Virus binding and entry

Virology lecture 2Dr. 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)– 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 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

From The Biology of Viruses, Voyles, McGraw Hill,

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-specific receptor•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 interactionFrom Introduction to Modern Virology, Dimmock & Primrose, Blackwell

• 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 - binding II

Entry of T-even bacteriophage - penetration

• Often referred to as a “hypodermic syringe” • 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

From The Biology of Viruses, Voyles, McGraw Hill,

DNA is under considerable pressure and seems to exit automatically once the base palte is opened upOther phage, e.g. T3, have a motor protein to reel out the DNA

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 LamB is 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• 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 DNAPhage 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• 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 Bacteriophages vol 2, ed Calender, 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

• Plants have a thick, rigid cell wall• Generally plant viruses do not have specific

entry mechanisms, but rely on A)introduction into the cell by a

vector (insect) – most commonB)mechanical injuryC)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• Occurs via receptors on the cell surface

Protein (glycoprotein)CarbohydrateLipid (glycolipid)

From Principles of Virology, Flint et al. ASM Press

Receptor utilization plays a major role in virus tropism / pathogenesis

Principles of virus penetration

• Viruses can penetrate directly at the plasma membrane, or via endosomes

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

• 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

Endosomes and virus entry

From Cell Biology, Pollard and Earnshaw, Saunders

• 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.g bafilomycin A)– 3) drugs that break down the proton gradient (e.g. monensin)– 4) exposure of the virus to a low external pHFusion can be induced at the cell surface by exposure to low pH

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

From Principles of Virology, Flint et al. ASM Press

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

Influenza virus binding - II• The major influenza glycoprotein,

hemagglutinin (HA) has a specific sialic acid-binding site on its “top domain” -

From Principles of Virology, Flint et al. ASM Press

HA mediates both binding and

penetration

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”

From Principles of Virology, Flint et al, ASM Press

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

• 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

From Principles of Virology, Flint et al, ASM Press

Fusion of an enveloped virus

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

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 chemokine co-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

• 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

• 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)