Influenza Attachment and Replication PM250

Author:

Aled Roberts

Keywords:

Subtitles:

so in the last video we went over the structure of the influenza virion and the genome focusing on influenza a as this is the main cause of influenza infections in humans now both of these aspects so the structure and the genome are key to letting the virus do its thing and its thing is infecting host cells replicating and then releasing progeny virions and this is a sort of life cycle if we can call it that as technically it is a non-living entity and i want to be clear here the function of the influenza virus is to replicate in a host cell and pass on genes to new progeny the disease associated with this process is almost a byproduct which just so happens to facilitate an increased transmission rate so in this video we are going to focus on three aspects of the influenza a virus how it infects a host cell how it replicates within the host cell and how it releases newly created progeny virions into the environment to restart this process so here is the entire process in a single image and we are going to go through each section now just a few things to make note of i've tried to color coordinate the diagram so everything that belongs to the host is in yellow or brown minus the arrows and everything relating to the virus is shown in blue so to start the entire process off we have host entry this is where the host needs to have briefed in what we call an effective dose of our influenza virus so this is the minimum number of virus particles required to initiate infection these virus particles then need to evade the host's innate immune system so that it can interact with host cells specifically those displaying the correct receptor for influenza on the surface of the cell okay so let's have a look at this attachment process and in the case of influenza virus it specifically recognizes cyalic acid receptors on the surface of a target host cell now there are many different receptors on human epithelial cells displaying cyalic acid but the key determinant here is the glycoprotein to which it is attached now there is a protein base which anchors the receptor into the host cell membrane as can be seen here in yellow there are then multiple chains composed of galactose molecules extending into the environment which can be seen here with these light orange circles and at the tip of the galactose chain bound via an alpha 2 6 glycosidic bond is a molecule of cyalic acid now the way in which cyalic acid binds to galactose via the alpha 2 6 glycosidic bond gives rise to a unique 3d configuration at the end of the receptor protein which is recognized by our influenza virus now there are other conformations such as an alpha 2 3 glycosidic bond which gives rise to a different 3d shape but there aren't as many of these in human cells and this configuration is more common in animals and this will be important to remember for our next video okay so now that our influenza virion has identified its target it needs to attach to it and this attachment process is mediated by our hemoglutinin surface glycoprotein which has a preferential specificity for this terminal cyalic acid with an alpha 2 6 linkage now as mentioned in the previous lecture the hemagglutinin glycoprotein is a trimer with a cell binding site sticking out into the external environment and this cell binding site contains a cyalic acid receptor so once there is a successful attachment of the hemoglutinin to the cyalic acid moiety the virion undergoes a process known as endocytosis this is where our influenza virion is surrounded by the phospholipid bilayer of the host cell until it becomes fully enclosed by it now this process can be mediated by the proteins clathrin and dynamin or it can occur via a micropinocytosis which are the two main mechanisms of endocytosis within our cells now the result of either of these processes is the formation of a membrane-bound structure that contains our virion known as an endosome and the key thing to remember here is that because our virion is contained within the endosome it is physically unable to do anything however the host cell can stalk to act on the virion so in a defensive move the endosome fuses with a lysosome which contains various digestive enzymes along with hydrogen ions making it very acidic the sole purpose of the lysosome is to digest whatever foreign substance is located within the endosome by fusing with it now whilst this is usually an effective defense strategy it is the worst strategy for the cell against the influenza virus why well remember back to our last video when we were talking about the various components of the virion the m2 ion channel protein is a proton selective channel so following the fusion of the lysosome to our endosome the protons lower the overall ph of the endosome resulting in the flow of protons through the m2 ion channel acidifying the inside of the virion now what this does is disrupt a wide range of protein protein interactions specifically protein interactions between the m1 matrix proteins that cover the inside of the virion and the nuclear proteins associated with the viral rna these detach from one another signaling the start of the uncoating process in addition to this the acidification of the virion causes the hemoglutinin to undergo a conformational change now this is an extremely complex process more complex than i'm going to talk about here but in general terms a protease from the host is inserted into the endosome by the lysozyme and it cleaves the hemogluten protein into two subunits now the acidic ph of the endosome and virion causes these subunits to part exposing what we call a fusion peptide which is then inserted into the phospholipid bilayer of the endosome and through various conformational changes the phospholipid membrane of the virion and the endosome are pulled together until they fuse and it's essentially at this point the contents of the virion gain entry to the cytoplasm and migrate from the virion into the host cell as they are no longer held in place by the m1 matrix proteins or cut off from the cell via the phospholipid bilayers so all in all from attachment to fusion it takes about 10 minutes for this process to occur now at this point we've hit a crucial point in the replication cycle of our influenza a virus the genome of the virus is currently located within the cell but it isn't where it's needed to be to make use of the host cell machinery it therefore needs to be trafficked to the nucleus so that it can replicate and produce viral proteins now again this is a complex process and one that i'm only going to go over briefly so as we can see our influenza genome is covered in nuclear capsid proteins and associated with pa pb1 and pb2 proteins which form the rna-dependent rna polymerase these segments make their way to the nucleus using the exposed nucleocapsid proteins which act as a nuclear localization signal that interact with the host's nuclear import proteins so a protein known as import in alpha will bind to the viral nuclear protein which is then recognized by imports in beta and the protein complex is then transported through the nuclear pore complex into the nucleoplasm and this process takes about one hour to complete so a lot longer than the initial attachment and cell entry stages now this process has been shown to be host specific so different hosts identify different nuclear proteins making nuclear protein adaptation crucial for productive influenza infections okay so moving on our negative sense viral rna is now located within the host cell nucleus ready to be transcribed into positive sense rna so that it can be translated and replicated so that progeny variants can be made so first we're going to look at the transcription of our viral rna so that we can create some viral proteins now our viral rna is negative sense and needs to be converted to a complementary strand of positive sense mrna and this process occurs by what we call a primed process and to do this the viral rna is a bit sneaky as it identifies host pre-messenger rna within the nucleus that contains a 5-prime 7-methylguanine cap now this cap is crucial as it helps signal the rna-dependent rna polymerase associated with our viral rna to start transcribing and hopefully all of this makes sense from the question that i asked you to go away and look at in our last video so via a process known as capsnatching the rna-dependent rna polymerase literally snatches the five-prime cap from the host messenger rna via its endonuclease activity then it starts to synthesize a strand of positive sense messenger rna using the viral rna-dependent rna polymerase host nucleotides and the negative sense rna as a template the result is a strand of positive sense viral messenger rna which has a five prime cap and then due to the genome structure of the influenza virus these strands gain a poly a tail so lots of adenine residues are strung together these are crucial as they help to increase the stability of the viral messenger rna whilst also acting as a signal for nuclear export however before our positive sense viral rna can be exported some messenger rna segments specifically segments 7 and 8 need to undergo splicing allowing us to get all 10 different proteins encoded by the viral genome so once this is all completed the positive sense viral messenger rna makes its way to the cytoplasm where it can be read by the host cell ribosomes and it can do this because of the five prime cap allowing ribosome binding now our viral messenger rna binds to free-floating ribosomes and is translated into various viral proteins that go within the virion so our pa pb1 pb2 ns2 and nps or it binds to ribosomes attached to the rough endoplasmic reticulum in order to translate membrane proteins so our ha na and m2 proteins and essentially this is where our story splits first we are going to follow our pa pb1 pb2 ns2 and nps back to the nucleus then we will come back and look at our other membrane proteins being produced in the rough endoplasmic reticulum now the reason why these specific proteins re-enter the nucleus is so that negative sense viral rna can be replicated many times over so that multiple progeny virions can be released into the environment so first we need to get our negative sense viral rna which can be used directly to create positive sense rna via an unprimed process which is different to the primed process we saw earlier using the cap snatching mechanism so in this process the rna-dependent rna polymerase creates uncapped positive sense viral rna which is coated with nuclear proteins that were synthesized within the cytoplasm and transported into the nucleus two things are then allowed to happen firstly the original negative sense rna is recycled and secondly the newly created positive sense viral rna is used as a template by the newly synthesized rna-dependent rna polymerase proteins to efficiently replicate this template again and again and again creating multiple copies of each genome segment which will be packaged into new progeny virions now the only thing left for our newly replicated negative sense viral rna segments is for them to be exported from the host nucleus to do this rna is covered in the nuclear proteins associated with the rna-dependent rna polymerase and then complexed with matrix protein m1 and the non-structural protein 2. this complex acts as a nuclear export signal which is identified by host crm1 proteins and exports the segments out through the nuclear pores and once in the cytoplasm the host crm1 proteins deposit our viral rna segments on microtubules which transport the segments towards the surface of the cell where it will start to be packaged into new virions now before we move on to the assembly process we need to take a step back and rejoin our viral surface proteins being synthesized by ribosomes associated with the rough endoplasmic reticulum so as i said earlier our hemiglutinin neuraminidase and m2 ion channel proteins are synthesized by rough endoplasmic reticulum ribosomes and they are then deposited into the membrane where they undergo various processing in order to create functional proteins now like most host proteins that are synthesized by ribosomes of the rough endoplasmic reticulum they are passed to the golgi apparatus or golgi body via the host's budding mechanism so this is where small vesicles detach from the surface of the endoplasmic reticulum and fuse with the membrane of the golgi apparatus where the proteins become finalized and bud off one final time and so the final vesicle which buds off from the golgi apparatus contains all of the membrane proteins for our virion and whilst in this circular conformation you might think it is similar to our virion it's not it doesn't contain any of the internal proteins or viral rna and therefore isn't considered a virion and these elements can't simply be injected in this vesicle needs to fuse with the cell membrane depositing the viral membrane proteins onto the surface of the cell which becomes key to forming our new virion now by this point in time our replicated viral rna segments have made their way over to the cell membrane using the microtubule system in order to start the assembly process for our new progeny virions the various segments of viral rna start to interact with m1 matrix proteins whilst non-structural proteins 1 and 2 start to accumulate in close proximity this allows them to make their way to the cell membrane where it starts to protrude out from the cell in a similar way to that seen in the rough endoplasmic reticulum and golgi apparatus finally there will become a point when the newly created virion detaches from the host cell membrane however it still remains attached somewhat to the host cell and that is because the cyalic acid receptors on the surface can still bind to the hemoglutinine glycoproteins of the virion and it acts as a sort of anchor tethering the virion to the host and this is where our neuraminidase surface glycoproteins come into effect they act as a mechanism to cleave the cyalic acid receptors rendering them inactive this does two things firstly it releases the virium from the host cell and secondly it stops reinfection of a host that has already been infected as these cyalic acid residues are required for internalization of the virion at the start of the process this now means our variant is free to enter the host airway where it can infect adjacent cells or be expelled from the host to be breathed in by another person and that is the replication cycle for our influenza virus now i don't expect you to get all of that after one viewing of this video it will take multiple viewings for you to fully understand the replication process however i hope that has been clear enough and you shouldn't need to get any more additional information from alternative sources i've tried to put everything in one place for you now just before i finish up this video i said i was going to go over various antivirals and how we can use them to inhibit the replication cycle mainly because the cycle is an attractive target for the development of influenza specific antiviral drugs now i'm actually not going to go through them in any great detail mainly just list the drug names the target and a basic mechanism because what i would like you to do as part of this video's self-directed learning is to apply this knowledge to what we learned throughout the video and determine the knock-on effects of these drugs and what they might do to the replication process as a whole so the first drug i'll mention are the m2 ion channel inhibitors rimantidine and amantidine both of which are not really used anymore as there is widespread resistance to these two drugs but back in the day when they were effective they inhibited the function of the m2 ion channel once in the target host cell and an interesting fact about the m2 ion channel inhibitors is that when they were effective they were only effective against influenza a infections not influenza b or c which can also cause human infections as we learned in previous videos the next group of drugs are the neuraminidase inhibitors and the most commonly known is tamiflu now these drugs stop the release of the virus from host cells and can therefore stop the spread of viral infection within a host so stopping it from spreading to adjacent cells unfortunately just like the m2 ion channel inhibitors there is a lot of resistance to these drugs as is always going to be the case when we apply a selection pressure to something and our final drug baloxavir moboxyl inhibits viral mrna synthesis by targeting the endonuclease activity of the rna-dependent rna polymerase effectively meaning it is unable to snatch the five-prime cap from the host pre-messenger rna and again just like the other drugs there is now a fair amount of resistance out there to this drug rendering it relatively ineffective and that essentially brings us to the end of this video in the next and final video of the series we are going to focus on how change occurs in the influenza viruses and the processes that facilitate this change leading to drug resistance epidemics and pandemics you