Molecular Mechanisms of Virus Entry PDF

Molecular Mechanisms of Virus Entry Ryan Noyce, PhD Research Associate Evans Laboratory [email protected] 1 Lecture Outline • Barriers that viruses encounter • Mechanisms of virus entry • Direct virus-cell membrane fusion • Endocytic network • Cellular receptors used by viruses • Measles virus •...

Molecular Mechanisms of Virus Entry Ryan Noyce, PhD Research Associate Evans Laboratory [email protected] 1 Lecture Outline • Barriers that viruses encounter • Mechanisms of virus entry • Direct virus-cell membrane fusion • Endocytic network • Cellular receptors used by viruses • Measles virus • Cellular receptor usage dictates host tropism and viral pathogenesis • SARS-CoV-2 • Entry of SARS-CoV-2 into cells – tissue tropism • Known cellular receptors/host factors/entry pathways • Emerging variants - Only focusing on ACE-2 receptors there are others 2 what are some cellular barriers that viruses encounter while trying to get into the cell? Cellular barriers to virus entry Grove & Marsh. 2011. JCB - Glycocalyx  umbrella term for outside cell layers Plasma membrane  just underneath the glycocalyx Today  focusing on passing The direct fusion is Ph independent or receptor mediated endosome Glycocalyx = umbrella term for this layer over the cell of carbohydrates etc,? - Plasma membrane sits right under this Actin cortex is under 1. Direct fusion at plasma membrane pH independent 2. Go through RME -> pH dependent 3 what are some major stages of virus entry? Major stages of virus entry 1. 2. 3. 4. 5. 6. 7. Binding to cell surface Lateral movement Activation of signaling Virus endocytosis/membrane fusion Penetration Intracellular transport Genome uncoating (Marsh & Helenius. Cell 2006) - Binding to cell surface  usually lateral diffusion - Activation of signaling  fusion  penetration Trick the cell into lettig them in 1. Envelope virus a. Direct plasma membrane fusion 2. Non enveloped Endocytosis has many ways Example -> Macropinocytosis = poxviruses use phosphadityl serine which tricks cell into thinking its apoptotic and tricks cell 4 Give examples of how viruses have evolved ways to enter the cells? Viruses have evolved many ways to enter cells • https://viralzone.expasy.org/936 1. Enveloped viruses can do direct plasma membrane fusion  endocytosis vs non pores 2. Pox use micropinocytosis  trick into thinking its an apoptic body Ex. ACE2 expressed on MANY surfaces More options the virus has the better -> evolutionarily conserved receptor gives it more optioins Essential for survival of host -> hard for it to downregulate Ex, here binds heparin sulfate proteoglycans -> usually non specific binding response -> engage cellular receptor HveA which is its true attachment protein on the surface 5 What makes a cellular receptor useful for a virus? What makes a cellular receptor useful for a virus? • Ubiquitous expression • Evolutionarily conserved across species • Essential for cell/host survival Jayawardena et al. 2019. Oncolytic Virother Ex. ACE2 expressed on MANY surfaces More options the virus has the better -> evolutionarily conserved receptor gives it more optioins Essential for survival of host -> hard for it to downregulate Ex, here binds heparin sulfate proteoglycans -> usually non specific binding response -> engage cellular receptor HveA which is its true attachment protein on the surface 6 What are some cellular receptors used by viruses? Cellular receptors used by viruses Maginnis. 2018. JMB Sialic acid = first discovered viral receptors (influenza) Measles -> use cell adhesion molecules because they are found on a lot of cells (IgSF and integrins) PtdSer (phosphodityl serine receptors ) _____is the first discovered viral receptor for influenza 7 Endocytic pathways used by viruses Mercer & Helenius, Annu. Rev. Biochem . 2010 Examples: - Clathrin mediated endocytosis - Viruses have evolved many ways to get into cells 8 Measles •rash develops 14d post MeV exposure •one of the leading causes of death among young children – IMMUNE SUPPRESSION •Schwarz vaccine developed in 1965 •In 2017, ~110,000 MeV-associated deaths, mostly among children under the age of 5 •80% drop in MeV-related deaths from 2000 to 2017 due to vaccination Has 3 receptors Takes 14 days for rash to develop -> but you are contagious that whole time STRONG immune suppression within the host -> leads to death in kids Schwarz -> vaccine preventable disease, very good and safe 9 The Measles Virus • Single stranded RNA genome • Negative sense RNA • Enveloped • Replication occurs in the cytoplasm • MeV P, C, and V proteins block innate antiviral responses within host cells (Noyce et al 2012. Curr Opin Virol.) - H protein  responsible for attaching onto the cell receptor - F  fusion protein which allows the virus to fuse into the receptor of the host cell - Different from other viruses such as SARS which has one receptor called spike protein function? The spike protein does both of the things it attaches and diffuses - Anything on the left side has more viral RNA when compared to the last region of the genome  less amount of polymerases Because it is a single stranded RNA, it transcribes in this long region - anything on the left side has more viral RNA compared to right side gene here which is polymerase, so there is least amount of polymerase expressed in the cells 10 Measles Virus Life Cycle SLAM/CD150 MCP/CD46 PVRL4/Nectin 4 There are three different receptors for the viral entry into the host cell: 1. SLAM/CD150 2. MCP/CD46  compliment binding protein 3. PVRL4/Nectin 4  Depending on the cell type measles is trying to infect, it will use these receptors differently  SLAM and PVRL4 are only used by clinical isolates of measles which has been propagated through tissue culture, cells it has adapted a mutation in hemagglutinin to use CD46 as well these two receptors  Only clinical isolates can used CD150 and PVRL4  These receptors allow the virus to get into the cell and then there is replication occurring and budding out SLAM/PVRL4 are only used by clinical isolates of measles - measles that is going to infect you if you are unvaccinated. The vaccine strain of measles which has been propagated through tissue culture cells it has adapted a mutation in hemagglutinin to use CD46 as well as these two receptors. 11 Measles Virus Receptors CD46/MCP • Regulator of complement activation • Expressed on all nucleated cells • Used by other pathogens as a receptor to infect cells • Human herpesvirus 6 (enveloped virus) • Adenoviruses (non-enveloped virus) • Streptococcus pyogenes (bacterium) • Neisseria (bacterium) • Target for gene therapy/oncolytic virus applications MeV strains: cell tropism: • WHY?? vaccine only ubiquitous - Only used by the vaccine strains  not used by clinical isolates or wild – type measles - Found everywhere What makes a good receptor? 1. Ubiquitous 2. Conserved Other viruses also use this as a way to get into the cells. This receptor is a target for gene therapy/ oncolytic virus application why is that? - CD46 is upregulated in cancer cells and clinical isolates don’t use it but because it is upregulated in cancer cells it can potentially replicate in cancer cells. 12 Measles Virus Receptors CD150/SLAM • Signaling Lymphocyte Activating Molecule (SLAM) • Expressed on a subset of immune cells (dendritic cells, macrophages, activated T- and B-cells) • Contain Ig-like domains (Variable or Constant) MeV strains: wt and vaccine cell tropism: Immune cells - Can be used by both wildtype and vaccine strains of measles - Tropism: immune cells - Not found on epithelial cells in the lungs 13 Measles Virus Receptors Nectin4/PVRL4 • Expression is normally restricted to placenta in humans (lower levels in trachea, skin) • High level expression associated with poor prognosis in NSCLC (Takano 2009) • Tumour-associated marker for breast, lung, and ovarian carcinomas (Fabre-Lafay, 2007; Takano, 2009; Derycke, 2010) • Mutations in Nectin4/PVRL4 lead to ectodermal dysplasia –syndactyly syndrome (Brancati, 2010) MeV strains: wt & vaccine cell tropism: Epithelial cells - Got IG like domains similar to SLAM - Upregulated in cancer 14 MeV dissemination & viremia MeV exit via Respiratory epithelium Nectin 4/PVRL4 Takeda, M. 2008. JCI. - Measles virus is an aerosolized virus - The virus comes through the respiratory route and infects the tonsil typically and disseminates to the lymph nodes and once in the lymph nodes it spreads throughout the lymphatic nodes  goes to sinus and secondary lymphoid tissue and it starts to replicate everywhere in the body using SLAM as the receptor - It wasn’t clear how the virus actually exited the host because SLAM receptor is not found anywhere in the lung tissue - There is an epithelial receptor found in the lungs called Nectin 4 which allows the virus to exit the host and is allowed to pass onto next host SLAM receptor is not found anywhere in the lung tissue. 15 MV enters respiratory tract Early infection respiratory tract • DC-SIGN is a measles virus attachment co-receptor • Virus attachment sphingomyelinase activity, SLAM receptor clustering Takeda, M. 2008. JCI. Noyce et al 2012. Curr Opin Virol. - The virus enters via the respiratory route and uses the DC sign as a co-receptor  binds to DC sign on the surface of the dendritic cells which will be found in the respiratory tract - There is no SLAM on the surface but activates a protein called smigonomylase (Smase) which causes the SLAMs found in the vesicles, on the surface of dendritic cells to translocate to the cell surface and the virus uses the SLAM on the surface to infect the dendritic cell - Once the dendritic cell is infected or the macrophage  the virus can travel to the lymph nodes where there are a lot of T and B cells present and here it can infect the immune cells 16 MeV enters respiratory tract Infected lymphocytes enter blood stream Immune cell infection lymph node Takeda, M. 2008. JCI. Noyce et al 2012. Curr Opin Virol. - These cells typically have SLAM already on their surface and an infected dendritic cell comes by and this infects a T cell 17 MeV dissemination & viremia MeV exit via Respiratory epithelium Late infection (virus spread) airway lumen Takeda, M. 2008. JCI. Noyce et al 2012. Curr Opin Virol. The virus disseminates through the body but now somehow needs to get out of the cell or the host - The virus fuses into nectin 4 for exit - Nectin 4 is present between adherent junctions which are present between two adjacent cells in the periphery in the respiratory tract - T, B and dendritic cells can come and migrate to the respiratory cells  to the lungs - The virus within these cells will bind to nectin4 and gain entry into epithelial cell and now the virus exists 18 Summary of Measles Virus Receptors & Disease Progression • MeV has both attachment (receptor binding) and fusion glycoproteins • Wild-type MeV uses at least two receptors (SLAM & Nectin-4) to infect and exit its human host • SLAM-mediated MeV infection of T and B cells results in virus dissemination and immune suppression • Nectin-4/PVRL4-mediated infection of lung epithelial cells (basolateral surface) is necessary for virus release - Mev has true attachment receptors  receptor binding and fusion glycoproteins H protein  attachment F protein  fusion Literature suggests there is neurotropism associated with measles virus People have been looking for neurotropic receptors and neuropile has been considered as a receptor but there is not evidence of that - There is a cool paper where they looked at the antibody repertoire in blood and they showed that people who have never been vaccinated for measles and then they got measles they lost immunity to other viral pathogens that they had previously generated immune responses to. This happened because the measles virus will go and kill all the B cells 19 Lecture Outline • Barriers that viruses encounter • Mechanisms of virus entry • Direct virus-cell membrane fusion • Endocytic network • Cellular receptors used by viruses • Measles virus • Cellular receptor usage dictates host tropism and viral pathogenesis • SARS-CoV-2 • Entry of SARS-CoV-2 into cells • Known cellular receptors/host factors/entry pathways • Emerging variants - A lot of this was discovered in last 3 years 20 Genomic organization of SARS-CoV-2 All Non-structural Proteins doi.org/10.3390/pathogens9050331 - This is a positive sense RNA virus - Makes one giant polyprotein - Has proteases in the genome that are involved in cleaning of the polypeptides into individual proteins - NSP3  Non-structural protein - ORF1B all non-structural protein - The structural protein lies in the end we will only talk about the SPIKE protein NS stands for non-structural proteins and ORF1A/B all non -structural proteins 21 SARS-CoV2 Entry into cells doi.org/10.1038/s41580-021-00418-x Unlike measles virus there is only 1 true attachment protein on the surface of the SARS COV2 virion  spike 1. It can either enter through endosomal entry  virus binds to ACE2 and enters via an endosome which leads to activation of cathepsin L which cleaves the spike protein and allows the virus to fuse using endosome membrane and release its contents into the cell - this happens in a number of different cell types - This depends on whether there are co-receptors along the surface of these cells like TMPRS2 is also a protease - The cathepsin L is found within an endosome we have TMPRSS2 on the surface of the cell to ACE 2 via - When TMPRSS2 Is present, the virus can bind via spike 2. TMPRSS2 causes cleavage of the S2 subunit These two are the pre-dominant pathways of SARS- cov2 entry into cells 22 Comparing the structure of SARS CoV-2 Spike with other -coronaviruses PPC – proprotein convertase site ( FURIN) doi:10.1073/pnas.2003138117 - The study compared the spike COV2 spike to other coronavirus virus including BAT and SARS COV1 - SARS cov2 in the S1/S2 area had a RRAR residue which is a coat protein converting site this site is recognized by furin and furin can cleave at this site - SARS cov1 doesn’t have this and doesn’t use this furin as a cleavage site - In cov2 furin cleaves between the S1 and S2 domains and this occurs in the virus producing cell Virus enters the cell  replicates  as it is packaging and releasing that is when the furin will work The virus has been pre-cleaved at the S1/S2 site upon release of the virus particle from the human cell Now the virus can attach to a target cell that has ACE2 on the surface and a cellular or endosomal protease will go and provide a second cleavage  either using TMRPSS2 or cathepsin L and this secondary cleavage will expose the fusion peptide within spike to induce the fusion 23 Pseudotyping viruses to study virus entry doi: 10.7150/ijbs.59184 • Spike protein expressed from a plasmid & infected with modified lentivirus expressing GFP reporter virus (turns cells green) – only one round of infection • Newly-made pseudoviruses (PV) incorporate the Spike glycoprotein in their viral membrane Advantages? Safety Limitations of this assay? Only one round of infection Role of other viral proteins - The most popular way that scientists use to look at virus entry 1. Take a lentiviral vector + spike + for SARS cov2 and co- transfect cells which will express the viral vector decorated with the glycoprotein of our interest 2. In this case we are using spike as the glycoprotein but in theory we can use other viral proteins, the most common one is the VSG V pseudo type virus which uses which uses G glycoprotein on the surface which will target the cell to infect any cell that can bind to that gene protein 3. Take the viruses that are made in the system and infect cells that have no other receptor  You can see whether it is a pseudotype virus or not , get into the cell  Typically, lentivirus is modified to express some sort of reported such as GFP reporter  Once the virus gets in, it will do one round of infection and the cells that get infected will turn green  Using this method we can report on the efficiency of the infection of that virus These newly named pseudo viruses will incorporate glycoprotein into their viral RNA What are the advantages to using a pseudotype virus? We didn’t know that SARS cov2 is a level 3 pathogen so you need to have a level 3 facility but pseudo typing allowed many people to work on the SARS cov2 research “ Safety” Limitations: - You don’t know the role of other viral proteins  you made a virus that looks like SARScov2 but it doesn’t have rest of the genome (add more notes) - You can get false positives  24 Evidence of proteolytic cleavage of SARS-CoV-2 Spike doi.org/10.1016/j.cell.2020.02.052 - They wanted to know if the spike protein of SARS cov2 demonstrated proteolytic activity - At this time they didn’t know that it had EPC motif at the furin cleavage site at S1/S2 1. Expressed spike SARS cov2 in cells 2. Looked to see whether or not they saw cleavage  they could see spike being cleaved into S1and S2 3. SARS-S no cleavage when expressed into cells and SARS cov2 spike  cleavage 4. First evidence to suggest that SARS cov2 spike cleaved 5. Now they wanted to check not only in the cell lysate but also in the pseudotype virus whether or not there was cleavage  to show if furin cleavage is happening on the way out of the cell - in all the virus particles they can detect pre-dominantly the cleaved spike - furine is cleaving the pseudotype virus as it is exiting the cell Take lentivirus + a spike gene (spike gene receptor of covid) and transfect into cells You can take these viruses and infect cells that have known or unknown receptors to see is pseudotype virus can get into the cell Lentivirus modified to express some sort of reporter (GFP reporter etc. So in the vector you originally added has this fused) Cells that were infected turned green or glow by reporter gene Advantages = virus is not harmful (main advantage), sars covid 2 is a level 3 pathogen so this allows studying of it without harm. Limitations - False negatives (coreceptors not present on cell etc.) - One round of infection You do know role of other viral proteins that are no longer present in this. (you got rid of the rest of the virus genome ! This may be important for how it all works together) 25 SARS-CoV-2 enters similar types of cells as SARS-CoV doi.org/10.1016/j.cell.2020.02.052 What type of cells could the pseudotype virus can infect? - Positive control  pseudotype virus VSV-G  is able to infect a lot of cells - Each bar is a different cell type  human, animal, bat, hamster, mouse - When you put pseudotype virus into any of these cells you get some level of entry of the virus  clear reporter expression in all of the cells - SARS-S  saw similar to what they saw in 2002 when they were doing similar assays on sars cov1 that It was a subset of cells but most of the human cells are getting infected and there was a canine cell line - The receptor that is being used by SARS cov1 is also being used by the SARS cov2 infection 26 Structure of the SARS-CoV-2 virion and spike attachment/fusion protein RBD – ACE-2 binding NTD – HSPG binding doi.org/10.1371/journal.ppat.1008762 - SARS Cov2 on the surface of the virion is a trimer of S1/S2 subunits - The receptor binding domain  where ACE2 is going to bind to spike is on the tip of the surface under some confirmation - Traditionally, there is an N-terminal domain of spike that is involved in HSPG - heparin sulfate proteoglycan binding which is an inefficient attachment receptor binding - The actual interaction is between spike and cellular receptor - In theory, any cell expressing ACE2 a virus needs to get in  the virus doesn’t need to replicate in the cell 27 SARS-CoV-2 Membrane Fusion Fusion peptide proximal region (FPPR) clamps down RBD in prefusion S trimer RBD binding to ACE2 exposes the S2’ cleavage site (secondary cleavage by TMPRSS2 or Cathepsin L) Cleavage at the S2’ site releases structural constraints on the fusion peptide (major conformational changes in S2) Post-fusion structure of S2 forms, which brings the cell and viral membranes together, facilitating formation of the fusion pore and virus entry See Janet Iwasa of SARS-CoV-2 Entry if interested The FPPR region of spike it clamps down RBD in monomer of RBD  prefusion - On viral membrane we have RBD down confirmation  for spike we can have RBD in two confirmations - One up confirmation  one of the monomers are in the up confirmation or 3 down confirmation  none of the monomers are in up conformation - You need RBD to be in an up confirmation in order to be recognized by ACE2  exposes S2 binding site  TMPRSS2 (on cell membrane) + catheplsin 2 (endosome) to do secondary cleavage - The secondary cleavage is important to expose the fusion peptide  release the structural constrains on the fusion peptide and allow and causes a huge confirmational change in S2  the whole system gets punched into the cell membrane - Folding back of HR2 which allows these two membranes to come close to that position and you get a formation of fusion pore  the virus can come and fuse into the cellular membrane 1. First cleavage happens before the virus releases out of the cell  between S1/S2 2. The second cleavage occur when one of the RBD up conformation binds to ACE2 and falling off of S2 subunits after cleavage with TMPRSS2 s1 28 Architecture of S protein on the virion surface doi.10.1126/science.abd4251 - What is the experiment ? - People can see two versions of the spike on the surface of these virions : 1. S1+S2 in complex (trimer) 2. Post fusion confirmation - Because you are getting pre-cleavage between S1 and S2 as the virus is exiting the cell that there can be some spontaneous dissociation of S2 - The S2 spontaneously dissociates and the spike on the surface of the virus will adopt post fusion confirmation Can see 2 versions of spike 1. S1/S2 in complex (trimer conformation) Post fusion conformation Because you get pre-cleavage (S1 and S2) before virus leaves cell there ca be some spontaneous dissociation of S2 because you do not need a lot to go into post dusion form. S2 spontaneously leaves which adopts post fusion conformation 29 Architecture of S protein on the virion surface Evidence? • EM images of -propiolactone-inactivated SARS-CoV-2 contain only post-fusion S2 • EM of PiCoVacc (inactivated SARS-CoV-2 vaccine candidate) show needle-like spikes in 2020 • Binding Abs against S2 are detectable in COVID-19 patients • S2 may be more exposed to the host immune system Considerations for current/future vaccine development? doi.10.1126/science.abd4251 - This was found on the EM images of inactivated SARS cov2 - One would imagine when developing a vaccine when you are in the post fusion phase and recognizing post fusion viruses that have come through the vaccination strategy that your B cells will recognize post fusion spike as oppose to pre fusion spike - This can create an immunodominant effect that you will be making antibodies which are not useful in neutralizing SARS cov2 pre SARS cov2 but only post SARS cov2 - All of the mrna vaccines have prefusion locked state of spike in their surface so that they don’t have - Lock spike in its pre-fusion state so that when it is expressed, it is only existing in the S1/S2 complex --. There is no post fusion spike that is being made Only post fusion S2 on the surface of the vriions Saw ONLY needle like spikes In post fusion state -> your b cells will recognize post fusion spike -> could cause immunodominant effect not useful in neutralizing prefusion covid 2 only post fusion Is the reason for prefusion lock stage of spike in mRNA vaccine, they have locked SPIKE in pre-fusion state so its only in S1-S-2 and no post fusion antibodies made against the vaccine 30 Pathways that SARS-CoV-2 uses to get into cells 31 SARS CoV-2-cell fusion at the cell surface 1. The S1 and S2 exists in the trimer form and are recognized by ACE2 2. There is already a prefusion cleavage between S1 and S2 by furin but TMPRSS2 can also cleave but it predominantly cleaves at the S2 prime site and the S2 prime site is important for S1 fall off and expose major conformational changes in S2 exposes the fusion peptide which allows the virus to engage with target cellular protein 3. Fusion of viral post membrane and release of contents into the cytosol 32 SARS-CoV-2 is resistant to endosomal protease inhibitors in TMPRSS2 expressing cells Inhibitor: Cathepsin B/L Cathepsin L Cathepsin B Inhibitor: Cathepsin B/L Cathepsin L Cathepsin B doi.org/10.1371/journal.ppat.1009212 In cells that already have TMPRSS2 on their surface and ACE2  use psueodytpe virus in the presence of the inhibitors, they can see that as they increase the inhibitor, there is a lot of infection in the presence of TMPRSS2 and the virus is able to use it in the cell lines In TMPRSS2 negative cells, when they use inhibitors that have cathelpsin L and there is an absence of TMPRSS2  can’t fuse through cell membrane  inhibit cathelpsin L , you get no infection with Inhbition of cathepsin B had no major affect on the ability of SARS cov2 to enter cell This study showed that cathepsin L is important for cells that are TMPRSS2 + for SARSCOV2 to enter the cell TMPRSS2 - 33 The role of proteases in SARS-CoV-2 entry (furin cleavage inhbitor) (TMPRSS2 inhibitor) (lysosomal cathepsin inhibitor) doi/10.1073/pnas.2003138117 - Hela cells are predominantly expressing ACE2 in the presence of the furin cleavage inhibitor, TMPRSS2 inhibitor or lysosomal cathepsin inhibitor - In the presence of TMPRSS2 inhibitor forcing that virus to use endosome mediated entry and it can still go in but there is a decrease in the efficiency - When you use TMPRSS2 in the presence of cleavage furin inhibitor you decrease that even more and in these cells when you use cathepsin inhibitor, you drop the level of entry and then when you use the furin cleavage - In this assay we are looking the role of cathepsin L and TMPRSS2 and you can see there is an affect - The others are reference cell lines  respiratory cell lines  if there is a furin or TMPRSS2 inhibitor it prevents the pseudotype virus from gaining entry into the cell 34 SARS CoV-2 cell fusion within endosomes • Cells with low surface TMPRSS2 • SARS-CoV-2 entry via cathepsin-Lmediated endocytosis • Cells with high surface TMPRSS2 • SARS-CoV-2 entry at cell surface A role for hydroxychloroquine? hydroxychloroquine? Only in cells lacking TMPRSS2 Cathepsin L mediated endocytosis: 1. Virus binds to the ACE2 receptor on the cell surface 2. Virus becomes internalized into the endosomes --. Activation of cathepsin L 3. This allows for fusion at the secondary cleavage site Does hydroxychloroquine blocks this pathway? - Went into many clinical trials with limited efficacy and looking at early events - You cannot inhibit this specific event because there are other cellular processes - You can only use HC when there is no TMPRSS2 present 35 Inhibitors of SARS-CoV-2 entry •doi.org/10.1371/journal.ppat.1009212 - Don’t know if anything has changed as the variants change - HC will be working in the cathepsin mediated activation but if TMPRSS2 is present, HC is not going to be useful and you will have to use camostat which blocks TMPRSS2 36 Therapies that target SARS –CoV-2 entry mAb therapy (Bamlanivimab) Why aren’t these therapies clinically used anymore? www.biorender.com - These therapies are not being used anymore - Some of the strategies involve using soluble RBD mimetics  make RBD mimetic and binds to ACE2 and block the ability of SARS cov2 to bind 1. The downsides of targeting the entry mechanisms is that the virus is evolving ways to get in These are no longer being used Use soluble RBD -> binds ACE2 and block covid from binding ace2 But ace2 is important for other things, may block endogenous functions (it actually might be fine though, ace2 binding site may be different) a. Mutations occuring in receptor binding domain of covid and it mutates a bunch so it may not be able to work. Maybe combo antibody therapy could neutralize? COVID just changes too much (RBD mutates too much) none of the monoclonal antbodies are working on omnicron anymore i. Virus evolves ways to get away from this 37 The emergence of SARS-CoV2 variants 38 First SARS-CoV-2 adaptation in humans • D614G lineage - This data is from the first 5 months of the pandemic - The ancestral SARS cov2 circulating had a mutation in spike D614G and it was an aspartic acid - In February in Europe, the D614 mutation emerged and it overtook the whole population g614 Mutation D 614 -> G In animal studies and population, as you get G614 mutation there is more replication in upper respiratory infection and there was greater transmission - So somehow this mutation allows replication more in upper resp - Also there was increased antibodies to this mutant -> present in pop Before mutation Inbetween S1 and S2 loop, this loop leads to more unfolded strucutre (less energy required to keep it folded) allows to be in RBD up conformation most of the time (instead of 3 down) - Bad -> can get premature host fusion Mutation occurs - Now you need more energy to unfold that loop - So you need more energy to get RBD one up, you have 2 other RBD down - RBD one up more likely to get productive infection and not fuse prematurely Extra energy stabalizes the conformatio of the other 2 S1 monomers to make sure they do not dissociate and do pre-fusion stuff - More efficient -> prevent pre mature shedding of S1 on spike 39 Emerging variants of SARS-CoV-2 • D614G lineage energy •Increased replication in URT in animal models InfecttiroannswmitihssGio6n14 is more efficient because it prevents premature S1 shedding on Spike • neutralization Abs in naïve population doi:10.1126/science.abi4711 - Between S1 and S2 is where you are getting cleavage and you have a mutation from D to G - We saw that in animal studies as well as in the populations, when we start getting the G614 mutation, we saw more replication in the upper respiratory tract in animals and saw more transmission - In naïve populations there was an increase in the neutralizing antibodies - Original strain --. D614 and that is found right in between the S1/S2 and very close to the 630 loop that is formed close to the crystal structure. There is lesss energy required to unfold this loop and this allows RBD down to be in RBD 1 conformation most of the time  RBD one all the time  less energy required to unfold it then you can get premature post fusion pathway - If you have RBD1 up you can get dissociation of S1 and S2 - When the mutation occurred, the mutant required more energy to unfold the loop and needed more energy to get RBD1 up - You get two RBD1 down which were energetically more stable and this allowed RBD1 up to more likely bind to ACE2 and get a productive viral infection as opposed to having pre-mature post fusion infection with G614 was more efficient because it prevented pre-mature shedding of S1 40 Possible reasons for the emergence of SARSCoV-2 variants • In a naïve population, virus has very little selective pressure on it, and can enter its host and replicate freely - As the population gets more infected or potentially vaccinated there is an increase in the antibodies targeting spike - Increase in antibodies targeting spike lead to an immunodominant epitope and now the virus has to figure out a way to infect this population where there are more circulating antibodies which results in a selective pressure on the virus to try and escape - If SARS cov2 continuously mutated one of these mutations will allow it to escape selective pressure - Antigenic drift 41 Possible reasons for the emergence of SARSCoV-2 variants • As more of the population becomes infected/vaccinated – increase in antibodies targeting spike (immunodominant epitope) Population infected More Circulating Abs 42 Possible reasons for the emergence of SARSCoV-2 variants • This results in a selective pressure on the virus to try and ”escape” ANTIGENIC DRIFT (Variants Emerge) SARS-CoV-2 mutates to escape this selective pressure 43 Summary of SARS-CoV-2 entry • SARS-CoV-2 Spike is the viral attachment and fusion glycoprotein • The only known cellular receptor for SARS CoV-2 entry is ACE-2 • SARS-CoV-2 can enter cells via endosomes or fusion at the cell surface • Furin cleavage of S1/S2 on Spike is required for infection of humans • A second proteolytic cleavage step is necessary for CoV-2 entry • Human adaptation of SARS-CoV-2 (D614G) reduces S1 shedding, resulting in more Spike molecules ready for fusion (in the one-up conformation) - Uses proteases from the host to cleave it and get fusion - Others are looking at neurological receptors 44 For next class • Read the following paper and be prepared to discuss their findings 45