Virus binding and entry

9/9/2004

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9/14/2004

General points - 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

General points II - 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 (the virus in the Hershey-Chase experiment)

•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 interaction

From 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 - note 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 receptorlamB 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 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• 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 common– B) mechanical injury– C) direct cell-cell transmission (via plasmadesmata

and viral movement protein)

• This is fine if you are a 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 (plasma membrane)

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

• 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 • This is the “trigger” needed for virus entry

• 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 pH

Fusion 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)

The first virus receptor to be identified - principally due to the fact that there is a receptor-destroying enzyme associated with the virus

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

From Fields Virology. 4th Ed Lippicott Williams and Wilkins

• 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)

Entry of avian leukosis virus (a model, simple, retrovirus)

• Classically all retroviruses were thought to be pH-independent

• More recently ALV has been proposed to require low pH, but in addition to its receptor-induced conformational change

• Entry is occurring via endosomes in this case

Entry of vesicular stomatitis virus (VSV)

• Virus receptor is a lipid (phosphatidyl serine; PS)– a unique example ??

• Very wide infection range (all cells have PS) - one of the most promiscuous viruses out there

• Fusion etc is similar to influenza…..– Both VSV G and influenza HA are referred to as type

I fusion proteins

• with two main differences– The trigger is reversible– The pH threshold is less stringent (approx. pH 6.5).

Fusion is though to occur from the “early” endosome

Type I and type II fusion proteins

• Type I is the most common and understood fusion protein– Influenza, VSV, retrovirus

• Type II fusion proteins are not proteolytically activated, have internal fusion peptides and no “coiled-coil” form; they are principally β-sheet

• Flavivirus (TBE), and Alphavirus (SFV)

Comparison of type I and type II fusion proteins

From Principles of Virology, Flint et al, ASM Press

SFV and TBE - alternative ways to

expose fusion peptides

• In SFV the fusion peptide is protected by E2

• In TBE the flat E protein rotates and twists

Surface representation of dengue virus

Clathrin vs. non-clathrin

internalization• Most viruses were originally assumed to

use clathrin as a route into the cell• Used by SFV, VSV, adenovirus etc• Other routes of entry exist and can be used• Caveolae (as used by SV40) are the best

characterized)• Influenza and rotavirus are other examples• In most cases non-clathrin pathways are

ill-defined

Dynamin is a GTPase that acts to “sever” the necks of the endocytic vesicle

It is not specific to clathrin-coated vesicles

Dominant-negative mutant (K44A) inhibits endocytosis

Eps15 binds to AP-2, the clathrin adaptor protein

It is specific to clathrin-coated vesicles

Dominant-negative mutant (Eps15delta95-295) inhibits endocytosis

From Biochem. J. (2004) Immediate Publication, doi:10.1042/BJ20040913 Cargo- and compartment-selective endocytic scaffold proteins Iwona Szymkiewicz, Oleg Shupliakov and Ivan Dikic

• Detergent-resistant domains in cell membranes

• Enriched in cholesterol and sphingomyelin

Lipid rafts

Play a very important role in virus budding

Can also be important for virus entry , esp non-clathrin endocytosis e.g SV40

From Munro S. Cell. 2003 Nov 14;115(4):377-88. Lipid rafts: elusive or illusive?

Herpesviruses• A complex system• Herpesviruses have 10-12 surface glycoproteins• Binds initially to heparan sulfate (via gC)

– used by a multitude of different viruses - non-specific– An attachment or “capture” receptor

• Subsequently binds to a co-receptors that allows entry (via gD) - herpesvirus entry mediator - specific– A fusion receptor– HveA TNF-R– HveB Nectin2 (Prr 2)– HveC Nectin1 (Prr 1)– HveD PVR

• Different herpesviruses use different receptors• But very different viruses can use the same receptor – e.g. pseudorabies virus and polio virus– Another example = CAR - the coxsackie/adenovirus receptor

Poliovirus/Rhinovirus (Picornaviridae)

• Picornaviruses bind to a variety of specific cell surface molecules - these are specific proteins

– Binding occurs via canyons (depressions) in the virus surface

Similar viruses can have quite distinct receptors

From Principles of Virology, Flint et al. ASM Press

Penetration of non-enveloped viruses

• Rhinovirus/Poliovirus (Picornavirus)

• Although not pH dependent, poliovirus may still enter

through the endosome• Interaction of poliovirus with PVR causes major conformational changes in the virus - leads to the formation of the A particle -physically swollen (less dense)

From Principles of Virology, Flint et al, ASM Press

• A particles are now hydrophobic. Viruses have apparently lost VP4, and the hydrophobic core is exposed on the virus surface

• With a non-enveloped virus, fusion is not possible. Instead

picornaviruses form a membrane pore

From Principles of Virology, Flint et al, ASM Press

Penetration might be controlled by sphingosine, a lipid present in the “pocket” -- or (more likely) by the pocket allowing “breathing” of the capsid

Parvoviruses may contain a phospholipase A2 activity in their capsid protein

The specific lipid composition of endosomes may be crucial for some viruses

Picornaviruses as enzymes ?Virus entry as thermodynamics ??

Adenovirus

• A relatively complex

system

• Receptor and co-receptor

• Clathrin-mediated

endocytosis• Instead of forming a

discrete pore, adenovirus ruptures or lyses the endosomal membrane

• The trigger is low pH, via the penton base protein

• The virus undergoes proteolytic cleavage - by virus-encoded proteases

SV40

• Entry occurs via endocytosis but in a clathrin-independent manner

• Entry does not depend on low pH• The virus enters through “caveolae” - a

specialized endocytic vesicle that forms upon specific cellular signaling induced by virus binding

• Receptor is combination of a protein (MHCI) and a glycolipid (sialic acid)?

• The “caveosome” containing the virus is delivered to the ER

Caveolae are specialized lipid rafts

Reovirus

• The rare example of a virus requiring the lysosome

• Reoviruses have a complex double capsid, which is very stable to low pH (gastro-intestinal viruses; rotavirus)

• The lysosomal proteases degrade the outer capsid to form a subviral particle i.e degradation by cellular proteases

• The subsequent penetration step is unknown From Principles of Virology, Flint et al, ASM Press

Rotavirus entry

• Trypsin cleavage of VP4 (= spike protein)

• VP4 becomes VP8* and VP5*

• Transient exposure of hydrophobic peptide

• Trimeric coiled coil formation

From Dormitzer et al (2004) Nature 430:1053

Comparable to influenza HA ?

The problem of cytoplasmic transport

• Assume the virus in question has undergone receptor binding and penetration - ie the virus/capsid in the the cytoplasm.

• The cytoplasm is viscous and the nucleus is often a long distance from the site of entry.

• This is especially true for specialized cells such as neurons

From Sodeik, Trends Microbiol 8: 465

μm μm

Table box 5.2

1 cm

polio 61 yr

HSV 231 yr

Microtubules and virus entry

• To facilitate transport viruses often bind to the cytoskeleton and use microtubule-mediated motor proteins for transport, i.e. dynein

VSV/Rabies, influenza

Adenovirus

Herpesvirus

From Sodeik, Trends Microbiol 8: 465

Nuclear Import

• Why replicate in the nucleus?What are the “benefits?”

• DNA viruses - need cellular DNA polymerase and/or accessory proteins (eg topoisomerase) -

• All DNA viruses replicate in the nucleus• exception = Pox viruses (even these will not replicate in an

enucleated cells or cytoplast)

• Almost all RNA viruses replicate in the cytoplasm, and most will replicate in a cytoplast

• Principal exceptions = retroviruses (these have a DNA intermediate) and influenza virus (has a spliced genome)

What are the “problems” with nuclear replication?

• An additional barrier during genome transport

• The nucleus of a eukaryotic cell is surrounded by a double lipid bilayer - the nuclear envelope.

• The nuclear envelope is studded with transport channels - the nuclear pores

From Flint et al Principles of Virology ASM Press

Parvovirus• Possibly the simplest example of nuclear entry• Small icosahedral DNA virus (18-26nm diameter)• Enters through endosomes (pH-dependent)• VP1 contains a nuclear localization signal (NLS)

• Basic amino acids • The NLS binds to cellular receptors (karyopherins or importins) that carry

proteins into the nucleusBut,

the NLS is hidden on the inside of the capsid

Therefore a conformational change must occur to expose the NLS

From Flint et al Principles of Virology ASM Press

Adenovirus• Contains NLSs on its capsids, binds microtubules• But, • The functional size limit of the nuclear pore is 26 nm• The virus is therefore transported as far as the pore. • It docks to the nuclear pore and then undergoes final

disassembly, and the DNA is “injected” into the nucleus - with DNA binding proteins attached

Specific importins help disassemble the capsid

• After fusion the tegument (most of it) is shed - phosphorylation dependent• Contains NLSs on its capsids, binds microtubules via dynein

• The virus is therefore transported as far as the pore. • It docks to the nuclear pore and then undergoes final disassembly, and the DNA is “injected” into the nucleus

Herpesvirus

Note the capsid is “empty” - no dark center on EM

From Whittaker Trends Microbiol 6: 178

Influenza virus

• The nucleoprotein (NP) contains NLSs and the RNPs are small enough to translocate across the nuclear pore

• The key to influenza nuclear import is the pH-dependent dissociation of the matrix protein (M1) from the vRNPs.

• This relies of the M2 ion channel in the virus envelope, the target of amantadine

From Whittaker Exp. Rev. Mol. Med. 8 February, http://www-ermm.cbcu.cam.ac.uk/01002447h.htm

Retroviruses

• Simple + complex• Simple retroviruses (oncoretroviruses) can only replicate

in dividing cells, e.g. Rous sarcoma virus (RSV), avian leukosis virus (ALV).

• Nuclear entry occurs upon mitosis - the nuclear envelope breaks down and the virus is “passively” incorporated into the new nucleus

• This is relatively inefficient and restrictive for virus tropism

• Complex retroviruses (lentiviruses) have evolved mechanism for nuclear entry in non-dividing cells, e.g. HIV

HIV

• Once in the cytoplasm the RNA genome is reverse transcribed into a DNA copy - the pre-integration complex (PIC)

• There is (probably) a role for microtubules in cytoplasmic transport

• The PIC is a large (Stokes radius = 28nm) nucleoprotein complex that contains several proteins, including:– integrase (IN) matrix (MA) and Vpr

• Each of these three proteins seems to play a role in transporting the very large PIC to and across the nuclear pore

• Also a role for the “central DNA flap”

Further reading

• Chapter 5 of Flint et al.• Chapter 4 of Fields Virology - not particularly

good

• “Brief overview on cellular virus receptors”, Mettenleiter TC, Virus Research 82 (2002) 3-8

• Cool movies -- http://trimeris.com/science/hivfusion.html