[Music] so now I want to go through the infected cell for a number of viruses and show you how they make RNA and what is their strategy for expressing The genome because all these viruses well you know there's seven classes of RNA genome of genomes in general and the RNA genomes have specific ways of expressing their RNA so the first we'll look at plus strand RNA viruses we're going to look at picorna viruses as an example polio virus and then we're also going to look at another kind of plus strand RNA Alpha viruses and the
first the peorn is these have plus strand RNA genomes right they're positive stranded in the Von they're naked they come in the cell uh they're translated and then once they're translated the enzyme the RNA dependent RNA polymerase is produced and that can replicate The genome and replication is very simple you make a complete copy of the plus strand you make a minus strand copy that's shown right here minus strand full length complement so that's what the enzyme does and the enzyme can only do that after it's made so translation has to occur first and then
it's copied again to make more plus strands and these plus strands can be either mrnas or genomes so at some point in infection they can be translated protein and then later in infection they can be packaged uh into verons so this is relatively simple the MRNA is the genome couldn't be simpler and so let's take a look at how this happens uh again these picorna viruses poolik viruses they bind receptors like every other animal virus that we're going to talk about we talked last time how we think the RNA gets in the cytoplasm and once
the RNA comes out of the viron from a from a vesicle then it can be immediately translated because it's plus stranded RNA has doesn't have to go to the nucleus doesn't have to go anywhere else in the cell it just gets translated so ribosomes come on translate it and make viral proteins among the viral proteins that are made are the is the RNA dependent RNA polymerase which is shown in blue here and this will copy the viral genome it takes it from plus stranded to minus stranded to plus stranded now this whole process interestingly happens
on membranous vesicles doesn't just happen floating around in the cytoplasm this happens to be the case for all plus strand RNA viruses they induce the formation of vesicles they actually take over the whole lipid Machinery of the cell and they divert all the membranes to their own use so the the ER and the Gogi actually dissolve in cells infected with these viruses and you get uh these small vesicles made and the RNA synthesis occurs on their surfaces we think that's to increase the efficiency of the process because it's probably easier to have things happen on
a surface and where all the components can be concentrated in one area right so that's how the replication Works make genomes on vesicles some of these rnas that are made can go back into the translation pool if it's early in infection you need to build up a lot of capsid proteins but then later on they can go into new verons which then leave the cell so that's a pretty straight forward uh picture let's look at the genome in a little bit of detail so this is the RNA Genome of polio virus at the top again
it's plus stranded it's about 7500 nucleotides long and it has some characteristics that we've mentioned briefly it has a five Prime untranslated region a three prime untranslated region it's polyadenylated so these are all characteristics of messenger rnas but what is different from a message is that there's a protein linked to the five Prime n so where a messenger RNA would have a cap these genomes have a protein and that protein is part of the primer for RNA replication as you will see now uh as you'll see later when we talk about translation RNA viruses have
a problem in making multiple proteins Because unless you can make multiple rnas there's no way to make more than one protein so in general in eukariotic cells one mRNA typically encodes for one protein and as I've told you already the viral Genome of polio is is an mRNA a single mRNA so the problem here here is how to make multiple proteins from that and there are a couple of different strategies that you'll see one of them is very simple you make a long protein initially you translate the whole genome into a long protein is like
250,000 Dalton in size and then you process it with proteases and the proteases are actually encoded within the long protein so for example there's a protease here called 2A and another here called 3C and as those are being made they can start to nibble away at the at the polyprotein which is what we call the precursor and eventually you have about a dozen or so viral proteins made all by the action of this prote these two proteases so that's one solution to being limited by the eukaryotic cell and not being able to make more than
one protein per messenger RNA now here in the genome we have capsid proteins of course we have our proteases we have vpg right here which is the protein linked to the end of the genome there's coding region for PPG and here is our um RNA dependent RNA polymerase it's called 3D paw and as soon as that's made in an infected cell and it's processed by the protease that can start replicating the genome now the Genome of the virus remember I I've showed it to you already as a straight line or a squiggly line but it's
never straight it always has some structure and this is in fact uh what the POR cornavirus genome looks like it has a lot of secondary are structure there is a clover leaf at the five Prime end so a three stem Loop type structure uh there's a uh stem Loop structure somewhere in the middle of the genome called the cre element we'll see what that does and then at the three prime end there's a pseudo knut remember ponut is that funny Twisted RNA secondary structure these are believed to be signals to tell the RNA polymerase to
replicate this molecule because we know that in an infected cell no cellular rnas are ever copied by the polymerase as soon as that polymerase is made it goes right for viral RNA so there must be some signal there and that makes sense of course you don't want the polymerase copying cellular rnas that would be a waste of time so uh these signals this Clover leap decree and the ponut probably make the pimary specific for the viral genomes now remember at the five Prime end of this RNA there's a little protein called vpg shown right here
there about 20 amino acids or so long it is linked to the first base in the RNA which happens to be a u shown here is uracil it is linked by a phosphodiester bond to a tyrosine in the protein you know tyrosine is one of those amino acids with a hydroxy and that's how it's linked uh to uh the protein so vpg is linked to it and then Here Comes our clover leaf and as you will see vpg is a primer for RNA syn synthis now vpg in order to be a primer has to have
two bases attached to it just two and then it serves as a primer and that is vpg uu and then it can sit down on the viral RNA and be a primer and the way the U's are added to vpg is on that cre element so if you remember we go back two slides in the middle of the genome is a stem Loop called the cre element this is a very specific sequence whose function is is to serve as a template for adding two U's to vpg so you can make it a primer so here
we have the cre element in the genome the viral polymerase this is actually a precursor of the polymerase with the protease attached recognizes the cre element uh then vpg comes in and sits into the polymerase you see there are three of them here three polymerase molecules those are the u-shaped blue molecules and then at the top of this Loop in cre there's a stretch of A's and those serve as a template for adding two U's to vpg so this is called a uid dilation assay or a uid dilation reaction we're putting two u's on vpg
just so it can be a primer for RNA synthesis so this is really unusual not many viruses do this all the pnas do it but not not others among the DNA viruses you'll hear about one next time that also uses a protein primer so the virus makes these vpg p puu and then it uses them to Prime RNA synthesis now for these viruses RNA synthesis is very unusual not only does it happen on membranes and it uses a protein primer but the template is a circular RNA so remember I've showed you so far these picornavirus
genomes as linear RNA molecules when they're copied on the surface of those vesicles they're actually circles so it can show that here here is here is one part of one of these vesicles on the upper left there's a viral protein which when it gets made gets embedded in these membranous vesicles so this protein 3ab is quite hydrophobic the viral genome then is attracted to that protein through the interaction of a cellular protein called pcbp with part of the Clover Leaf and part of the 3ab molecule the other end of the RNA the polyadenylation the polyadenylated
sequence finds a cell protein called p ABP polya binding protein these are cellular proteins that function in mRNA synthesis and this ends up circularizing the RNA because PP binds to a molecule of the viral polymerase 3 3cd which inter turn binds to the Clover Leaf so you see you have this interesting set of interactions the RNA is brought to the membrane by a protein RNA interaction and then it's circularized by an RNA protein protein interaction and that is the template for RNA synthesis if you disrupt this structure this circular structure in any way genetically biochemically
you prevent RNA synthesis so what happens here we've got our remember our vpg is is ID delated on this molecule then somehow this uid delated vpg gets transferred to the three prime end of the genome it hybridizes and then the polymerase goes with it and now the polymerase is beginning to copy the genome all this is happening on the membrane but we've taken the membrane away to make it simple So eventually this polymerase will copy this plus strand you get a double stranded product a minus and a plus strand together and then the same thing
basically happens to make more plus strands from the minus strand so it's a very unusual membrane bound protein Prime circular RNA based uh replication system now in infected cells remember I told you that the virus these viruses these plus strand RNA porna viruses induce vesicle formation so this is an electron micrograph that shows that so here's an uninfected helila cell uh here's the plasma membrane here and ER and mitochondria typical normal looking cell but a few hours after poo infection um the e r and GG are gone and the cytoplasma is full of these double
membrane vesicles the smaller black dots by the way are new virus particles this is quite Laden infection so you've already got new virus particles made these these tend these turn out to be double membrane vesicles and if any of you know about autophagy you know that these are generated by the autophagic process in the cell autophagic process in the cell so the virus autophagy is is a response to stress so when the virus infects the cell the cell responds by inducing autophagy the virus says I'm going to take these vesicles and use them to make
my RNA and that's what it does so what turns out to be a stress response and the virus end up utilizing for RNA synthesis so that's the way the plus strand RNA viruses replicate their genome the overall picture is quite simple although the details that I've told you are they seem complex but they're really not and they're quite interesting Alpha viruses also have plus strand genomes um but for reasons that are not clear except that it works the way these work as you will see they translate a little bit of this genome but not all
of it and to access the rest of the genome here they have to make a sub genomic mRNA but they also replicate their genome through a minus strand complement just like the porna viruses and so these viruses synbus virus sem leiki Faris virus these are alpha viruses they get into the cell by endocytosis the viral RNA again ends up in the cytoplasm this is a plus strand RNA it does not need to bring in anything with it comes in the Cell It's naked it gets translated you can see it's being uh ribosomes are attaching to
it and you're making some viral protein and that Protein that's made the first protein made is the viral RNA polymerase shown here all these NSP proteins and that will go on and replicate The genome make a negative Strand and a plus strand now as I told you before in order to access the rest of the coding region of the RNA and you'll see this better in a moment uh you have to make a subgenomic mRNA that's done by the viral polymerase here from negative strands you make a small RNA so that you can make proteins
that are encoded at the C Terminus or the three prime end of the viral RNA so let's see how that works so here's the schematic of the genome on the top this could be a porna virus except that instead of the vpg it's got a cap so now it's more of a typical mRNA as a polya at the three prime end it has utrs at both ends and it has a cap so this RNA REM remember it's in verion it gets into the cell it's translated immediately uh to make these proteins these are the RNA
polymerase and accessory proteins now there happens to be a stop codeon right here this red dot is a stop codeon so it can't translate the proteins Downstream from that I don't know why this is existing this way because pakoras don't put a stop Cod on here they translate the whole genome and they process it it just works doesn't make sense to us but we're looking at it from a human View Viewpoint and that's always wrong in order to translate these proteins the virus has to First make a negative strand so this RNA polymerase copies the
plus strand it makes a full length negative Strand and then it recognizes a sequence in the negative strand to make an mRNA so it's called subgenomic mRNA synthesis that's a captain polyadenylated RNA and from those the virus can make other proteins that are encoded down here so it's weird but you know it works and so whatever works in evolution uh can be Ed for all right those are two kinds of plus strand RNA replication schemes right polyprotein they're both actually polyproteins except the second one has an mRNA a subgenomic mRNA thrown in [Music] [Applause]
