Virus Attachment, Entry and
Uncoating
Virus Properties
• Virus is defined as a nucleoprotein
complex which infects cells and uses
their metabolic processes to replicate
• Smallest known infective agents
• Metabolically inert - no metabolic
activity outside host cell; must enter
host cell to replicate
• Most are highly species specific
Virus versus Virion
• Virus is a broad general term for any aspect
of the infectious agent and includes:
• the infectious or inactivated virus particle
• viral nucleic acid and protein in the
infected cell
• Virion is the physical particle in the extracellular phase which is able to spread to new
host cells; complete intact virus particle
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
Overview
Name
Process
1
2
3
Attachment
Entry
Uncoating
4
5
Biosynthesis
Maturation
Virus attach to cell membrane
By endocytosis or fusion
By viral or host enzymes, separates NA from
protein coat
Production of NA and proteins
NA and capsid proteins assemble
6
7
8
Release
Shedding
Infecting new
host
By budding (enveloped viruses) or rupture
6
Viral Replication
i) Adsorption (attachment):
• random collision
• interaction between specific proteins on viral
surface and specific receptors on target cell
membrane (tropism)
• not all cells carrying a receptor for a
particular virus can be productively infected
by that virus
Viral Replication
i) Adsorption (attachment):
– some viruses may use more than
one host cell receptor (e.g. HIV)
– able to infect a limited spectrum of
cell types (host range)
– most neutralizing antibodies are
specific for virion attachment proteins
Viral Replication
ii)
Entry (penetration):
• 2 mechanisms - endocytosis
- fusion of virus
envelope with
cell
membrane
iii) Uncoating:
• release of viral genome
• cell enzymes (lysosomes) strip off the
virus protein coat
• virion can no longer be detected; known
as the “eclipse period”
Attachment: Cell Receptor
• Virus may bind up to three different cell
receptors in succession:
– Low affinity receptor - in high abundance,
virus contacts cell surface
– Primary receptor - in lower concentration
– Co-receptor – follows binding of primary
receptor
Attachment: Specificity
• Host Range - the organism(s) that the virus
is able to infect (narrow or wide) i.e. plant,
animal, human
• Tissue Tropism- the cell type(s) a virus is
able to infect i.e. skin, oral, GI, CNS
Attachment: Binding
• 3-D fit between viral ligand and cell receptor
• Mainly weak electrostatic charges.
• Evidence for this is interaction may require:
– specific pH
– specific ionic strength
– presence of specific ions i.e. Ca++, Mg++
Attachment: Nonenveloped
Picornavirus
• Virus ligand - a deep cleft (“canyon”) in
triangular face of capsid (viral proteins VP1,
VP2, VP3)
• Binds to cell receptor ICAM –1 (intracellular
adhesion molecule 1), normal function is to bind
cells i.e. WBC
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
From Introduction to Modern
Virology, Dimmock & Primrose,
Blackwell
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
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
• 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 up
Other 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 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 (Phi 6)
•
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-, rhabdofamilies
– 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
Entry / Uncoating
• Entry is the mechanism used by the virus
to penetrate into the host cell
• Uncoating is the separation of the nucleic
acid from the capsid, and refers to changes
that occur to make the viral nucleic acid
ready for expression
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)
Figure courtesy of
A. Helenius
The very high particle:pfu ratio (approaching 1:1) of SFV ensures
that all the virus particles are part of the “real” entry pathway
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
• 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
Specific
requirement for α23 and α2-6 linkages
- gives different
tropism for avian
vs. human cells
(pigs have both)
From Principles of Virology, Flint et al. ASM Press
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”
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
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
Fusion of an enveloped virus
Model for viral membrane fusion mediated by
class I fusion roteins. Influenza virus, which is
internalized into an endosome, is shown as an
example. In the native state of the fusion
protein — which is a trimer — most of the
surface subunit (green) is exposed. Part of the
transmembrane subunit, including the fusion
peptide, is not exposed. Following fusionactivating conditions, conformational changes
occur to 'free' the fusion peptide (red) from its
previously unexposed location. In the case of
influenza HA, this occurs by a 'spring-loaded'
mechanism. The 'pre-hairpin' intermediate
spans two membranes — with the
transmembrane domain positioned in the viral
membrane and the fusion peptide inserted into
the host-cell membrane. The pre-hairpin
intermediate forms a trimer of hairpins, and
membrane fusion occurs, which leads to pore
formation and release of the viral genome into
the cytoplasm.
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
Attachment: Enveloped HIV
Virus
•
•
•
Host cell protein in virus envelope
(cyclophilin A) initially binds HIV
to low affinity receptor (heparin
sulfate) of the cell
Followed by binding of viral ligand
(gp120) to primary receptor (CD4)
on T helper cells, macrophages, and
glial cells
Binding of gp120 to CD4 results in
conformational change of gp120,
which then binds to chemokine coreceptor CXCR4 on T lymphocytes
or CCR5 on macrophages
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 coreceptor (CCR5 or
CXCR4)
•
HIV virions are able to gain access to their host cells by way of viral
host-cell membrane fusion (Gallo et al., 2003).
•
The viral envelope gp120 first recognizes its primary receptor on
host cells, CD4 (Dalgleish et al., 1984; Ugolini et al., 1999), using a
binding motif contained within its second constant (C2) region
(Kwong et al., 1998).
•
This interaction gives rise to a conformational change in gp120
which exposes its third variable loop (V3) which contains a
consensus amino acid motif that allows for binding to a seven
transmembrane-spanning chemokine co-receptor (Jones et al., 1998;
Kwong et al., 1998; Berger et al., 1999).
•
Both interactions are necessary for viral fusion and entry (Sattentau
and Moore, 1991).
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)
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
Entry: Nonenveloped Virus
• Receptor-mediated endocytosis
• Clathrin coated pits (seen by EM)
• Invagination and pinching off of the
membrane
• Forms an intracellular endosome
containing the virus
• Endosome becomes acidified
Uncoating: Nonenveloped Virus
• Low pH causes
conformational
changes in capsid
protein (hydrophobic
region interacts with
membrane forming a
pore)
• Viral nucleic acid
released
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 clathrincoated vesicles
Dominant-negative mutant
(K44A) inhibits endocytosis
Eps15 binds to AP-2, the
clathrin adaptor protein
It is specific to clathrincoated 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
Lipid rafts
•
Detergent-resistant domains in cell membranes
•
Enriched in cholesterol and sphingomyelin
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
HveB
HveC
HveD
TNF-R
Nectin2 (Prr 2)
Nectin1 (Prr 1)
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
From Principles of
Virology, Flint et
al. ASM Press
Similar viruses can have quite distinct receptors
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
Penetration might be controlled by sphingosine, a lipid
present in the “pocket” -- or (more likely) by the pocket
allowing “breathing” of the capsid
•
With a non-enveloped virus, fusion is not possible. Instead
picornaviruses form a membrane pore
From Principles of
Virology, Flint et
al, ASM Press
Parvoviruses may contain a phospholipase A2 activity in their capsid protein
The specific lipid composition of endosomes may be crucial for some viruses
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
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
μm
μm
1 cm
polio 61 yr
HSV 231 yr
Table box 5.2
From Sodeik, Trends Microbiol 8: 465
Microtubules and virus entry
VSV/Rabies,
influenza
Adenovirus
From Sodeik,
Trends Microbiol
8: 465
Herpesvirus
• To facilitate transport viruses often bind to the cytoskeleton and use
microtubule-mediated motor proteins for transport, i.e. dynein
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)
From Flint et al Principles of Virology ASM Press
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
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
Herpesvirus
• 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
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://wwwermm.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