Virus binding and entry

Virology lecture 3 Dr. Sadia Anjum

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

Virus entry

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

Clathrin mediated internalization

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

Clathrin vs. non-clathrin internalization

Influenza Entry

Herpes viruses• A complex system

• Herpesviruses have 10-12 surface glycoproteins

• Binds initially to heparan sulfate (via gC) – It is - non-specific and is attachment or “capture”

receptor

• Subsequently binds to a co-receptors that allows entry (via gD) - herpesvirus entry mediator - specific• 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

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

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

Rhinovirus/Poliovirus

• 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

Adenovirus

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

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 ?

• 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 SV40From Munro S. Cell. 2003 Nov 14;115(4):377-88. Lipid rafts: elusive or illusive?

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

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 nucleus

But, 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”

For more detail

• Chapter 5 of Flint et al.

• Chapter 4 of Fields Virology

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

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