encoded in the viral genome, some of which is encoded that is not in the genomes. we don't have the complete protein synthesis apparatus in any viral genome, even the 2-pam virus still lacks the ribosome, we could find a virus someday with it, it would still be a virus because there are many other things that the cell provides that are necessary and not in the viral genomes, there are no genes that encode proteins involved in membrane biosynthesis.

I used to have neither jeans nor metabolic genes on this slide, but now they have found photosynthesis genes, they have found complete photosynthesis systems in some oceanic viruses so that they can produce more energy in the cell for their reproduction, especially the viruses that are found in sunlit waters infecting sunlit water hosts, there are no centromeres or telomeres, so you know our DNA is organized and we have structures called telomeres at the ends, we have centromeres that are not. They are normally found encoded in viral genomes, the proteins that make them up, but as I always like to say, we probably haven't found these genes because every time we sequence a new giant virus genome, 90% of the genes are new and not We recognize them.

What its proteins encode we have never seen before, so someday we might find more and more, of course, if a viral genome wants to at some point encode everything, then it is no longer a virus, because a virus needs to enter a cell. that's our definition of a virus, something that would have genes that code for everything that needs to be done would be a cell, sorry, I suspect we'll get close, but we'll never get there, so here's a couple of tables in a row to show you. the largest and smallest viral genomes just to give you an idea of ​​a size range.

The largest viral genomes known and the largest ever discovered is the Pandora Salinas virus, which has two million four hundred seventy-three thousand bases of double-stranded DNA which is incredibly huge. I can't emphasize how big the genome of Haemophilus influenzae is here which is a bacteria a free living bacteria the missing genome is 1.8 million base pairs it's smaller than this viral you know down here at the bottom here's a bacterial genome that this bacteria needs? It lives inside another bacteria, so it can't do everything on its own, but it can still live on its one hundred and twenty twelve thousand base pairs of DNA, so these viruses are huge, so in the middle column is the length and to the right the number. of proteins, this Pandora virus encodes 2500 proteins and there is another Pandora virus which is 1.9 to be fair, now there are like six different Pandora viruses that would take up most of this table and I have left them out because they are actually all the biggest ones , but I want to show you some other viruses that are big, there are two Pam viruses that encode the entire protein synthesis apparatus except the ribosome and then we have a virus called boto South Hands is a virus that infects protists in the oceans and then the mega virus mom.

Mimi virus virus okay so these are the French again so they found the Mimi virus first and Mimi means mimicking microbe and then they thought they would be clever and they found mom and muumuu and so on and you can name your virus so they stay, but when you go to meetings people make fun of the French for doing this and they laugh because they think it's funny and then all the way here in the cafeteria Rouen Bergens virus another virus infecting protists in the ocean 600,000 now to give you some perspective. We thought the largest virus genome was the smallpox viruses, which were about 300,000 base pairs, so it was a big jump when we found the Mimi virus. 1.1 million people were amazed and the more you look, the more you find them and many of these are just found.

By taking water samples and sequencing all the nucleic acid you can find, or even many people take the water and put it in cultures of amoebae. Many of these viruses infect amoebae and use amoebae as reading and you can isolate. I know a story. from a french biologist, smart jean-michel read that he told me that he was in australia at a meeting and he looked out the window and saw a pond, he took his water bottle, he took some water from the pond and he brought it back to france and They isolated him. I think one of these Pandora viruses came from a muddy pond in Australia, so if you're willing to look everywhere, you can find new ones.

Those DNA viruses, there are no RNA viruses in that larger graph because RNA viruses are. It's so far behind in size that it fits, but I wanted to show you the largest RNA virus genomes that we know of. The biggest one so far is this 41.1 KB 41,000 bases of these are single stranded positive strand RNA genome and this is a planarian virus they just shredded it into the sequence of the genomes and found viral sequences in them and then there's another one of 35.9 KB which is a virus please, iya, it's a mollusk. If you do any neuroscience, you know this is commonly used in neuroscience research and then there's a ball python virus that had virus 33.5, then the corona virus that we've talked about a little bit, the one that's circulating in China and other SARS and Mercer about 29,000 bass because we used to think bass were the biggest. but they are no longer the largest, they are larger and again they are found accidentally by people who simply sequence nucleic acids from different organisms, so there may be larger ones, but they never reach a million bases because RNA cannot sustain a larger size.

The reasons are complicated, but RNA polymerase is that RNAse copy makes a lot of mistakes and never corrects itself, so because of the mutation rates, the RNAs can't get very long, they have too many mutations, these viruses are the ones that have the Larger RNA. The viruses are corona viruses, they encode an error-correcting protein that allows them to become so large and most RNA viruses don't, which is why they are much smaller. I believe the average size of an RNA virus genome is about eleven thousand bases in length. They are the smallest known viral genomes, this is quite interesting, so the smallest would be a viroid that has one hundred and twenty nucleotides of RNA, that is, it is naked, it does not encode any protein, but when it is introduced into a plant it replicates , makes the plant sick, we will talk about this and at the end of the course, that is what we summarize, they are what we call satellite viruses and these will only replicate in a cell infected with another virus, the replicas will be called viruses with the ability to replicate, so they have small genomes that do not encode any protein and then the first genome that encodes a protein is that of hepatitis Delta.

This is a small RNA that encodes aprotein 1,700 bases long and will only replicate in cells infected with hepatitis B. The virus and people get infected with it, so if you have hepatitis B, which about 350 million people worldwide have infections with the virus of hepatitis B, some of them also have the Delta virus and it seems to make the infection more severe and then we get a little bit larger circle virus or single-stranded DNA virus with two proteins encoded in the genomes and they they are also single stranded DNA most of us have these viruses in us the blood supply is full of these viruses they don't seem to do anything and every pint of blood we have contains these viruses so we can't not use blood because they are present, we wouldn't have a blood supply.

The Gemini virus is a plant virus with proteins, the 7 quite small proteins of hepatitis B, which is a DNA. These smaller viruses are RNA viruses. Here we have some DNA viruses and then at the bottom there are other RNA viruses with fairly small genomes, so RNA viruses are usually small, but I'm surprised that this one here with less than 20 bases is notable, but I think it's a relic of an RNA world. Well, our next question is what information can be encoded in a viral genome. Genetic products that catalyze membrane biosynthesis. Genetic products that catalyze energy production.

Complete protein synthesis systems. Centromeres or telomeres. Enzymes to replicate the viral genome. Well, how can we? Most of you got the last one there, but not a large percentage of enzymes to replicate the viral genome of all these things? That's the only right thing. Genetic products that catalyze membrane biosynthesis. It's not on one of the slides. Energy production. I guess it's true. Yeah, so 15 percent of you got that and that makes up the rest of the 77. Yeah, I told you there are some photosynthetic genes. They are right. I have to change this next year. Okay, so those of you got B or C right, no. right, complete protein synthesis systems, they have everything but the ribosome, so that's part of it, there are no centromeres, they are telomeres, so let's talk about some viral DNA genomes and some examples, DNA viruses predominate in bacteria , they do not predominate in us, as you will see there very few RNA viruses from bacteria, their reasons for that and when we talk about evolution, we will talk about that, many DNA viruses, of course, emulate the host because we, the host of all viruses, we have DNA, but almost all viral genomes are not like cellular chromosomes.

So the chromosomes in us are chromatin eyes, they are wrapped in histones and wrapped around them in a very specific way and most of the viral genomes in the particle are not like that and there are a lot of different things that happen in DNA synthesis that don't happen in the cell and you'll see them when we talk about that specifically here are some examples of double stranded DNA genomes. First, again double-stranded DNA is very simple because it can be transcribed to produce mRNA which would then be converted into proteins translated into proteins. Of course you have to always replicate DNA to produce more DNA to put into these particles and that happens using minus one protein encoded by the virus.

They are the smallest viruses, as you will see, they do not encode much of the DNA synthesis apparatus, but they do have a protein. which is used to hijack the host and here I have the DNA genomes, the double-stranded DNA genome is divided into two parts, we have DNA genomes that are copied by the host's DNA polymerase, as you know, we have DNA polymerases that replicate our genes and the smallest ones. of the viral genomes do not have enough coding capacity to ultimately encode a polymerase, so they are copied by the host's DNA polymerase. An example of the polyomavirus is that there are five thousand bases of double-stranded circular DNA, which we will talk about quite a bit. in this course they do not code for a polymerase, but they do code for a protein that hijacks cellular polymerase, as you will see, larger double stranded DNA genomes can code for DNA polymerase, as the ones they code for include adenoviruses and you can see the size.

The differences here are herpes viruses and poxviruses. These are obviously not drawn to scale, but you look at the size and you can see that they get bigger. These used to be what we thought were the biggest viruses, the smallpox viruses, but now the giant viruses are much bigger. so double-stranded that they are copied or not by the host's polymerase some examples of viruses with double-stranded DNA genome Given there are no viruses that cause a variety of human infections respiratory gastrointestinal infections herpes viruses that infect almost everyone on the planet and we will Speak about them in some detail, then we have the papillomaviruses in the polyomavirus.

Papillomaviruses cause warts. There were hundreds of more than 150 different types of papilloma. If you have ever had a wart on your hand or feet, it is caused by these viruses if you are an athlete. and you walk through the locker room you will get the papillomavirus pop on the bottom of your feet because they come off like your skin, but that's how they spread and you will pick them up in the cracks on the bottom of your feet. You'll get warts on the bottom of your feet, some of them are sexually transmitted and cause cancer, and we'll talk about polyomavirus being another virus that most of us are infected with and apparently it has no consequences unless you're immunosuppressed and, finally, poxviruses.

Among them are quite large DNA viruses, the smallpox agent, and there are also many others that people are using for gene therapy and we will talk about that later. I put here a concise virus that is the largest physical size particle that we know of from teeth. of iris that I became this came out of an ice core in Siberia it's thirty-five thousand years old, it's not like that, it doesn't have the largest genome, so it wasn't even on that table, but it's five times bigger than the poxviruses that I tried to approximate. that here so you can see how there are midgets, everything else is fine, so we have another kind of Baltimore class with double stranded DNA with gaps.

Why it has a gap, you'll find out when we talk about reverse transcription, but the way the genome looks. It is actually a circle, it is partially double stranded, as you can see here, it has a space where there is only one strand, the negative strand, it also has a protein attached to one end of the DNA and a piece of RNA on the other, and like I said we'll figure out how this happens, but that can't be transcribed into mRNA, it has to be double stranded so you have to remove the protein, remove the RNA, repair it, that seems to happen by the host cell and then that DNA is can transcribe. to produce mRNA and that cycle is shown here, the gap in the DNA is repaired and can then be transcribed into mRNA which can be converted into proteins.

Now these viruses are unusual, they also encode a reverse transcriptase, an enzyme that takes RNA and makes a copy of DNA. and so what happens here? DNA is transcribed. Proteins are produced. One of the proteins is a reverse transcriptase, an enzyme that copies some of the mRNA and makes a copy of the DNA. And it is during reverse transcription that the spaces and these proteins are produced in the RNA. attached to the genome the hepatitis B virus is an example of this, this is a serious human pathogen, it is transmitted sexually and blood, blood transfusions are transmitted sexually and the problem with it, there are 350 million people infected and that long term infection can give you liver cancer and that's why we're trying to prevent this we have a vaccine we have some antiviral approaches very well single stranded DNA genomes another kind of Baltimore these viruses can package the negative or positive strand and again that doesn't can be transcribed it has to be double stranded the host cell when a single stranded DNA enters the nucleus the host cell repairs it makes it double stranded and that can then be transcribed to produce proteins that will then produce new virus particles, we will talk about how all of these are replicate individually later there are two examples on this slide, one is a virus with a fairly small circular single stranded DNA genome, these are the Sirico viridi, including the TT virus, which again infects most of us with no apparent consequences to the right is the parvovirus they are single-stranded linear genomes with an unusual structure shown here there is a human parvovirus called b19 that causes the disease this is one of the childhood rash diseases, let's see if I can eliminate them from measles chickenpox mumps rubella and v disease would be this and measles mumps rubella chickenpox we have vaccines that prevent them all you don't have one for this but if you have a dog or a cat you know that there are canine and feline parvoviruses that can kill your pets so we have vaccines to prevent them one year when I was teaching this Winfrey okra course I don't watch the show but someone told me that her dogs died from parvovirus infection why because she didn't vaccinate them if they would have been fine then you should vaccinate your pets okay.

One question, our next question. Which DNA genome upon entering the cell can be immediately copied into mRNA? all of the above is fine, how did we do most of them? You got double stranded DNA, which is correct, only double stranded DNA can be transcribed, nothing else, gap, no, circular, no, linear, no, okay, RNA genomes, like I said, RNA viruses dominate the fear of eukaryotic viruses, the fear of viruses is All the virus genomes that we know of are very rare and bacterial. The key point here is that cells do not have RNA polymerases, they cannot copy RNA genomes.

RNA-dependent RNA polymerases are DRPs and are unique to viruses, so all RNA virus genomes, except the small ones that encode nothing of course, encode Rd RP and these a-- polymerases produce mRNA and replicate the genome to produce more, so this is an important difference between RNA viruses and DNA viruses. Let's double review some of the classes. double stranded RNA virus, as I said, the double stranded RNA genome cannot be translated even though it has a positive mRNA and must be copied into mRNA which can then be translated or replicated to create new genomes. An example is a virus called rotavirus which is a cause of human gastroenteritis and these viruses have segmented genomes, the virus particle is in pieces, between one and twelve segments, depending on the type of Rio virus and they are all packaged in the virus that carries them. needs all. to start an infection, but they are all in one virus particle, single-stranded RNA with sense Plus, the strategy that makes the most sense to me, the viral genome is more stranded, it enters the cells and is immediately translated into protein, it is not necessary replicate it.

Initially, the viral proteins can then build capsids, they can also move on to one of those proteins, it has to be the RNA polymerase, which will copy the plus through a minus intermediate to produce more genomes. We have many viruses with extra. sense RNA and corona viruses there we have been talking about flavy virus yellow fever virus Zika virus West Nile virus Pokorny virus Poliovirus Rhino virus and toga virus many of the encephalitis viruses we hear about encephalitis viruses transmitted by mosquitoes are toca visors I can see that the genomes have different lengths and different configurations, some of them are probably toothed and plugged in the corners.

It is unusual for them to have a protein and we will touch on some of these as we look at individual viruses. Some examples in the corner I have. I already told you that polio and rhinos, tripes are also an additional chain that I have not mentioned much, although I did talk about whales that become infected with them, these also cause human gastroenteritis and are mainly responsible for cruise ships returning when they have an outbreak in them, we'll talk about that later are the corona viruses, the three SARS MERS epidemic strains in their new and then our Flavie viruses and the toga virus, for example, the equine encephalitis virus and the rubella, one of the childhood rash viruses is a The toga virus plus RNA viruses can also have a DNA intermediate which is a separate class from Baltimore these have positive sense with the DNA these are the retro viridi there is a family and that family has two human pathogens HIV of course one two types one and two we will dedicate a Later we talked about HIV and AIDS and the human lymphatic virus, another virus that causes cancer, and these viruses are unusual because the RNA Plus in the particle is not translates when it enters the cell, it is copied into a copy of DNA that is then integrated into the cell. chromosome of the host and there it is transcribed into mRNA which then produces proteins and eventually some of those RNAs are packaged into new virus particles.

We will cover this replication cycle in great detail, but again the genome is a plus RNA, but it is not mRNA because it is not translated that when it enters the sound we also have RNA viruses with negative polarity, so the negative RNA when it enters the cell cannot translate, so the only way toTo make proteins is to make mRNA from it, but the cell cannot do that. that cells have no way to produce mRNA from a - RNA, so the viral particle must contain the enzyme RNA polymerase in the particle, so it is different from positive-strand RNA viruses because the positive strand can enter the cell and translate, so you don't need an enzyme in the particle of a positive strand RNA virus, but for these viruses you need to have the enzyme in the particle, so some of them are segmented, some of them, such as influenza viruses, some of the individual Mearns molecules, such as the measles virus and the rabies virus, and examples of these viruses include measles and mumps, rabies, sensation viruses Ebola and Marburg, which are quite large compared to these others, are drawn roughly to scale with each other, of course, the influenza viruses and the latest virus, and I have to tell you this.

The Book Fever was written in the 70s about the first Lassa outbreak in Africa. Some of this takes place in Columbia. It's very interesting, but I read this. I had just graduated from college. I didn't know what I wanted to do with my life and I read. This and I decided I wanted to be a virologist, so I have it here in recognition that when you have a genome segmented into pieces you can do something that no virus can do. You can reaffirm that you can have two influenza viruses that are genetically diverse. When you infect a cell and the viruses that come out have mixed the genomes of the two input particles, we call that rearrangement.

You can see the red and blue genomes here and then we have the parental viruses and here is one with a red segment of a virus and the rest. of the others that is very special and allows viruses to experience a variation that is above normal mutation rates and we will talk about this they are ambi sense genomes like the arena virus is the last virus that I liked so much and here the RNA has both Components plus and minus, so the green part here is plus and minus is negative and these viruses have a polymerase in the particle to copy this into an mRNA when it enters the cell, that's what MB sense means, now It has both RNAs.

I've been drawing lines for you, but actually in virus particles they're not just simple lines, they have complex structures, they have secondary structures shown here, caused by base pairing between complementary regions, there are local base pairings, there are long-term interactions, so for the red can base pair here and we know that if we solve the structures of these RNAs we get things like this shown in panel D, where they are very different from the linear diagrams that we We draw often, so I want you to keep that in mind. I do this for simplicity, but in reality all RNAs are folded inside the virus and inside the cell.

We have one last test here: which statement about viral RNA genomes is correct and can also be translated to produce proteins. A double strand can be directly translated to produce. Plus protein RNA virus replication cycles do not require a negative strand Intermediate RNA genomes can be copied by the host cell RNA-dependent RNA polymerase is all of the above, well let's see how we did it most of you got genomes from positive RNAs that can be translated to make the viral protein maybe you are correct some of you said that the double strand can be translated directly, they can't, you can't access the positive strand, it has to be converted into an mRNA, the cycles Positive RNA virus replication does not require an intermediate negative strand, right?

We need to do: to get more positive strands, there is no host cell RNA polymerase, so that is not true. The development of the plaque assay allowed us to do

genetics

with animal viruses to manipulate their genomes. We could choose a plate and characterize it, but today we design it. mutations in two genomes using DNA copies of all viruses, this includes DNA and RNA viruses, we make a DNA copy, we propagate the plasmids in bacteria, we can purify them and make all kinds of deletions, additions or changes and perhaps most importantly , we can create viral vectors. make therapeutic applications that we will talk about in the last lecture of this course: the process of putting DNA in a cell viral DNA to obtain viruses it is called transfection it is first done with lambda phage bacteria the word comes from infection by transformation you transform the DNA in a cell and you start an infection we can do this with DNA or RNA molecules, put them in the cells and the result is viruses, the way it is done is different According to the virus, here is an example of a poliovirus with an RNA virus very simple 1 plus chain, where the RNA itself is infectious.

If you just put it in the cells, you can make a copy of DNA and put it in the cells, it will also give rise to the virus. and you can modify the DNA however you want, someone has modified it, for example, this virus causes paralysis, but you can genetically modify it so that it does not paralyze and they are using it to cure glioblastoma by injecting it directly into the tumor and that is what you can do with copies of DNA from genomes you can manipulate viruses so that they are beneficial to us this is how you would do it with the influenza virus there are eight different RNAs of the influenza virus you create a plasmid that codes for each of the RNAs and this plasmid has two promoters one that drives the production of mRNA to get proteins, the other drives the production of viral RNAs, the negative strand is incorporated into the virus particles, so you take eight plasmids, you put them all together in cells and you generate influenza viruses.

Now I want to tell you something. very short story about how this was used in the 1918 pandemic, the global influenza epidemic was horrible, millions and millions of people died, but we didn't have the virus, we didn't isolate the influenza virus until 1933, so we couldn't work with he. In this 1918 pandemic, in 2005, several researchers isolated RNA from formalin-fixed sections, so many of the people who died were from the military, the military took fragments from their lungs and froze them. In 2005, someone came in and extracted the RNA and determined. the viral sequence of the RNA another group also went up to Alaska and they went to graves that had been frozen all year and they dug up one of a person who had died of 1918 influenza, they took a lung biopsy, they extracted the RNA and did the sequence, so between these two studies they got the complete sequence of the eight segments, put them on plasmids and recovered the virus so that now we can work with it, and this was a terrible flu that killed a lot of people, so we want to know why was particularly virulent, we have the ability to do this and do whatever we want with a viral genome and, in fact, it is no longer even necessary to clone the genome into a plasmid.

Here's an example from horsepox, an extinct virus for which we had the sequence of more than 200,000 units, a group in Canada chemically synthesized that overlapping DNA. You can synthesize them through them. About $150,000, they put this DNA into cells and the Horsebox virus emerged, the virus that went extinct. The moral is a virus that has never been eradicated. As long as you have the sequence, this experiment scared some people. Here is an article on phys.org. Scientists recover extinct horsepox, raising important biosafety questions. So, this is all about synthetic biology and biosafety. You can do experiments. You can do experiments that couldn't be done before with a virus, you could change its properties, we couldn't do that, that's why the US government has formed this Biosafety Advisory Council to regulate these experiments, if you write a proposal to modify a virus. dangerous virus, they will review it and make sure that it is worth doing and that you do it under proper containment so that the virus does not get out and cause damage next time, we will start to analyze how viruses are created by looking at the structures of different types that