[Music] all right good afternoon and welcome back I have to show you this water bottle this is the coolest look at this it's got a biohazard symbol so nobody's going to steal it from me right and it says got pox on it this was from a a summer meeting here at Columbia they gave it to us so my talking about not stealing things my my license plate is viruses on my car so I applied for a parking permit once and they say put your license plate I put viruses and weeks and weeks went by and they weren't sending it so I emailed and the lady said Oh I thought it was a virus so I didn't open it oh gosh that's why we need to teach the world about virology okay there is water in here actually and I hope I don't leave it here because I really like this orange bottle let's talk about virus's making RNA today and this Story begins uh in the 60s let me give you some RNA history so viruses are discovered remember around 19 00 uh by 1935 people started to make crystals of viruses to solve their structures they were interested in what they were made up of so this was first done by a guy named Wendell Stanley in 1935 he crystallized tobacco mosaic virus and found that there's only 5% RNA in the crystals the rest was protein so he said the protein is nucle is the genetic material this guy got a a Nobel Prize for that they should take it back right 1944 we learned that DNA is genetic material 1952 the Hershey Chase experiment which we talked about showing DNA is the genetic material of viruses and 1953 the structure of DNA is solved by the way that's the year of my birth that's why I am a Scientist because big discovery that year actually there are other things that happened that year too 1956 RNA in tobacco mosaic virus shown to be genetic material we talked about these experiments and then by 1959 many people found RNA in multiple animal viruses and so they realized this was a thing that that it is genetic material and so they start to ask how does this RNA get replicated is it the virus is it something in the virus or is it the cell that that does it what we're going to talk about today is is shown here in the in the Baltimore scheme we're gonna remember mRNA is at the middle that's of course RNA we're going to talk about viruses that have RNA genome so we're going to talk about viruses with doubl stranded RNA single stranded negative sense RNA and viruses with plus RNA now there's another class of viruses with RNA but they go through DNA intermediate the retroviruses and they're going to have their own lecture the first experiments that were done to try and get at this question what is making the RNA uh are like this experiment in this experiment you take a cell this is a cell it's not a fried egg it's a cell and you infect it with polio virus and you you then make cell extracts at different times after infection you break the cells open and you that's a cell extract and then you add triphosphates the four triphosphates and one of them is usually Radioactive in the old days we used radioactivity people don't do that anymore but we had no choice back then you add radioactive triphosphate and then you can measure the incorporation into RNA so it's a way of measuring RNA synthesis and the the result of this experiment shown on the right here so this is a graph we have hours post infection 1 through six so very short reproduction cycle for this is polio virus and then on the left hand uh the y- axis is the RNA polymerase activity so this is incorporation of the label into RNA and then on the right a y AIS is the tighter of polio virus type to so this is pfu Prill right so they did a plaque assay here to measure the tighter so those are the square boxes so you can see between two and three hours you have an increase in infectivity it increases and eventually plateaus and at the same time the RNA made increases those are the circles and so uh you can see there is an something is making RNA in these infected cells um and of course people wanted to know what it was and it turned out to be viral in origin um there are a lot of experiments that went into doing that but I'm just going to leave you with the identification of it being viral and not cellular many people thought cells could make RNA from RNA but that turns out not to be the case all right so um subsequent to that experiment people found that the enzyme that made that RNA in poliovirus infected s we call it an RNA dependent RNA polymerase could also be found in the particles of negative strand RNA viruses and so that as we'll see in a moment the polymerase the RNA polymerase has to be in the particle of negative Strand and double stranded rnas for positive strand rnas the polymerase does not have to be in the particle nowadays if you sequence a viral genome you can identify polymerases by certain amino acid combinations so here is one gly asp asp gdd is a signature for an RNA polymerase we'll see why in a bit and then you can make you can take the gene encoding that proc you can synthesize it on its own and show that it has polymerase activity then you can also solve the structure of the protein I have actually Crystal structures here that's um not necessary anymore you can do cryoem molecular so that could be crystallography or cryoem real time updating of lectures is great you know in the old days we had to make slides actual physical slides I don't know if you remember that and how many people went to a talk and dropped their tray of slides minutes before their talk and had to oh my gosh stories I can tell all right so here's a summary of this idea about whether or not you need the pmer in the particle so for viruses with negative strand RNA genomes the polymerase is in the particle because the negative strand on its own when it goes into a cell the cell cannot do anything it cannot be translated it's the negative strand it's the wrong polarity right so the polymerase is in the particle here's vsv and influenza virus the polymerases are actually complexed to the RNA they're part of that R nuclear capsid RNA coded with protein the positive strand RNA viruses are typically naked there's no protein coating them there's no RNA polymerase because the plus strand can go right into the cell and be translated now of course there are always exceptions in biology and the two exceptions here are the retroviruses which are plus strand RNA they have a reverse transcriptase in the particle because they're going to make DNA from that plus strand so it does not get translated and the corona viruses which do not have a polymerase in the particle but they do have a nuclear capsid so here is the Corona virus third from the left the RNA is complex with protein so it's a nucleo capsid turns out the same with retroviruses they are also complex with protein they also a nucleocapsid as well so all the other plus strand viruses the drna is naked we're not sure why they're not in these two kinds of viruses probably the corona viruses they're so the RNA is so big that protecting with protein is probably needed and again the double stranded RNA genomes the RNA is naked there's no protein on it but there is a polymerase in the particle because if a double strand RNA gets into a cell it sounds like a joke right if a double strand RNA gets into a cell um it's not translatable because the plus RNA in the double strand is blocked by the negative strand so nuclear capsids we've talked about before as being substructured of of a virus particle right they're typically nucleic acid protein complexes that were within a membrane they can also be within a icosahedral capset here are just two examples of what they look like on the left is vascular stomatitis virus which has a bullet-shaped nuclear capsid right the protein a single protein nuclear capsid protein repeated many times it interacts with the viral RNA and it interacts with itself to form this coil on the bottom is actually the structure of the single protein and a piece of RNA you can see there's a Groove in the middle of the protein and the RNA is bound in there and on the left is a structure of a few turns of this nuclear captured protein you can barely see this Darkly colored area in dark blue that's equivalent to one of these uh subunits here so that's and of course there would be RNA uh WRA C up in this as well but it's not shown there on the right is the influenza virus nuclear caps here there are eight of them eight RNA protein complexes and on the right is the structure of one so we have a nuclear nuclear protein the the orange circles there bound to each other and to the viral RNA and then at the one end of this ribonuclear protein is the RNA polymerase which is the negative strand virus so it has to bring in an RNA Cates into the cell and on the bottom is again the structure of the nuclear protein and you can see an RNA fitting in a Groove in the middle of it as well and on the bottom a space filling model of the ribonuclear protein and you can see one of the molecules colored dark so this is a side view which is kind of analogous to the drawing up at the top there and then this is a we've turned it 90° yeah and we're looking down the center so you can see there's a hole in the center so those are what nuclear capsids look like and remember within these structures even naked RNA they're always structured they have secondary and tertiary structures these are Illustrated here so here's RNA in green and even single stranded RNA can base pair with itself so you can see we can form stems by having sequences within not too far away from each other base pairing you can have multibran Ed stem loops and so forth so these can get very complicated you can also have something that's called a pseudo knut which is shown in panel B it's called a pseudo knut because it looks like a knot but it's not and pseudo knut you have a stem Loop and then a few base pairs in the loop can base pair with some bases Downstream of the loop that's what the dotted lines mean and it forms a structure shown in the next panel uh so those are the base pairs actually happening and then it actually twists a little bit and on the upper right is what the structure actually looks like so it looks like a n but the the RNA is not threading through itself and pulling itself as your shoelace would and so for example here's a here's an RNA of HIV here and um it's highly structured here's the the depiction of this structure on the top many stem loops and that's the actual three-dimensional structure of the RNA these rnas are important these structures are important they interact with proteins they they have regulatory roles and so forth they're not just there to to look nice so today we're going to talk about RNA synthesis and there are rules so it's RNA based RNA synthesis in other words copying RNA into RNA and there are rules for that first of all that's an RNA molecule it's in green so we have color schemes for our molecules in this course they're taken from the virology text green is RNA and blue is DNA so that's an RNA molecule and that's a plus strand RNA molecule because it's a light green and a dark green is a minus strand uh first of all the first rule is the RNA has to be copied from end to end you can't lose any sequence whenever you copy it right because then you will eventually not be able to make a virus if you lose the sequence it's there for a purpose and secondly you have to make mrnas that can be translated by the host cell and we know this already from the Baltimore scheme right that the cellular translation Machinery translates these mras so they have to be compatible with that and there are a number of features that ensure that continuing with the rules for RNA synthesis uh typically the synthesis begins and ends at a very specific place on the template so if you have a template AR and you're copying it the initiation is is very specific and the determination is sometimes it can be the ends but not always the synthesis of nucleic acids may or may not need a primer so on the top for example is a piece of nucleic acid called the template and it's going to be copied by an RNA polymerase which is that protein and brown there and some polymerases need a primer a short aligon nucleotide maybe 10 to 20 bases in length that tells the polymerase where to begin so the primer base pairs to the RNA and then the polymerase sits down and begins to add bases but not every polymerase needs a primer and throughout this coures we talk about different polymerases I'll tell you when they do or or when they don't other besides the RNA polymerases you need other viral proteins you typically need cellular proteins for this whole process and then the actual polymerization the polymerization of course is based on the template the polymerase is reading the template if it sees an a it will put in a u right if it sees a g it will put in a c and so forth so that's template directed incorporation the product is made in a five to3 Prime Direction so five Prime end and the three prime ends of the RNA are shown here and this primer is shown in the five to3 Prime Direction and The polymerase will add bases to the three prime and only because there's a hydroxy there that can be used to add more bases and then it will synthesize in a 5 to3 Prime Direction but the template is red in a 3 to five Prime Direction okay here's the template 3 to 5 Prime so it's red into 3 to 5 Prime synthesis is always in the opposite direction 5 to 3 Prime and so this is called templated RNA synthesis because obviously there's a template that's Direct ing what the polymerase should make but sometimes there is non-templated synthesis for example we'll see today polymerases can stutter they can get stuck at a base and just keep churning out bases without a template and that actually has some some role all right so let's start with initiation of RNA synthesis there are two general modes there's either denovo which means you don't need a primer so the word denova in the con denovo in the context of RNA or DNA syn is means you don't need a primer so for on the top here these are the last two bases at the three prime end of the RNA so the synthesis will begin at the three prime n and the polymerase can just start adding ntps that are complementary to those and then we have cases of polymerases that need primers they will not copy the template without a primer some polymerases utilize primers that are linked to a protein we'll see an example of that today and some polymerases utilize primers that are capped so cellular mrnas have a cap at their five Prime end it's a chemically distinct Group which we'll talk about later and some polymerases require a capped short oligonucleotide to to use as a primer we'll see examples of these today there are Universal rules actually for both DNA and RNA synthesis that are based on this mechanism discovered many years ago it's called the two metal mechanism of catalysis you may have encountered this in biology courses but here it's shown how it works here's a template strand on the right showing all the detail atoms so here's the five Prime end and then we have a series of bases and the three prime n and of course when I say bases it's not entirely correct the base is just the T or the a or the G or the C the base is linked to a ribos and then the the ribos is connected by to the next Base by a single phosphate linkage and these these bases of course come as triphosphates those are the precursors for polymerization uh and here for example um so here is a t being added to this a by the polymerase and you can see it is it's got three phosphates 1 two three so it's a a TTP thymidine triphosphate but during the polymerization the last two phosphates are removed and you end up with just one phosphate in between the bases so here's where the two metals come into play the um in the polymerase which we'll see in a moment there are amino acids that hold in place two magnesium ions and they're they're typically aspartates for RNA polymerases so you can see aspartate a and C here they are holding those magnesiums in place and the magnesiums are needed to help catalyze the reaction of adding a new base so in this particular synthesis we're we're synthes synthesizing five to three prime so the t is the next base to be added we have um a hydroxy typically at the end of the last base there uh and that oxygen will will attack this particular phosphodiester Bond and uh cause that phosphate to attack the oxygen and then then the two phosphates are released and all of that is coordinated by these two magnesiums which you can see are forming non-covalent interactions with the oxygens here one two 3 four so the magnesiums hold these bases into place so that this catalysis can happen and of course the polymerase helps this catalysis proceed by lowering the energy requirement so that's the two metal mechanism and we'll see how this actually works physically today when we look at the structure structures so our first question today is which is a universal rule about RNA directed RNA synthesis a the rdrp RNA dependent RNA pmer May initiate denovo or require a primer RNA synthesis initiates randomly on the template it's synthesized in a 3 to five Prime Direction RNA synthesis is always template directed so which is a universal rule all right let's see how we did so most you got the right answer the the correct answer is rdrps May initiate Den noo or require a primer that's correct initiates randomly no always initiates specifically on the template nobody got that that's good it's synthesized in a 3 to five Prime Direction no it's synthesized in a 5 to3 Prime the template is copied in a 3 to five Prime Direction it's always template directed no it's not and I'll show you the slide where I said that there are some non-templated synthesis which will we'll actually see today now all the structures of the four different kinds of nucleic acid polymerase have been discovered so what are they So today we're talking about RNA dependent RNA polymerases and so here's a structure of the poliovirus polymerase on the right and then the other three kinds of polymerase there's reverse transcriptase which is RNA dependent DNA polymerase there's um DNA polymerase which is DNA dependent DNA polymerase and then sorry this this t7 RNA polymerase is actually DNA dependent RNA polymerase the one that makes messenger rnas and then finally is um DNA dependent DNA pmer so these are the four classes of nucleic acid polymerase RNA RNA RNA DNA DNA RNA DNA DNA as far as we know there aren't any others than that and the structures of many of these have been solved and they all look very similar they look like a right hand with fingers thumb and palm domains the fingers and the thumb kind of encircle the active site which is the Palm domain shown in that picture uh as a little red dot and we'll explore that in a moment on the top of the the amino acid alignments of these four classes of polymerase and the colored areas are are similar sequences between the different polymerases the most similar sequences you can see this Motif a is is in red it's conserved in all of the polymerases that you can see and so the green and the yellow and the purple and so forth and so we think these probably originated from a a common ancestor maybe most likely an RNA dependent RNA plase but of course that's a long time ago precellular so we don't really know the yellow domain is where the catalysis occurs so the yellow domain is in the palm and here you can see the in the polio polymerase two beta strands that are making the yellow domain that's where catalysis happens the same for all the other polymerases and in that yellow domain the active site that's where there are amino acid that are crucial that hold those magnesiums into place right I told you about these before like gly a ASP the two aspartates coordinate those two magnesiums to allow the catalysis to happen and you can see in other viruses you have gasp ASP sometimes you have Ali ASP Asin little variations on each one but that's the function of this triplet is to coordinate those magnesiums so there's some conservation obviously among these so here as a closer look at the poliovirus RNA dependent RNA polymerase again it's a right hand this is the fingers domain and the thumb on the right they're they're touching for poop polymerase they touch they don't touch for all the polymerases sometimes there's an open hand configuration and again you can see the yellow the two beta strands that constitute the active site and these two amino acids where the side chains are shown as balls and sticks those are the two aspartates that are coordinating those magnesiums and they're completely required for catalysis if you change one of those to something else the polymerase is inactive just one amino acid change so that's where they're located and that's where the catalysis happen so let me show you a different view so you get an idea of how this works so these are now space filling views where we have all the molecules as spheres against the polio rdrp and the yellow again is the um active site where we have those two aspartates and um in fact the um the two aspartates are shown here in purple and the uh the template enters on one side of the molecule so here's a top view of the polymerase we're looking down we've removed some amino acids here in the front so you can see inside the the RNA comes down the top it passes over the active site it comes in single stranded and then it's made double stranded and then the double stranded RNA EX so you can see here the green initially came in uh well actually on this picture they're putting blue in should have been green but it's single stranded blue and then the second strand that's made is green and the double stranded RNA exits here's the front view so you can see the single stranded template going in the top it kind of it goes it turns to a 90 degree angle and comes out double stranded on the front so the top in the B and the front are where the nucleic acids enter an exit and of course as they're entering they're passing over the catalytic site and they're made double stranded there's one other important part of this polymerase that's the back and the ntps actually enter the back so if you go in the back you have access to the active site which it's yellow here so the ntps come in the back it's a big enough channel for that and then they're part they're put in at the Active sign the way this works actually is let's say there's um an a at the active site that needs to be polymerized next you need its complement which would be a u the there's no way for the enzyme to know how does it know to get the right triphosphate it doesn't it tries all four so there's some triphosphates floating around and they randomly come in and if it's the wrong one it leaves it doesn't have a high enough affinity and the right one finally comes in it base pairs and it's polymerized so that's happening really really fast but it's a random thing it's not like the polymer says Ah it's an a give me a u no doesn't happen that way they're all four in there and they just it's trial by error trial and error now I want to go through this process of RNA synthesis for plus minus and double stranded RNA viruses so for plus we're going to look at polio viruses and for and also Alpha viruses because they're slightly different so polio virus is the the the genome is is a plus stranded RNA and it is copied into a negative strand which simply serves as a template to make more plus strands this negative strand does nothing else except to serve as a template seems like a waste but that's what you have to do these are M these are both genomes and messenger rnas so for flavy viruses West Nile dangi the RNA is actually capped at the five Prime M but for polio virus it's not capped for the alpha viruses chicken virus synbus is also plus Str Ed RNA viruses they're capped and by the way you see they're also polyadenylated at the three prime n they're Bonafide messenger rnas these viruses also synthesize a subgenomic RNA in other words an RNA that's not the complete plus Strand and uses that for making proteins as well right so let's take a look at this in some detail so polio virus binds its receptor which we talked about last time it's taken in to the cell the receptor catalyzes the formation of a channel in the particle and the membrane so now the RNA is in the cytoplasm since it's plus stranded RNA can immediately be engaged by ribosomes the ribosomes make proteins these are actually made as one long protein for this virus which is chopped up by proteases encoded in the genome in fact that's how the corona viruses work also Anda loid the antiviral inhibits one of those proteases among the proteins that are main include the prase which copies the plus RNA into double stranded RNA is a plus and a minus Strand and then the plus strands come off and are packaged into new virus particles so very simple scheme plus to minus back to plus and this happens this replication happens on vesicles membrane vesicles that are induced by Virus Infection we'll talk a little bit more about these later on in the course this is a schematic of the viral genome so you can see the RNA at the top it's about 7,400 bases long there is a protein linked to the five Prime end and not a cap but a protein called vpg there's a polya at the three prime end and when ribosomes engage this or an A they make a very long protein which is called a polyprotein and then it's processed to the individual proteins by two viral protases and you get capsid proteins and the rest of the proteins involved in RNA synthesis including the polymerase which is here 3D Paul at the very five Prime end the protein is linked to the RNA by a a phosphodiester linkage to a um tyrosine so tyrosine is one of the amino acids that has a Hy hydroxy right so it that hydroxy can be linked to the phosphate of the first base and you can see that here this is a urine so phosphor diester Bond and then the rest of the genome is shown here there's a clover leaf structure here very at the five Prime which is involved in RNA synthesis as we'll see and then the rest of the genome follow so that's a coent linkage that that occurs between the viral protein that is linked to the RNA so this is another kind of view of the viral RNA which shows some important secondary structures remember I said secondary structures happen in RNA and they are functional so for polio RNA we have a clover leaf at the five Prime n we have a stem Loop somewhere in the middle it doesn't actually matter that it varies where it is and this is called a cre element and then at the three prime end we have a pseudo knut these are all signals for RNA replication that distinguish the viral RNA from cellular rnas when polio infects a cell it the polymerase does not copy any cellular RNA whatsoever it only copies viral RNA and it's because the viral RNA is only have these signals in them so to start RNA synthesis that cre element that I just showed you it actually serves as a template for the polymerase which is shown here as 3 CD polymerase binds to this cre element and then it takes a free molecule of vpg which is the Protein that's going to end up at the five Prime end of the genome and it adds two U residues to it uu which is going to be the primer for RNA synthesis so you could guess the first two bases of the RNA are AA they're going to base pair with that uu those U are added because this Loop in the cre element has a series of a residues so it's a templated reaction okay now we have a primer for RNA synthesis and that primer is going to sit down on the RNA so here's a of VAR plus strand RNA that's about to be copied by the polymerase the uh the polymerase is at here's the cre element in the middle so the polymerase says you're a delated vpg adding two use and somehow it gets from the cre element to the three prime end of the genome which is shown there the polya sequence and then the polymerase will start to elongate from that primer and eventually make a full length minus strand of the whole genome so that's the primer step now this whole Affair occurs on membranes this is a cellular membrane at the top left and in addition the RNA is circularized so the five and three prime BS are brought together by protein protein interaction so there is a protein in the cell called polya binding protein pabp it binds polya it also binds a molecule of the polymerase that is bound to the C Clover Leaf and that interaction requires the Clover Leaf to be bound to a cellular protein called pcbp so all of that does is to circularize the RNA brings the five and three prims together so this polymerase that's bound to the Clover Leaf is the one that's going to start copying the viral RNA and it does so and makes a um a double stranded copy this this process is carried out as I said on membranes those membranes that are induced by Virus Infection we'll talk more about those I think in lecture 11 our next question is wh which is a part of the Polar virus replication strategy the production of subgenomic mrnas denovo without primary initiation of RNA synthesis circularization of template for initiation of RNA synthesis all of the above which is a part of the poliovirus replication strategy now you may be thinking why does anyone need to know any of this stuff right Pax lovid is a proteas inhibitor we wouldn't know about the ability to inhibit a proteas if we didn't know this kind of thing and there are other antivirals that inhibit polymerases which you need to know there's an an antiviral that inhibits an endonuclease that's involved in influenza RNA synthesis so it's only when you know the basics that you can really devise antivirals and even vaccines that that work so you know if you're a doctor one day and you give you give your patient a prescription for PAX loid and they say what is an M protas which is the target you're going to know because of this course they'll think you're really smart and they'll keep coming back to you which is what you want okay let's find out how we did so the right answer is uh 51 52% of you circularization of the template you don't make sub genomics it's not without a primer nobody picked that it's not all about it's just circularization so let's have a look since not not everybody got that let's take a look at what what what's going on so there's circularization of the template right there's interaction between polya poly binding protein in the polymerase brings the five and three prime NS together that's what uh that's what that's all about and and so it is a primer dependent reaction and then you you make full length copies of the plus strand which are the minus strands intermediates are the other plus strand virus we'll mention briefly are the alpha viruses these include chunu virus which you may have heard of because the name is so cool but it's it actually CA causes an arthritis likee disease and it's mosquito born and these viruses uh make a subgenomic RNA that's why I want to tell you a little bit about them so these viruses bind receptors they're they're envelope viruses they are taken into endosomes pH drops you have Fusion catalyzed confirmational changes in the RNA ends up in the cytoplasm so now it's a plus stranded RNA it is there's no polymerase in the particle so that's translated directly to make a single protein which is the RNA polymerase you got four subunits to it but that's the RNA polymerase this this nsp1 2 3 4 so that RNA polymerase will then copy the incoming plus RNA to make negative Strand rnas and um then some of those negative strands are going to be copied to make a subgenomic RNA so the input RNA is not fully translated and and it to get to the rest of it you need to make a subgenomic RNA and that sub genomic encodes the structural proteins the capsid protein that makes up the capsid shell and then glycoproteins that end up in the membrane of the virus so here's a schematic of what's going on here here's the plus stranded RNA when it gets in the cell the first half or so is translated to form uh the the polymerase protein and this there's a termination codon here in the genome that stops translation and then the polymerase makes the negative Strand and then the polymerase makes from the negative strand a subgenomic mRNA so sub genomic simply means it's smaller than the genome right so here it is it's the right hand half of the genome the red arrow is the initi initiation site for synthesis of that mRNA and that is then translated into proteins now you may say what's the function of this PO virus basically makes one long polyprotein why isn't it the same here it works right evolutionarily it's it's competitive and this is just another bifurcation on The evolutionary pathway you can have a a genome be translated completely or you can make a a subgenomic RNA that's the only thing I can tell you about why that happens now Corona viruses are a Twist on this strategy there are plus stranded RNA viruses they do not have a polymerase in the particle because the RNA is plus stranded but they make lots of subgenomic rnas so in a Corona virus infected cell so there's the Corona virus binding to its receptor you know some of them fuse at the surface Remember by after the spike protein is cleaved others confused within endosomes either way the RNA is in the cytool first third of it or so is translated to make the polymerase enzyme and that polymerase can either reproduce the genome to make full length genomes that you want to make new particles with and we'll talk about a sembly of particles separately later or it can make mrnas and the the polymerase will take the plus stranded genome and make negative stranded mrnas and then from those it will make plus stranded mrnas which are then translated to make a variety of structural proteins and these mrnas are unusual because they're nested in other words you have a big one and then a smaller one within it and a smaller and a smaller one nest in Latin is Nido it's the name for the order of viruses that includes the Corona virus is Nido virales that's why and the nested nature is shown on the right here on the left is this is the genomic RNA and these boxes are all the proteins that are encoded probably you recognize s the spike protein right because it's the basis of all the vaccines against SARS kov 2 but there are other proteins as well when this RNA comes in the cell the left half is translated to make the polymerase which then goes on to copy the genome as I've said and you end up with with nested subgenomic rnas and you these are very interesting because the biggest one encodes all of these proteins to the right of the polymerase but only the first one is translated the ribosomes buying to that RNA the first protein so here 2A would be translated here H this is the spike mRNA and all the way down to n the last protein the nuclear capsid protein is made from a tiny mRNA that only encodes that protein and if you ask you ask what's the function of this strategy can't give you a good answer it just exists and it works how it works is very interesting here's the genome at the top here and it contains a number number of ingenic sequences the polymerase will recognize the three prime end of the plus Strand and remember it can make long Min minus strands or it can make subgenomic rnas and the pyas begins to initiate synthesis here at the three prime end uh and then at this first region it will stop and make one particular subgenomic mRNA and it does so by copying and stopping going a little further and a little further that's how you get that nested set of mrnas and these are all negative strands initially and then they're copied to make the plus stranded rnas now what's unusual here is that there is a leader sequence which is shown here in gray and blue which is added to each one of these mrnas and the mechanism is that the the leader sequence is encoded at the very five Prime end of the genome and so for each mRNA to have that leader sequence requires the genome wrap around and be copied by the polymerase in this kind of configuration so here's a linear version polymerase is copying when it gets to this intergenic sequence trsb apparently the RNA is folded around and then the plase copies from the five Prime end of the same molecule it does that for each of the mrnas that are made and the reason I'm telling you this is because this is a kind of Rec combination when the polymerase changes template actually not template but it's going from the thre Prime n to the five Prime n and this ability is why Corona viruses have high recombination rates if you look at the SARS K2 genome it is a mosaic of many different SARS CO2 like viruses because recombination occurs at high rates and we'll probably talk about that later minus strand RNA viruses here the problem is you cannot be translated so you have to have the polymerase in the particle so here the r the negative strand RNA is shown in the the darker green we're going to talk about viruses with one RNA unimolecular like vsv and we're going to talk about viruses with segmented genomes like influenza virus on the right this in all cases in both cases the strategy is going to be the negative strand RNA comes in with the particle with a polymerase and then it is either copied to form mrnas or it is copied to form uh fulllength minus strand genomes to make new particles with so let's go through that for vsv vsv is one strand of negative stranded RNA it's not messenger RNA right it's negative strand so it has to come in with a polymerase the virus enters by endocytosis the RNA protein complex is released into the cell the polymerase on the part on the genome copies it to make subgenomic mrnas as shown here those are engaged by ribosomes and they encod code for a variety of proteins and among those proteins structural proteins to build new virus particles and of course the RNA polymerase that is going to take that negative strand it's going to make a plus stranded copy of it and then it's going to copy that back to New negative strands which could either go into new particles or be recycled into making new proteins now there's a special situation here and that is when the viral genome is not mRNA there has to be a switch from making mRNA which happens early in infection when you need a lot of proteins to be made you need to make mRNA you have to switch from that to later in infection making a lot of genomes and we're going to look in in these cases that we go over where that switch is occurring and for a virus like polio virus where the genome is mRNA there doesn't there's no switch doesn't have to be because it's the same molecule but here there's a switch because the mrnas are different from the genome so here is the viral genome for vsv remember bullet shaped particle related to rabies virus negative stranded RNA so no cap comes in these it's in the virus complexed with the nucleo capsid protein and a molecule of RNA polymerase which consists of two proteins L and P and as soon as that RNA is released into the cytool the polymerase makes subgenomic mrnas from it you know 1 two 3 four five that encode all of the viral proteins so the mrnas are made they're translated into cytosol and here L is the polymerase protein G is the glycoprotein spike and so forth so that's the overall strategy how does it happen so here again is is a look at the negative strand here in the middle minus strand genom AR it's Ed with nuclear protein nuclear capsid protein as it comes out of the particle the polymerase is attached to the three prime end it will copy this negative Strand and it makes one two three four five subgenomic mrnas what it does is the prelimary starts at the three prime n it makes the nmna it stops it then starts again it makes the P stops and starts again and so forth all the way down to the very end at some point in replication you have to have a switch from mRNA synthesis to making genomes because towards the end of a cycle you need to make viruses you need genomes for that how do you do that so you have to have some way of making fulllength plus strands as the intermediate these mrnas cannot serve as an intermediate they're pieces and they're not full length copies so the pme switches modes it begins to make fulllength plus Strand and the switch is controlled by the nucleocapsid protein the nend protein as you make more and more n protein in an infection the nucleocapsid protein coats the plus strand RNA that is being made and it forces the polymerase to keep copying without terminating and making individual mRNA so the N protein acts sort of like an anti Terminator the mrnas are made by terminations start Stop Those are eliminated by the nucleocapsid protein now you get a full length plus Strand and the polymerase copies it to make a full length minus strand which is also coated with nucle capsid of course because it's going to go into the particle and here's a look at how this is happening here's our negative strand at the top there's the NG Gene the P Gene the M gene and it goes on there inter genic regions that have the start stop signals for the polymerase the polymerase is here at the left it initiates it makes the nmna then the pyase stops at the ingenic region and starts again and makes the P mRNA so they're these stop start sequences of events and again to make a full length plus you just need enough end protein to antagonize those starts and stops the other thing that happens at this intergenic region is some non-templated RNA synthesis to make the poly tail so here is the end of an mRNA being made this is an ingenic region where we have all of these bases shown so there's the RNA polymerase it hits this U stretch there's a stretch of one two 3 four 5 six seven U's here and the P stalls and begins to stutter it just keeps cranking out A's without moving until it's added about 200 A's and it stops doing that and it moves on to the next Gene and that then releases an mRNA with the polyat tale so the polyat tales of these viruses are not templated they're added by non-templated RNA synthesis where the polymerase Encounters this stretch of use begins to stutter we call it stuttering we add lots of a residues and then stop at about 200 and then the Pat will reinitiate uh at the next mRNA and do the same thing over and over and so the all right so that's vsv this a similar event happens in influenza virus infected cells you have those events but on a scale of eight different mrnas eight different RNA segments so the genome is negative stranded but it's in eight pieces and again when the virus infect effects out we saw the other day how the rnas get out they actually go in the nucleus where they are copied to negative strands and then the negative strands are copied into mrnas the Mr mrnas are exported to the cytoplasm to form virus particles and we'll talk about how that works uh later but today I just want to focus on the RNA and again there's a switch from mRNA to genome because the genome is not mRNA it's a negative stranded RNA so this is what the genome looks like eight segments of negative stranded RNA from them one or two messages are made for most of them one message is made from two of the segments the primary mRNA is actually spliced to give a different RNA that encodes a different protein but essentially the same series of events happens on each segment that we're going to talk about now and this should look just like vsv with a few twists here's the negative strand excuse me RNA that's coming into the cell the polymerase copies it to make an mRNA shown at the top there and the M of course is used to make protein now this synthesis is very unusual first of all it is primary dependent and the primer is a capped fragment of host mRNA our mrnas are all capped and there's an enzyme in the influenza virus polymerase that Cleaves our mrnas to yield a cap in about 12 or 13 bases and that's the primer for influenza virus RNA synthesis so every influenza virus RNA has some sequence at the five Prime end which is non-viral it's not coding but it's nonviral the enzyme that does that the endonuclease is the target of a brand new antiviral baloxavir for influenza treatment which is actually better than the neuraminidase Inhibitors but we'll talk about that later and at the end the the RNA is poly dentil but it falls short of the end of the RNA so it's not a complete copy and so at some point you have to switch from mRNA synthesis to full length plus strands because the mrnas are not suitable for copying to make genomes and again the nucleocapsid protein is the key for that when enough nucleocapsid proteins are present in the cell the polymerase will no longer make mrnas it switches to making full length plus copies of the negative Strand and those can be copied into negative strands to make more virus particles okay so uh the MRNA is not the same as the genome so we have a switch the switch again is controlled by nucleocapsid protein the priming event that I talked to you about is initiated by this this endonuclease so here's the negative strand RNA of the virus those are the last bases and the endonuclease of the virus takes a cellular message which has a cap on the five Prime n and then a a number of bases and Cleaves it about 13 bases from the end and then that's used as a primer for RNA synthesis so you can see the MRNA that's made by the polymerase has about 13 bases plus a cap from host cell Mr and there a couple of other viral RNA polymerases that do this as well and again the enzyme that does this cleavage is inhibited by an antiviral booe polyadenylation of these mrnas is also similar to that of vsv with a Twist so on the top is the polymerase complex that is actually copying these mrnas it's got three subunits pb1 pb2 and Pa the five Prime end of the RNA the viral RNA binds to a specific site on the polymerase there shown with red and then the polymerase of course starts copying at the three prime end and it it pulls the RNA through the protein so the protein doesn't move down the RNA as you you would think it might but the RNA is attached to the polymerase and then the thre Prime n is drawn through the active site and then the mRNA is made that's shown here capped and coming off there's a stretch of view near the five Prime end of the viral RNA as you can see there and when the polymeris gets to that it can't pull through it right because the five Prime end is attached so it's like a string tied down so the active site has a stretch of view in it and of course the polymerase begins to synthesize a bunch of A's until that mRNA is polyadenylated so it's a sort of similar mechanism for polyadenylation as VV although the details are different but again an example of non-templated RNA synthesis there's just seven Ed and you end up with 200 or so on the MRNA so that leads to the question how are influenza virus and vsv RNA a synthesis similar so um a is the switch from mRNA to genome synthesis is controlled by an RNA binding protein polyadenylation occurs at a short stretch of U viral mrnas are shorter than minus genome RNA or all of the above which are similar let's see what we did so most of you got all the above which is right the switch is controlled by an RNA binding prot which is NP poly dentil occurs at a short stretch of view and the mras are shorter than the genome right for vsv they're all those tiny subgenomic mrnas they're clearly shorter for flu it is short of the end of the genome let me show you that here the the MRNA is about 20 bases short of the five Prime n of the genome so that can't be a template for RNA viral RNA synthesis so both cases The genome the mrnas are shorter so for vsv they're clearly shorter right influenza more subtle and finally let's talk about double stranded RNA viruses Rio viruses you have a doubl stranded RNA in the particle no polymerase I'm sorry there is a polymerase in the particle but not complex to the RNA the polymerase makes a plus strand that is translated into protein and it makes plus strand that are eventually copied and used for new genomes so let's take a look at how that happens so there are three six 7 8 nine 10 double stranded rnas for real virus these viruses can have different numbers of double stranded RNA and the particle comes in it enters the endocytic pathway that pH drops and there also proteases in the endosome and those proteases strip off the outer shell of the virus remember there are two icosahedral shells and what ends up in the cytoplasm is a core so the core is hydrophobic and can push its way in through the endosome membrane into the cytool so the core has the double stranded rnas in it and the core also has the RNA polymerase the RNA polymerase in the core begins to synthesize mrnas the rnas never leave the core they remain in there's one example where the RNA is not in the cytool and those rnas come out from each fivefold axis there is a a kind of touret structure at each five-fold axis which is open to the interior and that's plugged by the outer shell in the intact viron so it's not accessible but in the cell where you have this core the RNA the mrnas can come out and those go into the cytool they're of course translated into protein they can be used used to assemble new virus particles so here in Step eight we have a a viral particle being assembled which has mrnas in it that particle also has polymerase so th that polymerase can make those double stranded and eventually the other proteins can be added and you have a mature virus particle so the function of the outer shell is to keep those T like structures plugged and to cover the hydrophobic parts of the core and then you strip off that outer shell the core is now hydrophobic and leave the the endosome and then the MRNA is made in the core can go out through the openings at the five-fold axis so where's the switch to genome synthesis it's here in the newly assembled core where these mrnas no longer can be translated when they're in the core right so as soon as you put them in the core you're now switching to genome synthesis because in that core the polymerase will copy them and make them double stranded so that's where the switch occurs um this this is a schematic of the genome should have showed you this earlier the double stranded rnas each encodes an mRNA that encodes one or a few proteins and here's the virion and then an intermediate where the outer Pro proteins are removed and then all of the outer proteins are removed to give you the core particle and that's the one where the Mr Ras can be made and they can come out from these turrets now here's a cryoem structure of a core of a riov virus core which is assembled in the cell in the process of making RNA and so you see RNA molecules exiting at each five-fold axis that's where the touret would be you don't see it on this reconstruction and the idea is that there's a polymerase underneath each fivefold axis that is synthesizing the MRNA from the double strand and then the mrnas go out and those mrnas are shown here with caps and and they're in Gray all right finally last last topic for today arti viruses are unusual they undergo a lot of mutation and Rec combination where does that come from first of all mutation comes from misincorporation of nuclear tides by the polymerase so all polymerases make errors but RNA polymerases don't have error correction mechanisms to remove the error and so the error frequency of of a polymerase in an RNA virus is one mistake in 10,000 or 100,000 bases so in a 10,000 base genome a mutation frequency of 1 in 10,000 means one mutation per genome so in any cell where you have tens of thousands of viruses made every every position can be mutated DNA viruses are different as we will see later they have error correction to get around this but RNA viruses do not there is an exception though the Nido alyss which includes the corona viruses have a protein that is error correcting it's called xon it's an exonuclease which basically looks for mismatches the wrong base and it cuts it out so this um it gives you about a 15 or 20 fold decrease in mutation rate because if you remove this protein you you get a virus with a 15 to 24 fold increase in mutation rate and we believe we think that these viruses have a error correction protein because the genome is Big it's not the right order they're big because they have um error correcting protein in them they genomes can be up to 40 KB in length and no other virro genome is that big because they don't have AR correction mechanisms if you don't correct errors a genome of that size will simply sustain too many errors on each replication and it won't be viable now for for poliovirus polymerase it's been possible to identify amino acids that control Fidelity of polymerization in other words the incorporation of the wrong base that is completely controlled by how the template the primer and and the ntp interact at the active site so here's a structure of um a base interacting at the active site here are two aspartates in yellow 328 and 329 remember they're coordinating the magnesiums and this is a base that's just come in it's B it's hydrogen bonded with surrounding amino acids and it will be added by the polymerase and remember I said that all four ntps come in and out of the activite very quickly so what makes the one win well if only if it's correctly base paired does it transmit a confirmational change to the rest of the protein it reorients the triphosphate and allows phosphor transfer to to occur here's the triphosphate here so there's the ribos ring and the rest coming off is the triphosphate if it's the right base that triphosphate will be reoriented by the polymerase and it will be incorporated into the chain if it's the wrong base it will not reorient then it will fall out and the next one will come in so that's how it samples the bases continuously the right one induces the right confirmational change for polymerization to occur it's pretty cool now um a single amino acid in the poliovirus polymerase has been found that regulates the Fidelity of copying and this is a b this is amino acid 64 it's a change from G to S glycine to Serene if you make that one am has to change in the polymerase it makes fewer errors it makes fewer errors and we think this is a remote from the active site if you can see here there's the active site in yellow and this is on the back of the molecule and we think this change slows confirmational changes that the ones that occur on ntp base pairing so if that happens very quickly uh you may make a mistake and this position I somehow regulates that so it makes the enzyme more dependent on correct ntp base pairing in the active site and so basically if you change g64 to S it becomes uh less good at finding the right triphosphate in the active site um and you um you make you actually make sorry it makes it better at finding the the right confirmation and you make fewer errors but as we'll see when we talk about ever illustion you could these don't occur in nature viruses don't have better polymerases they all have polymerases that make mistakes it's evolutionarily beneficial to make errors that's how you evolve and we make errors too we make our DNA polymerases they make tenfold less error than RNA polymerases but they still make errors and that's why we get diseases but it's also why we can evolve as a species and why all all living things can evolve because their polymerases make errors now another contribution to diversity of RNA viruses recombination RNA can recombine and what happens is polymerase copying a particular RNA let's say it's copying uh the RNA here called the donor it can get to a certain point and then switch copying to another RNA so then in the end you have a Recon combinant RNA that consists of sequences from both genomes so this is exchange of nucleotide sequences among different rnas it's different from reassortment where segmented genomes can mix an infection and this is always creating new genomes in the RNA world and it can it can be very high frequency for polio virus can be up to 20% in a single replication cycle and again we've identified sequences in the polymerase that regulate RNA recombination and there are four amino acids not three you can see they're listed here but their their names are not so important for you to know four amino acids regulate Rec combination frequency so here's g64 right which regulates Fidelity the incorporation by the polymerase and here's the active site and here are four amino acids all in AOW here in cyan green red and brown they all control Rec combination frequency so that's located in the thumb domain and it's the RNA exit Channel and what we think is happening is that these cont if I showed you where the primer is in this structure they would the primer would be aligned along these four amino acids so we think that these amino acids in their wild type State allow the virus polymerase to distinguish between whether the right RNA is there or not and so you you end up with a base level of recombination frequency and if you change those amino acids you can um you can decrease the recombination frequency so we'll see some implications of this later on but those are two ways that the RNA virus World under goes change by mutation and by recombination and they're both regulated by the RNA polymerase okay so that is RNA dependent RNA synthesis next time we're going to talk about another kind of RNA synthesis where the template is [Music] DNA