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Virus binding and entry. Virology lecture 2 Dr. Sadia Anjum. Virus entry. The first event in any virus life-cycle - often limits infection to the “correct” cell Can be primary determinant of tropism Tissue tropism - e.g. measles (skin cells) vs. mumps (salivary gland)

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virus binding and entry
Virus binding and entry

Virology lecture 2

Dr. SadiaAnjum

virus entry
Virus entry
  • The first event in any virus life-cycle - often limits infection to the “correct” cell
  • Can be primary determinant of tropism
    • Tissue tropism - e.g. measles (skin cells) vs. mumps (salivary gland)
    • Species tropism - e.g. togavirus (both insect/mammalian cells), poliovirus (primate cells), T4 phage - (few strains of E.coli)
  • Binding - initially electrostatic, based on charge ± pH, specific ions - followed by local hydrophobic interactions
  • Initial binding is often low affinity, but high avidity (tight binding) due to multiple binding sites
  • The virus binds to a receptor on the cell surface - can be ubiquitous/specific, with variable density
  • Initial binding is followed by penetration and subsequent uncoating
virus entry1
Virus entry
  • Whether or not the virus is enveloped makes a big difference - at least for penetration
  • All viruses must cross a lipid bilayer, plant and bacterial viruses must also cross a cell wall
  • Uncoating means that the stable virus stucture must become unstable
    • -transition from extracellular (chemical) form to intracellular (biological) form
    • There must be some sort of “trigger” or regulated disassembly process
t even t2 phage structure
T-even (T2) phage structure

From The Biology of Viruses, Voyles, McGraw Hill,

entry of t even bacteriophage binding
Entry of T-even bacteriophage - binding
  • Best understood = T4 phage
  • Initial binding is reversible and electrostatic - the outer-most part of the long tail fiber binds to surface lipopolysaccharides (LPS) of the bacterium
  • (binding can occur in vitro, and is competed by specific sugars) - a non-specificreceptor
  • Binding is “additive” until all six tail fibers are bound
  • Binding of 3 fibers is needed to initiate infection
  • The virus may “browse” the surface –

looking for a suitable site for penetration (possibly sites of cell wall synthesis where the outer and plasma membranes are close together).

Note - this is a multi-valent interaction

From Introduction to Modern Virology, Dimmock & Primrose, Blackwell

entry of t even bacteriophage binding ii
Entry of T-even bacteriophage - binding II
  • The receptor binding sites of the short tail fibers are now exposed and bind (also to LPS)- now binding is essentially irreversible
  • Conformational change in the baseplate - hexagon to extended star-shaped conformation –
  • Initiates sheath contraction ( to 37% of its original length)
entry of t even bacteriophage penetration
Entry of T-even bacteriophage - penetration

• Sheath of the helical tail slips and forms a shorter helix.

• The tube itself stays the same with the end result that the tube is pushed down and contacts the membrane

the tail does not directly punch through

• Lysozyme molecules are releases which forms a pore through which DNA enters

  • Often referred to as a “hypodermic syringe”

DNA is under considerable pressure and seems to exit automatically once the base palte is opened up

Other phage, e.g. T3, have a motor protein to reel out the DNA

From The Biology of Viruses, Voyles, McGraw Hill,

entry of other phage i
Entry of other ‘phage - I
  • Phage λ (DNA) - a virus with a longer, but simpler tail than T4

From The Bacteriophages vol 1, ed Calender, R., Plenum Press

The single tail fiber (J protein) binds to lamB (the maltose transporter) - an example of a specific receptor LamBis inducible.

This means the virus only infects in the presence of high nutrients. Also needs Mg2+ - binding is electrostatic - an example of tropism

Penetration requires the bacterial pts protein (also part of the maltose transporter) - the co-receptor

attachment and penetration require different viral proteins

entry of other phage ii
Entry of other ‘phage - II
  • PRD1 - an icosahedral phage with an internal membrane
  • For gram -ve bacteria (with two layers of lipid separated by peptidoglycan) phage entry is a challenge
    • 1) Binding
    • 2) Conformational change -> dissociation and opening of 14 nm hole in the capsid
    • 3) Second conformational change converts internal envelope to tubule, which delivers the DNA

Phage encodes 2 proteins (P5 and P17) that have peptidoglycan-hydrolyzing activity

- equivalent protein in T-even phage = gp5 (lysozyme activity)

From Rydman and Bamford (2002) ASM News 68 330

entry of other phage iii
Entry of other ‘phage - III
  • Enveloped RNA phage - φ6
  • These phage bind to pili, the pilus then retracts down to the outer membrane, the virus undergo fusion, enzymatically destroys the peptidoglycan cell wall (p5 protein) and then penetrates the plasma membrane

From The Bacteriophagesvol 2, edCalender, R., Plenum Press

The Hershey-Chase experiment is now no longer valid, as (most of) the protein (35S) has entered the cell along with the nucleic acid (32P).

plant viruses
Plant viruses
  • Plants have a thick, rigid cell wall
  • Generally plant viruses do not have specific entry mechanisms, but rely on
  • introduction into the cell by a vector (insect) – most common
  • mechanical injury
  • direct cell-cell transmission (via plasmadesmata and viral movement protein)
  • This is fine for non-enveloped virus, but enveloped plant viruses do exist (bunya-, rhabdo- families
    • These viruses must fuse their envelope
binding of animal viruses
Binding of animal viruses
  • Occurs via receptors on the cell surface

Protein (glycoprotein)

Carbohydrate

Lipid (glycolipid)

From Principles of Virology, Flint et al. ASM Press

Receptor utilization plays a major role in virus tropism / pathogenesis

principles of virus penetration
Principles of virus penetration
  • Viruses can penetrate directly at the plasma membrane, or via endosomes
penetration of enveloped animal viruses
Penetration of Enveloped Animal Viruses
  • Envelope = fusion
  • Semliki Forest virus (SFV) a togavirus - the classic virus for entry studies
  • Early experiments (early 1980s) by electron microscopy showed entry into vesicles - now known to be clathrin-coated (clathrin-coated vesicles, or CCVs)
    • The virus then enters the endosome (“early” endosome)

The very high particle:pfu ratio (approaching 1:1) of SFV ensures that all the virus particles are part of the “real” entry pathway

Figure courtesy of A. Helenius

slide15

Endosomes and virus entry

  • Endosomes are used by cells for nutrient and growth factor uptake
  • The virus “hijacks” the cellular pathway
  • One key feature of endosomes is their progressive acidification - due the the action of the vacuolar H+/vATPase
  • Endosomes do much more than provide low pH
    • Deliver through cortical actin and microtubule-mediated transport in the cytosol
    • Specific redox/ionic environment
    • Defined lipids for fusion/penetration

From Cell Biology, Pollard and Earnshaw, Saunders

slide16

• The lowered pH causes conformational changes in the spike glycoprotein, and the exposure of a fusion peptide

  • • In most cases a pH of around 6.2-6.5 is sufficient for fusion
  • - fusion occurs in the early endosome
  • • Entry and infectivity (in cell culture) can be blocked by :
    • – 1) addition of a weak base (e.g. NH4Cl) that neutralize the endosome
    • – 2) drugs that target the vH+/ATPase (e.gbafilomycin A)
    • – 3) drugs that break down the proton gradient (e.g. monensin)
    • – 4) exposure of the virus to a low external pH

Fusion can be induced at the cell surface by exposure to low pH

influenza virus binding i
Influenza virus binding - I
  • Binds to cell surface carbohydrate - sialic acid
  • Ubiquitous/non-specific receptor
  • In principle, this can be present as part of glycoprotein or glycolipid

Specific requirement for α2-3 and α2-6 linkages - gives different tropism for avian vs. human cells (pigs have both)

From Principles of Virology, Flint et al. ASM Press

influenza virus binding ii
Influenza virus binding - II
  • The major influenza glycoprotein, hemagglutinin (HA) has a specific sialic acid-binding site on its “top domain” -

HA mediates both binding and penetration

From Principles of Virology, Flint et al. ASM Press

penetration of influenza virus
Penetration of influenza virus
  • Influenza virus requires a lower pH (5.0-5.5) and enters the “late” endosome, but fusion occurs before entry into the lysosome (this avoids degradation)
  • The acid-triggered fusion event is well understood - a conformational tail forms a rigid “six-helix bundle” or “coiled-coil” of α-helices, which flips the fusion peptide out and allows insertion into the membrane
    • Note the fusion peptide is “external”

The “trigger” is irreversible - this means that exposure of virions to low extracellular pH will destroy infectivity

From Principles of Virology, Flint et al, ASM Press

slide20

From Principles of Virology, Flint et al, ASM Press

• The low pH has a second very important role for influenza entry - the virus contains an ion channel in its envelope (M2).

• The presence of M2 allows acidification of the virus interior, and promotes uncoating of the M1/vRNPs

• Drugs that block M2 block infection - amantadine. This is highly specific for the viral M2 ion channel, with no effect on the cellular H+/vATPase

slide22

Fusion of an enveloped virus

From Dimitrov (2004) Nature Reviews Microbiology 2:109-122

retrovirus hiv
Retrovirus (HIV)
  • A classic example of a receptor/co-receptor requirement
  • A specific receptor
  • Binds initially to CD4 - present on immune system cells (T-cells) - gives the virus tropism for the immune system
  • This is not enough - the virus also binds to a chemokineco-receptor (eg CCR5, CXCR4) present on a sub-set of cells (macrophages / T-cells)
  • Gives even more precise tropism
  • The virus binds to both receptors via the gp120 glycoprotein
penetration of retrovirus hiv i
Penetration of retrovirus (HIV) - I
  • HIV enters by a quite different route
  • Entry is not low pH-dependent (no inhibition by NH4Cl etc), and fusion occurs directly with the plasma membrane

From Principles of Virology, Flint et al, ASM Press

penetration of retrovirus hiv ii
Penetration of retrovirus (HIV) - II
  • If pH is not required for fusion, what is the trigger ??
  • Following receptor binding a conformational change (also the formation of a coiled coil) occurs in the HIV-1 gp120 molecule - exposes its fusion peptide (present on gp41 - the second half of the gp160 Env protein)

From Principles of Virology, Flint et al, ASM Press

HIV has a receptor (CD4) and a co-receptor (CCR5 or CXCR4)