Virus Entry: Attachment and Receptors

Virus Entry

• Virus entry consists of attachment, penetration, and uncoating.

Part I: Attachment

• Viruses attach to cells via cellular receptors like proteins and carbohydrates.

Function of Cellular Receptors

• Cellular receptors mediate virus attachment, trigger conformational changes for membrane fusion or penetration and Interact with other receptors.
• Targeting of virus occurs to low pH compartments, resulting in penetration or fusion

Affinity and Avidity

• Affinity is the strength of a single molecule's interaction with another.
• Avidity measures the overall strength between a virus and a receptor.
• Avidity is more than the sum of individual affinities, and affinity is often low while avidity is high.

Identification of Viral Receptors using Monoclonal Antibodies

• Immunizing a mouse with a target cell containing membrane receptors generates antibodies from plasma cells.
• Fusing a plasma cell with a cancer cell makes a hybridoma, each producing a specific antibody.
• Monoclonal antibodies are used for neutralization assays to prevent infection.

DNA Cloning and Transformation

• Genes can be isolated and cloned, leading to a loss or gain in infection susceptibility.

• cDNA is isolated from cells sensitive to a virus.

• Transformation: cDNA introduced to insensitive cells makes them express the receptor and become sensitive.

Verifying Successful Infection

• Cells are transformed with the virus containing a drug resistance gene.
• Cells expressing the receptor become infected.
• Adding the drug kills non-sensitive cells.

Alternative Infection Indicators

• Inserting a GFP indicator gene into the virus genome allows for tracking of viral replication with fluorescent markers.

• Antibodies against the cellular receptor can also be used as fluorescent markers.

• One type of receptor can be used by different virus families.

• CAR functions as a receptor for both Coxsackievirus (an RNA virus) and Adenovirus (a DNA virus)

• Viruses from the same family may use different receptors: Coxsackievirus uses the CAR receptor, while Rhinovirus uses the LDL receptor

• Viruses can bind to multiple receptors: Herpes Simplex Virus binds to five receptors. This includes gB and gC (bind to heparan sulfate proteoglycan), gH/gL (bind to integrins), and gD (bind to Nectine-1, HVEM, or 3-OS-HS).

• Ebolavirus attaches via its glycoprotein (GP) to the host cell.

• The GP contains a mucine-domain and glycan cap that aid binding and endocytosis.

• GP is cleaved in the late endosome by cysteine-proteases into GP1 and GP2 subunits, exposing a binding site for the NPC1-receptor.

• Binding to NPC1 leads to fusion of viral and endosomal membranes, releasing the viral genome into the cytoplasm.

• Niemann-Pick type C1 disease, lacking NPC1, confers resistance to Ebola infection.

• Naked viruses use capsid proteins to bind receptors.

• Enveloped viruses use transmembrane glycoproteins to bind receptors.

• Poliovirus is an example of Picornaviruses.

• The receptor, also known as poliovirus receptor has 3 Ig domains, which uses it’s N-terminal Ig domain to bind to VP1.

• 60 VP1 proteins are present which allows poliovirus to bind to 60 CD155.

• Rhinovirus is another picornavirus which can bind to LOL receptors or ICAM-1 receptor.

• Adenovirus is a naked virus that has external fibers. These fibers have trimers which are anchored in the penton base which possess knob, shaft and tail.

• Fiber knobs mediate contact with the receptor (CAR) via the AB loop.

• Some viruses use gangliosides to attach.

• Gangliosides concentrate in lipid rafts. They are glycosphingolipids with sugar and sialic acid residues. An example is GM1, found in lipid rafts.

• Caveolae are membrane invaginations stabilized by Caveolin and Cavin-1 in cholesterol-rich areas called lipid rafts

• Influenza viruses bind to negatively charged terminal sialic acid molecules on cell surface glycoproteins or glycolipids.

• Sialic acid’s presence enables attachment to various cell types.

• The interaction between influenza and sialic acid is low affinity, but high avidity due to multiple hemagglutinin (HA) molecules

Neuraminidase

• Neuraminidase cleaves glycosidic bonds of sialic acid, which is needed for the release of viral.

• The Hemagglutinin protein binds to sialic acid.

• HA-monomer is glycosylated and cleaved into HA1 and HA2 subunits.

• Each HA-monomer has a helical stem (HA2, membrane fusion) and HA1-globule (sialic acid binding).

• Influenza A strains vary in their affinity for sialylooligosaccharides

• Human virus strains prefer sialic acid linked via an α(2,6)-glycosidic bond to galactose found in the human respiratory epithelium, while avian strains prefer α(2,3)-linkages, common in duck intestines.

• Amino acids in the sialic acid binding pocket determine host tropism

Receptor Interaction

• Bepaald virus tropisme: de mogelijkheid voor een virus om een bepaald celtype te infecteren.

• Bepaald virus host range: de mogelijkheid voor een virus om een bepaald organisme of celcultuur te infecteren.

• Coronavirus binds its spike protein to the ACE2 receptor, with adaptation allowing for interspecies transmission.

• Human SARS-CoV has a higher affinity for ACE2 receptor.

• HIV-1 binds is a retrovirus that possess a gp120 receptor binding domain known as the surface unit; SU - CD4 binding.

• The HIV-1 receptor bindings takes place when CD4 binds to the cavity of the SU.

• The CD4 has a phenylalanine 43 which is an essential component for binding.

• Resistance to HIV infections can result from mutations in the co-receptor CCR5.

• Stepwise receptor binding for successful HIV binding:

• CD4 (primary receptor)

• CCR-5 (co-receptor for macrophages)

• CXCR-4 (co-receptor for T-cells)

• SU binding to CD4 induces conformational changes in gp120, exposing the V3 loop for co-receptor (CCR-5/CXCR-4) binding.

• Co-receptor binding triggers further conformational change in the TM, exposing the fusion peptide.

• The fusion peptide is hydrophobic and can insert into the host membrane.

• Successful viral infection requires susceptibility and permissivity.

• Susceptibility: the host cell has the correct receptors that allows entry in to the cell.

• Permissivity: Host cell contains all machinery for replication, assembly and virion freedom.

Part II: Penetration + Uncoating

• Enveloped viruses either fuse with the plasma membrane or enter via endocytosis followed by fusion with endosome/lysosome.

• Naked viruses enter via endocytosis or pore formation.

• Fusion requires energy, fusion proteins and a regulated process by membrane proteins.

• Viral fusion proteins are membrane-anchored glycoproteins located on the viral envelope.

• Fusion proteins have fusogenic peptide that facilitates cell-membrane binding and destabilization.

• HIV fusion peptide inserts into the cell membrane.

• Fusion proteins undergo conformation changes upon binding to a receptor or exposure to low pH.

• Some viral glycoproteins can induce fusion only after cleavage by a host protease.

• Viruses can use host-cell factors to activate membrane fusion by receptor binding.

• Example: HIV-> CD4 receptor leads to activation of the fusion proteins.

• Examples where Viruses use gastheercelfactoren can be triggered when pH is low in endosomes.

• Example: Influenza -> low pH in the endosoom activeert fusie.

• Sindbis virus and VSV) used in the mechanism.

• Kombination of factors required for membrane fusion where receptor binding and low pH are needed.

• Avian leukos virus (ALV) has both: Receptor binding + low ph is needed to start fusion.

• Paramyoxviruses use only receptor interactions where the kapsel fuse directly with the plasmamembrane. Die have 2 main enzymes:

• F-enzym=Fusion enzyme.

• Hemagglutinine neuraminidase (HN) eiwit, this is receptor binding eiwit.

• As soon as the enzyme has been cleaved by protease the F0 binds with the sulfide binding on the other cell membrane.

• Follow has a 2 step process

• Linkage of N-terminale kant van het F1 bestaad uit 20 hydrofobe aminozuren die het fusie peptide vormen.

• Then Binding is place by the HN with the new host that leads to Enzyme conformation.

• The new Conformation in F1 causes direct Membrane fusion.

• HA = trimer + alfa helices

• Influenza virus required fuse lage pH

• Hemagglutine (HA) glycoproteine – siaalzuur bevattende receptoren

• Clathrine afhankelijke endocytose

• HAI: This component hold the fusiepeptide loop, and inside this lopo the cleavage takes place.

• Protease has been created the HAI LOOP: loop has been created than to has 2 steps and finally it connect on the fusiepeptide.

• Laag pH (5) conformationeele verandering -> hairpin is created.

• Fussiepeptide will go to a direct acces. And lastly it creates a fused Pore and created viralev ribonucleoproteins (vRNPs), RNA (ribo) and finally the complex proteins.

• All the influance has 8 vRNPS

• Uncoating Influenza virus

• Influanca does pack everything Inside the vRNVS, also the the eiwit M1= matrix. Uncoanting is require the the dissiciation with the enzym

• There voor M1 is important

  • Bindt vRNPs -> maskeert nucleaire lokalisatie signaal (NLS) Stabaliseer the structuuuuur door de interactie aan te gaan met Neuraminidase (NA) en Hemagglutinine (HA).

M2 pompt H+ in het virusdeeltje zodra het endosoom verzuurt. Verlaging van de pH zorgt dat het M1 eiwit een conformationele verandering ondergaat en loskomt van envelop en vRNPs.

• Therefore fusie first requires a cleavage , than de Ph must decrease to de M2 enzum gets on. Where dose the A repplication takes place? It reqauires also the the RNA vyrusussen de the the cytosma.

• Semilixi Forest Virus: cellulaire eiwitten -> uncoating

  • Alfavirussen, RNA, envelop
  • Endocytose the fuse de envelop with the endosoom

• The klasse II creates a new fuse enzyme the Nucleocapside -> ctoplasa

• And finally the coating wil been created aan the eiwit

• HIV - lentivyrusen the has direct the fuse action with the correct CD enzyme And de correct enzyme are been created inside the the fuse.

HIV structuur:

• Envelop Capside Nucleo capside
  • Fuse of the HIV has been created

CD4 + gp120 = conformatie verandering V3 loop -> CXCR-4/CCR-5 De Conformatie has changed naar de gp 41 direct en het fusie peptide action.

• Enzyme Cyclofiline is been used to de the binding on the capside eiwit.
  • Entry vaan makete vurusen:GEEN FUSSI
• The capside will change also their formation and can interact diract aan and cell and therefore perforeren the mebraanen .
• The newentry is required the the and de enzymes in that cell.
  • For poliovyrws the receptor binding takes place

For Picornavirus to happen the step has to be fallowing

• Pur, 1VP1 must bing with eiwit's that normallise the kapasiid to get and disabize the new capsilde.
• With 4 with a changed will then shedding
• Membrane, pore formaltion with th the that plasma.
• Sfingoise kan be verplaast if there are drugs that ha a better and higher affiteit and dus the infection has failed.
  • Ademvirusser use Recepttor aan lage PH and de enzymen die het helpen:
• Fiber knob- carb receptor.
• Enzimine- pentoon base.
• De cathrin will also work together also the rest can been moved easily if not reqioored
• De endosomen must also have a large PH so that the PE can work.
• Proteins Vi, insertia of the mesbraa aan rupture endosoom
• 7. microtubuli, De nuclaire poor ->kern

Buitenste capside shell wordt in endosoom + lysosoom door cathepsine proteases geproteolyseerd

• Binneste shell capside is hydrofoob en medieerd penetratie lysosomale membraan

• DNA viruses require replication for is is where al the meshinery is.

• RN viruses , are replication on cytoplasma How gets the vurus en de kern the steps

• De gerhele viron: paruvirus. RNQ: de virmus and herpus can only been broken by the virus.