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Principle of molcular virology > Lecture 4 - Viral entry into living cell > Flashcards

Lecture 4 - Viral entry into living cell Flashcards

1
Q

Define a viral target

A

Viruses have unique targets - their ability to enter, multiply and disseminate from a cell depends on cell specific characteristics they exploit ‘killing of the target’

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2
Q

Define a viral host

A

Viruses have to enter the cell without damaging th host needed for replication

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3
Q

Why does the mechanism of entry of a virus vary?

A

Depends on the type of host and type of virus

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4
Q

What host cell properties of animal cells affect the specific entry mechanisms of viruses?

A
  • Have to overcome the lipid membrane (fluid membrane good for viral entry).
  • Specific characteristics affecting entry mechanisms: Surface molecules, membrane fusion (enveloped viruses) and transport processes
  • Entry is receptor mediated and can result in the delivery of nucleic acid and protein shell
  • Animal viruses can be enveloped/not-enveloped
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5
Q

What host cell properties of bacterial cells affect the specific entry mechanisms of viruses?

A
  • Have to overcome the cell wall (protects inside membrane of bacteria, not as rigid as that of plants)
  • Specific characteristics affecting entry mechanisms: Surface molecules, the plasma membrane, and cell wall
  • Bacterial viruses use receptor mediated entry resulting in the delivery of nucleic acid (and less often the protein shell)
  • Bacteriophages are mostly non-enveloped.
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6
Q

What host cell properties of plant cells affect the specific entry mechanisms of viruses?

A
  • Have to overcome rigid cell wall
  • Specific characteristics affecting entry mechanisms: cellulose cover and intercellular channels.
  • Uses passive entry, vectors (insect) injection, mechanical damage
  • Plant viruses are non enveloped
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7
Q

How is bacteriophage entry mechanisms related to their structural organisation?

A

Bacteriophages come in many structures, mostly have a well structured head containing genetic material and a tail to inject genetic material

May be enveloped and so use different mechanism of entry

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8
Q

What is the aim of a virus?

A

To enter a live target cell to release their genetic material and allow replication

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9
Q

How do enveloped/non-enveloped viruses enter a cell?

A

Both need a strong enough attachment that the virus knows it is attached to a target cell.

Enveloped:

  • by fusion.
  • Envelope from previously infected cell used to get inside by lipid fusion and release capsid in cell.
  • Capsid can contain genetic info and other proteins, e.g. enzymes to help with replication

Non-enveloped:

  • by lysis/pores.
  • Attached to target then use a mechanism to create pore and insert target protein inside cell.
  • only genetic info inserted into target cell with maybe one or two accessory proteins
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10
Q

How does a virus achieve attachment to its target cell (G-, G+, Mammalian cells)?

A

Bacteria:

  1. First virus needs a non specific general attachment to cell surface molecules.
    • Gram - membrane protected by lipopolysaccharides, virus only has access to LPS or membrane spanning protein.
    • G+ has peptidoglycan layer containing lipoteichoic acid (virus needs to find a way to bind to these as not many specific receptors on G+)
    • Mammalian cells: there are a lot of carbohydrates and sugars that are exposed and give a good non specific target for viruses to attach to.
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11
Q

What general membrane components can a virus attach to?

A

Animal cells:

  • Plasma membrane
    • Sialic acid (has lots of sugar branchings),
    • Heparan Sulfate proteoglycan (HSPG) (proteoglycan moves around and allows cells to catch nutrents floating around.)

Bacterial cells:

  • Lipopolysaccharide (GRAM-)
  • Teichoic aids (GRAM+)
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12
Q

What needs to occur after non-specific viral attachment to a target cell?

A

Specific attachment

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13
Q

What proteins can a virus bind to for specific attachment to animal/bacterial cells?

A

Animal:

  • Protein could be part of the immunoglobulin family
  • CD55, LDL receptors, integrins, small GRCRs, chemokine receptors, c-type lectins

Bacteria:

  • In Gram - (inner membrane, periplasm and outermembrane) virus uses the transmembrane protein in the outer membrane to attach specifically
    • OmpA (transporter protein)
      • Omp complex is a target of a lot of bacteriophages.
    • Can also bind glycolipids, flagellin proteins.
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14
Q

How is efficient attachment achieved by viruses?

A

By combining general and viral specific attachment

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15
Q

What general and specific targets does the HIV virus use?

A
  1. Sense and land through heparan sulfate then attaches to two receptors.
  2. Glycoprotein CD4 immunoglobulin protein, which is found only on a number of cells
    1. Can have stong HIV binding to CD4 but needs second receptor to progress
  3. Coreceptor (chemokine receptor)
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16
Q

What general and specific targets does the T4 bacteriophage use for attachment?

A

Initial binding through LPS, but interaction needs to be stabilised by the receptor.

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17
Q

Once a virus is attached, how does it get ready for entry?

A
  • Need specific conformational and/or structural changes of the virus (especially in bacteriophages)
  • Triggered by complex, strong and specific virus-receptor interactions
  • Leads to the formation of an activated viral intermediate ready to trigger entry
  • mechanisms are virus specific and can engage various cellular factors
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18
Q

How can blocking a virus be important in therapeutic approaches?

A

If can’t block attachment could instead block conformational changes of the virus and not get entry

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19
Q

How does the T7 bacteriophage get ready for entry?

A

Undergoes extensive structural remodelling during infection.

  1. Weak attachment to LPS, at which point the virus can come off.
  2. Followed by firm attachment to LPS and receptor.
  3. Tail of the bacteriophage recieves a signal when bound and begins to change.
  4. With the help of long extensions on its structure it contracts and allows puncture devise in the core to move forward.
  5. Further contraction induces changes in core head meaning the plug moves away so the head opens and releases nucleic acid.
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20
Q

How does the bacteria enveloped phage φ6 get ready for entry?

A

Enters by fusion

  1. binds to bacterial pili (glycan)
  2. pilus then retracts down to the bacterial outer membrane (example of cell helping the virus)
  3. Virus undergoes fusion, loses the outer membrane (of which it has two)
  4. uses protein under outer membrane to enzymatically destroy the peptidoglycan cell wall (p5 protein)
  5. then penetrates the plasma membrane to deliver nucleic acid
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21
Q

How do non-enveloped viruses get ready for entry into animal cells?

A

As can’t fuse, need to enter through a pore or membrane lysis to disrupt membrane integrity.

Animal viruses:

  • proteins and enzymes to aid process
  • alongside cellular factors activate the viral particle near its site of penetration
  • e.g. Polio virus, Reovirus, Rotavirus, Adenovirus, Polomavirus
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22
Q

What the process by which a pollovirus gets ready for entry into an animal cell?

A
  1. Requires PVR receptor, and recognition of the receptor starts to strengthen the interaction
  2. The interaction of the VP protein allows a pore formation and release of RNA.
  3. Muliple viral proteins, VP1 and VP4 interact with a receptor which changes structure to expose that which is necessary for pore formation. (VP1/4 channel for injection of RNA)
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23
Q

What the process by which a reovirus gets ready for entry into an animal cell?

A
  1. Uses enzymes (cathepsins) to activate virus
  2. Cathepsins start to break the outer structure of the capsid
  3. capsid releases peptides which break the membrane in the endosome
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24
Q

What the process by which a rotavirus gets ready for entry into an animal cell?

A
  1. Tripsin enzyme modifies viral shell
  2. generates a protein intermediate which will make a hole inside the membrane
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25
Q

What the process by which an adenovirus gets ready for entry into an animal cell?

A
  1. Uses pH inside the cell which changes the state of the shell, destabilises capsid
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26
Q

What the process by which a polomavirus gets ready for entry into an animal cell?

A
  1. Uses chaperone moecule to help change its shell for entry into ER
27
Q

How do enveloped viruses get ready for entry into animal cells?

A
  1. Enveloped viruses often covered with viral glycoproteins, these are compact and held together. (Fusion incompetent state)
  2. Following priming (binding), glycoproteins are in a fusion competent state
  3. This triggers the formation of a prehairpin structure, where the glycoproteins separate and a fusion loop is inserted into the host membrane.
  4. Glycoproteins then pull the membrane together (prebundle) to a point where the host and viral membranes are in close apposition
  5. Further structural changes form a hemifusion of the two membranes (bundle state)
  6. Membranes fuse forming a fusion pore (timer-of-hairpins)
28
Q

How does HIV prepare for entry? (animal cells and enveloped virus)

A
  1. Binding of gp120 (CD4 binding site) to CD4 leads to conformational changes in gp120 that involve the exposure of its CCR5/CXCR4 binding site and formation of the gp41 fusagenic hairpin peptide.
  2. Conformational changes in CD4 starts to change structure by moving the virus towards the cell membrane
  3. This extension allows domains of gp120 to interact with CXCR4/CCR5 forming a very stable trimeric structure
  4. Gp120 dissociates into subunits and gp41 engages with the target membrane (fusion peptide) leading to fusion
29
Q

How do influenza and HIV differ in their entry?

A

Influenza binds a receptor then undergoes conformational changes to insert a fusion peptide and get a rearrangement (as does HIV) but:

Don’t enter at the same place.

HIV enters at the surface wheras influenza fuses inside the cell.

30
Q

What are the events that occur in influenza up to the process of fusion?

A
  1. Initial binding non-specificly to sialic acid, (present in large numbers of the surface of cells in airways)
  2. This non specific binding is not enough, needs to be taken inside the cell to reach an acidic environment (fusion can only happen at low pH)
31
Q

How is fusion activated in influenza?

A
  1. Upon acidificaition in the endosome, protination of HA1 leads to the dissociated of HA1 monomers.
  2. Water can enter triggering the structural transition of the B-loop into a helix and liberating the fusion peptide
  3. Fusion peptide inserts into the endosomal membrane yielding the extended intermediate conformation of HA2
  4. Refolding of the amino acid residues of the helix into a loop mediates the apposition of the two membranes
  5. This triggers fusion by the hairpin conformation of HA
32
Q

Where can viruses fuse?

A
  1. Cell surface/plasma membrane
  2. Early endosome
  3. Late endosom/lysosome
  4. ER
33
Q

What viruses fuse at the plasma membrane?

A
  • Murine leukemia virus
  • Epstein barr virus
  • Human immunodeficiency virus
34
Q

Wht viruses fuse at the early endosome?

A
  • Hep C
  • Semliki forest virus
  • Vesicular stomatitis virus
  • Human immunodeficiency virus
35
Q

What viruses fuse at the Late endosome/lysosome?

A
  • Influenza
  • Avian leukemia virus
  • Human rhinovirus
  • Dengue virus
  • Reovirus
  • SARS coronavirus
36
Q

What viruses fuse at the ER?

A
  • SV40 polomavirus
37
Q

How can a virus internalise?

A

With or without its receptor

38
Q

How does a human rhinovirus internalise?

A

LDL receptor which is brought inside when the virus is internalised

39
Q

How does a human immunodeficiency virus internalise?

A

Binds to CD4 and CCR5/CXCR4

Internalised without receptors

40
Q

How does a Hep C virus get internalised?

A

Without receptor

41
Q

What viruses exploit the internalisation pathways and how?

A
  • Flu and adenoviruses use the clatherin dependent pathway (normal pathway of endocytosis inside the cell)
    • They are taken up through claterin vesicles to the early endosome to the late endosome (with low pH) to the lysosome (acidic pH) and this triggers a fusion event.
  • In adenovirus capsid is released in endosome and delivered to the nucleus to allow replication of new viruses.
  • SV40 uses caveoli and caveosomes (don’t have to be acidic) as need access to the cyoskeleton.
    • Microtubulin movement allows virus to be taken towards the centre of the cell (nucleus). Stops at the ER for passage before entry into the nucleus.
42
Q

How are signalling events used by viruses?

A

Interplay between the virus and the receptor necessary for entry

  • Binding stimulates signalling events to activate downstream signals that are key for the virus
  • In Influenza A and adenoviruses signalling could stimulate uptake by endocytosis.
  • In SV40 signalling allows the release of the virus into the cytoplasm from caveaosomes and transport.
43
Q

Why is transport key for viruses? and what are the three ways by which viruses can get transport?

A
  • Viruses want to exploit motor on microtuble transport (dyenin) to get to the centre of cells and to nucleus
  • VSV/Rabies, influenza: Vesicle containing virus uses dyenin
  • Adenovirus: Entry, release from vesicle and use dyenin for transport
  • Herpesvirus: immediate use of dyenin following internalisation
44
Q

What are the different ways by which viruses can enter the animal cell by viral endocytosis?

A
  1. Macopinocytosis - envelope stretches out and encompasses particles on the outside
  2. Clatherin dependent endocytosis (pit no more than 100nm wide)
  3. Cavolin dependent endocytosis
  4. Clatherin and caveolin independent pathways
45
Q

Why is phagoytosis not used by viruses?

A

Used for taking up big particles (over 200microns) virus often only around 50microns

46
Q

What is the mechanism of entry of Ebola?

A
  • By macropinocytosis (and some clatherin-mediated endocytosis)
  • This wasn’t thought to be possible because of large, worm like structure, but it can compact
47
Q

What can the Ebola virus glycoprotein bind to?

A

Evola glycoprotein has lots of potential receptors:

  • C-type lectin
  • TLR
  • folate receptors

Needs to get taken up inside the late endosome (low pH and cellular proteins in late endosome)

  • Gp1 cleaved by cathepsins in LE/LY, (protein was 130kd now 19kd)
48
Q

What happens to Ebola in the late endosome/lysosome?

A
  1. Ebola outer glycoprotein GP1 needs to be cleaved to activate fusion.
  2. Cleaved by cathepsins (enzyme resident of LE/LY)
  3. Cleavage of GP1 with the help of the low pH starts to initiate conformational changes which expose the fusogenic tail.
  4. factor X (proposed to be endosomal membrane protein NCP1, a 14 transmembrane domain protein) thought to help cleave gp protein to get fusion
49
Q

How does the internalisation of Ebola differ to that of Flu?

A

Ebola: Macropinocytosis happens randomly, ebola may not need to be specifically recognised to enter the cell Influenza: When binding stimulates downstream signalling for entry for specific internalisation

50
Q

What enters the cell following fusion for: Animal viruses (enveloped and non-enveloped) and Bacteriophages?

A

Animal viruses

  • enveloped: Capsid
  • non-enveloped: Nucleic acid (polovirus) or capsid (adenovirus)

Bacteriophages:

  • Nucleic acid (bacteriophages T4 and T7)
  • or capsid (RNA phage phi 6)
51
Q

What is uncoating?

A
  • Capsid dissassembly to release nucleic acid, relies on an interplay of complex virus-specific mechanisms and cellular proteins
  • e.g. adenovirus exploits microtubule for movement, capsid used to protect viral DNA until reach nuclear pore
  • Interaction of capsid element with nuclear pore protein induces changes in the capsid to break it open and allow the release of DNA
52
Q

How does adenovirus uncoat?

A
  1. adenovirus exploits microtubule for movement
  2. capsid used to protect viral DNA until reach nuclear pore
  3. Interaction of capsid element with nuclear pore protein induces changes in the capsid to break it open and allow the release of DNA
53
Q

What is the process of HIV uncoating?

A
  1. Capsid helps to direct movement inside the cell and deliver DNA directly at the port with no risk of degradation in the cytoplasm.
  2. Capsid gets progressively disassembled in a protective way to allow reverse transcriptase to act on nucleic acid inside
  3. Cellular proteins protect the capsid to allow the formation of DNA until it is ready to be transported inside
  4. Disassembly of capsid requires cellular factors: CPF, TNP, integrase for when DNA gets transcribed. These are attached to DNA and with TNP help to transport genome into nucleus
54
Q

What different ways do viruses have to get entry and prepare genetic material ready for transcription?

A
  • envelopes/non enveloped
  • endocytosis/not endocytosis
  • microtubule use
  • fuse at certain levels of internalisation
55
Q

What is a viral tropism dependent on?

A
  • The inital site of entry into the body (tissue)
  • Virus-cell specific interactions/recognition
56
Q

What is the first line of infection defense of the body?

A
  • Virus needs to bypass different mucosa
    • e.g. Eye mucosa (conjunctive)
    • skin (dengue, biting mosquito)
    • Vaginal mucosa
    • respiratory mucosa

All are barriers to infection

57
Q

What is the tropism of HIV?

A
  1. Initial entry through oral and vaginal mucosa.
  2. Mucosa is used a a landing site
  3. Target the immune cells survielling the tissue
  • T cells, Macrophages and activates monocytes are target cells. (express CD4 and CXCR4/CCR5)
  • HIV can enter dendritic cells but will not infect these cells
    • cells bind HIV via a lection (DC sign). Transmit HIV to target cells.
    • As they are antigen presenting cells interact with T cells and release virus to the T cell
58
Q

How has understanding HIV entry led to drug discovery?

A
  1. Inially called HTL3. Took blood samples, saw virus infecting mainly CD4 cells. If cells were treated with antibody that recognised CD4 or CD3 (T cell receptor) there was a small inhibition of infection. Anti CD4 antibody was very efficient at blocking the infection. (others showed a response but only because the antibodies are so big eventually end up blocking infection) Therefore CD4 imp for entry.
  2. Demontrating CD4 was interacting with the virus - used antibody against CD4, pulled down the viral envelope. If an antibody was added that recognised a specific area on CD4 the envelope would no longer pull as it prevented an interaction between CD4 and the virus. Show that the virus interacts with CD4 at a particular site.
  3. Cells incubated with envelope of the virus and observe what is bound. Antibodies that recognise the binding site of gp120 to CD4 inhibit binding, but if an antibody is used that recognises further away from this site there is no inhibition.
  4. Neutalising antibodies were discovered in the lab but were not efficient in the patient
  5. Serum from patient was isolated and found to contain a lot of antibodies against virus, these were isolated and cloned and found to be targeting HIV.
  6. Found that some patients were preventing the virus from multiplying as had v low levels of the virus after 3 days comparative to other patients. Results were that these patients had a loss of neutralising antibodies.
  7. Need something that would block the infection but not be related to CD4. Found that if treated with some chemokines, this could reduce the infection of some specific viruses. Discovered that the chemokine was binding a receptor. A cell was generated that could express the different targets for the chemokine (CCR1): 2B, 3, 4, 5, and fusin (CXCR4). Found that virus could get entry via CXCR4/CCR5
  8. Found could block entry by removing the chemokine receptor from the surface. If had cells expressing CXCR4 and CXCR4 ligase get removal of receptor and entry could no longer be induced.
  9. Problem with removing receptor is that it activates them, leading to side effects. Found small molecules that could block receptors and completely inactivate them.
59
Q

How are some people resistant to HIV infection?

A
  • deletion at delta 32 on CCR5
  • the receptor missing this domain can’t get to the surface and mediate entry.
  • recessive mutation in CCR5 gene
  • allele frequent in caucasian populations (8-10% maybe) but absent in other groups
  • recent origin 2000 years ago
  • If a person is a heterozygote, means get less receptor and more resistant.
  • If homozygote don’t get infected.
60
Q

What did the 1st HIV therapies entail?

A
  • Blocked coreceptor binding
  • small molecules insert into a crucial domain that the viral requires for fusion and this inactivates the virus.
  • possibility to block gp120 to prevent binding to CD4.
61
Q

Give two examples of anti HIV treatments

A

Entry inhibitor:

CCR5 inhibitor (antagonist)

  • Maraviroc
  • only for patients tested having on R5 viruses

Fusion inhibitor

  • T-20 (enfuvirtide, Fuzeon)
  • given by injection
  • fusion peptide mimics gp41
  • mainly for patients with drug resistant strains.
62
Q

What are the viral and cellular binding partners of HIV?

A

Viral:

2 glycoproteins joined together by a disulphide bond:

  • gp41 = transmembrane glycoprotein,
  • gp120 = masks N terminus of gp41 which induces fusion and gets inside surface of cell.

Cellular:

  • CD4 receptor
  • chemokine receptor (CCR5/CXCR4)
63
Q
A

Principle of molcular virology (7 decks)

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