Virus binding and entry - PowerPoint Presentation

Slide1

Virus binding and entry

Virology lecture 3 Dr. Sadia Anjum

Slide2

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

Slide3

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

Slide4

Type I and type II fusion proteins

Type I is the most common and understood fusion proteinInfluenza, 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)

Slide5

Comparison of type I and type II fusion proteins

From Principles of Virology, Flint et al, ASM Press

Slide6

SFV

and TBE - alternative ways to expose fusion peptidesIn SFV the fusion peptide is protected by E2

In TBE the flat E protein rotates and twists

Slide7

Virus entry

Slide8

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

Slide9

Clathrin mediated internalization

Slide10

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

Slide11

Clathrin

vs. non-clathrin internalization

Slide12

Influenza Entry

Slide13

Herpes viruses

A complex system

Herpesviruses

have 10-12 surface glycoproteins

Binds initially to

heparan

sulfate (via

gC

)

It is -

non-specific

a

nd 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

Slide14

Slide15

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

Slide16

Poliovirus/Rhinovirus (

Picornaviridae)Picornaviruses bind to a

variety

of

specific cell surface molecules - these are specific proteinsBinding occurs via canyons (depressions) in the virus

surface

Similar viruses can have quite distinct receptors

From Principles of Virology, Flint et al. ASM Press

Slide17

Rhinovirus/Poliovirus

Slide18

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

Slide19

Adenovirus

Slide20

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

Slide21

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

Slide22

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

?

Slide23

Detergent-resistant domains in cell membranes

Enriched in cholesterol and sphingomyelinLipid 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?

Slide24

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

Slide25

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

μ

m

μ

m

Table box 5.2

1 cm

polio 61 yr

HSV 231 yr

Slide26

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

Slide27

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)

Slide28

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

Slide29

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

Slide30

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

Slide31

• 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

Slide32

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

Slide33

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

Slide34

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”

Slide35

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