Transcript PICORNAVIRIDAE

Picornaviridae
Molecular Virology
Plus Strand RNA Virus Families
PICORNAVIRIDAE
•More than 200 viruses prevalent world-wide.
• cause many serious diseases of animals and man.
• Foot and mouth virus first animal virus described (1898).
• Poliovirus is an important model:
- first virus purified and crystallized.
- first inactivated vaccine used (Salk 1950’s).
- first picornavirus to be sequenced.
- first infectious cDNA clone of an animal virus.
- first picornavirus structure to be solved.
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PicoRNAviruses
ss, positive sense RNA, icosahedral capsid, 25-30nm, and no envelope
Pico= small
[Greek]
Positive sense
ICOsahedral
RNA genome
4 coat proteins VP1, VP2, VP3,VP4
simple capsid : 60 capsomere (composed of VP1,2,3)
The single-strand genome of 7500–8500 nucleotides
Properties
• Picornaviruses are among the smallest pathogens of
vertebrates and are responsible for many important
diseases in humans and animals.
• Picornaviruses are responsible for a wide range of
clinical diseases resulting from multiple factors such as
receptor specificity, tissue-specific susceptibility,
virulence and the mechanisms of transmission.
• Picornaviruses bind to cell specific surface receptors
and this interaction is an important factor in
determining host and tissue specificity of each virus.
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Properties
 replicate in cytoplasm
 Positive sense genome
(genome RNA is infectious)
directly as a messenger RNA to template
 encode viral polymerase
 mature viral proteins
generated through proteolytic
cleavage
 virions released on cell lysis
Properties
• Picornaviruses cannot gain entry to cells by membrane
fusion, as is the case for enveloped viruses, and require
special mechanisms to breach cellular membranes and safely
deliver the genome into the host cell.
• Genome replication occurs in association with virus modified
cellular membranes.
• Viral RNA templates complementary negative strand
molecules which in turn template multiple positive strand
copies.
• The synthesis of all RNA molecules is initiated by an
uridylated peptide primer.
• The mechanisms of transmission of infection play key roles
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in the epidemiology of picornavirus infections.
Evolutionary relationships
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Evolutionary relationships
A Phylogenetic Tree of the Picornaviridae
Coxsackie B3
Swine vesicular disease
Enterovirus 70
Poliovirus type 1
Bovine enterovirus
Coxsackie A16
7 Genera Recognized
0.5
0
Enteroviruses
acid resistant
acid sensitive
Human rhinovirus 14
Human rhinovirus 2
Rhinoviruses
Human rhinovirus 89
FMDV-A
FMDV-O
Aphthoviruses
EMC
TMEV (Theilers)
Aichi
Cardioviruses
Hepatitis A
Simian hepatitis A
Hepatoviruses
Echovirus 22
Parechovirus
Kobu virus
Evolutionary distance
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Classification
Genus
Diseae
Enterovirus
Poliovirus type member
Coxsackie A/B virus
Enterovirus type 71
swine vesicular disease
porcine enteroviruses
3 major types cause paralysis.
Rhinovirus
cause respiratory tract
infections
cause colds in humans (110
types) and pigs
Hepatovirus
Hepatitis A
avian encephalomyelitis
contagious liver infections
Apthovirus
Foot and mouth disease
most destructive in Africa
Cardiovirus
encephalomyocarditis virus ECMV
cause heart and brain
inflammation
source is a rodent reservoir
Parechovirus
Human Parechovirus 1 & 2
(HPEV1 & HPEV2)
EMC group
acid labile
yes
yes
Erbovirus
Koburirius
Teschovirus
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porcine teschovirus
Virion structure
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Naked icosahedral virus, 22-30 nm in size, with a dense core of about 16 nm.
sedimentation value is 160S
The capsid consists of a densely-packed icosahedral arrangement of 60
protomers, each consisting of 4 polypeptides, VP1, VP2, VP3 and VP4.
VP4 is located on the internal side of the capsid.
RNA is single stranded RNA ：
1. 35S, 2.4 X 106 daltons, 7-8 Kb, 30% of virion mass
2. 3' end 90 poly A nucleotides
3. 5' is not capped but terminated in pUp that is covalently linked to a
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2 kD (VPg) protein joined through an ester link to tyrosine
Virion structure
(a) Basic ‘jelly roll’ fold of proteins VP1–3. (b) Poliovirus VP1. (c) Poliovirus VP2. (d) Poliovirus VP3. (e) Icosahedron showing
locations of 2-, 3- and 5-fold axes of symmetry and VP1–3. (f) Cryoelectron microscopy-derived reconstruction of poliovirus.
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(g) Cryoelectron microscopy-derived reconstruction of complex of poliovirus and soluble form of cell receptor. Courtesy of Dr
J Hogle.
Genome
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Genome
1. linear, ssRNA(+) genome of 7.1-8.9 kb, polyadenylated, composed of a single ORF
encoding a polyprotein.
2. Viral genomic RNA has a viral protein (VPg) at its 5’ end instead of a methylated
nucleotide cap structure.
3. The long UTR at the 5’ end contains an internal ribosome entry site (IRES). The
IRES allows direct translation of the polyprotein.
4. The P1 region encodes the structural polypeptides.
5. The P2 and P3 regions encode the nonstructural proteins associated with replication.
6. The shorter 3’ UTR is important in (-)strand synthesis.
7. L is an additional N-terminal leader protein present in some genera that can either be a
protease (aphthoviruses, erboviruses) or have other function (kobuvirus, cardiovirus).
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Genome
polyprotein processing
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Attachment
1 Attachment.
Capsid
RNA
Receptor proteins
IG Like - Members of the
immunoglobulin family of receptors
found on cells of the immune
system.
Figure 4. CAR, coxsackie/adenovirus receptor; PVR, poliovirus receptor; ICAM-1, intercellular adhesion molecule 1;
VCAM-1, vitronectin cellular adhesion molecule 1; DAF, decay-accelerating factor; HAVcr-1, hepatitis A virus receptor
1; LDLR, low-density lipid receptor; 2 1, integrin; v 3, integrin; CBV, Coxsackie B virus; PV, Poliovirus; HRV, Human
rhinovirus; EMCV, Encephalomyocarditis virus; HAV, Hepatitis A virus; EV1, Echovirus 1; FMDV, Foot-and-mouth
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disease virus and CAV9, Coxsackie virus 9. Courtesy of Dr D Evans.
Rhinovirus binding and entry
1. The integral membrane protein, intercellular adhesion molecule 1 (Icam-1)
was identified as the receptor for the major group of human rhinoviruses.
2. Icam-1 is a member of the immunoglobulin superfamily.
3. Icam -1 is found on the surface of many tissues, including nasal epithelium
and lung epithelium.
4. The normal function of Icam-1 is to bind a ligand on the surface of
lymphocytes and to promote immunological and inflammatory functions.
5. This host response accounts in part for cold symptoms.
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Attachment To the Icam-1 Receptor
•A canyon is formed of VP1 and VP3 in the
capsids of rhinoviruses and some
enteroviruses like poliovirus.
•The canyons are the sites of interaction
with cell surface receptors.
• As the picornavirus binds to Icam-1, the
host receptor penetrates into the viral
canyon and this causes a change in
conformation of the capsid to permit virus
entry.
•Ab ：neutralize infectivity by blocking
entry of the receptor into the canyon.
•However, viruses quickly mutate to
change the shape of the canyon to prevent
antibody binding.
•antiviral drugs (WIN compounds) ：
inhibit uncoating and attachment of the
virus.
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Penetration and Uncoating
1
Attachment.
Capsid
RNA
Nucleus
Host cell
Cytoplasm
2 Entry
and uncoating.
Uncoating releases
viral RNA and proteins.
• Receptor mediated endocytosis, fusion with lysosome, lowered pH, loss of
VP4
• Loss of VP4 opens hole in centre of pentamer for RNA release
• Release of RNA into cytoplasm (about 1% of virus succeed)
• Fusion area in VP4 may create pore in phagolysososme to allow RNA out.
Entry into Cells
• Some non-enveloped virus inject their genome into the host cytoplasm
through creation of a pore in the host membrane. This is mediated by a viral
pore-forming peptide associated with the viral capsid.
• The icosahedric capsid of picornaviridae is able to create a pore either at
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plasma or endosomal membrane to inject their genomic RNA.
Entry into Cells
• During interactions of poliovirus with
its receptor major conformational
rearrangements occur in the virus
particle.
 The particles lose VP4 and the
hydrophobic N-terminus of VP1 is
displaced to the virion surface
 N-termini of VP1 forms a pore in the
cell membrane through which the
RNA is released into the cytosol.
 Some evidence suggests that virus
particles may undergo endocytosis in
some cell types.
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Replication
1 Attachment.
Capsid
RNA
Nucleus
Host cell
Cytoplasm
2 Entry
and uncoating.
3 RNA replication by viral RNA-
dependent RNA polymerase.
– strand is transcribed
from + viral genome.
Uncoating releases
viral RNA and proteins.
Viral
genome
(RNA)
mRNA is transcribed
from the – strand.
ssRNA;
+ or sense strand;
Picornaviridae
Viral
protein
Replication
Cytoplasmic
• VPg is removed from the viral RNA, then translated into a
processed polyprotein.
• In entero-, rhino, and aphtoviruses, shutoff of cellular capdependent translation through the cleavage of the translation
initiation factor eIF4G by viral protease.
• Replication of viral RNA takes place on membrane vesicles
derived from the ER.
• A negative-sense complementary ssRNA is synthesized using the
genomic RNA as a template.
• New genomic RNA synthesized is packaged into preformed
procapsids.
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Replication initiates by formation of a Covalent Bond
• Polioviral RNA is linked to the 22 aa VPg via a uridine-tyrosine
phosphodiester bond.
• During replication, mRNAs are
produced by cleavage of this
phosphodiester bond by a
cellular enzyme to produce viral
mRNAs containing a 5’ terminal
Up.
• Genomic RNAs incorporated
into virus particles contain the
5’ VPg.
• These steps occur in vesicles
derived from the smooth
endoplasmic reticulum.
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Replication
• vRNA translated to form a large polyprotein which is then
cleaved to yield 3 major segments
– P1- the capsid proteins are at the 5' end
– P2- regulatory protein in the centre
– P3-growth functional proteins at the 3' end
• Polyprotein is cleaved in a cascade process with
early cleavage by protein 2A (a nascent protease),
intermediate cleavage by 3C,
maturation cleavage of V0 by VP2 to yield VP2 and VP4 .
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Replication
1. 2Apro
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Poliovirus genome contains a single ORF, which encodes a 247 kD polyprotein.
•
It has VPg at its 5’ end and a poly(A) tail at its 3’ end
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Processing occurs in 3 steps.
1. The first is to cleave the P1 capsid protein precursor, which is catalyzed by 2Apro
2. The second step is to process the noncapsid and the capsid precursors catalyzed by
3Cpro and 3CDpro
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3. The third step is the processing of VP0 into VP4 and VP2
Replication
•The organization is as follows:
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Replication
Polyproteins are Proteolytic Processed During Translation
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Protein synthesis is required for RNA synthesis
RNA replication requires:
– 3B (VPg) as a primer (VPg-pUp-)
– 3D to serve as the polymerase
– 2B and 2C for vRNA synthesis
– host factors
– synthesis takes place on host membranes
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Polymerase complex copies +vRNA to -cRNA and then -cRNA back to
+vRNA
– Polymerase makes 5-10% cRNA and >90% vRNA
– Most early vRNA ends up as mRNA and don't have VPg associated;
50% of later vRNA is packaged and has VPg
•
Late mRNA lacks caps because the 2A protein cleaves the cap binding
protein of ribosomes so host synthesis is blocked but naked vRNA is
translated
•
Translation reaches peak 3-4 hours post infection
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Polyproteins are Proteolytic Processed During Translation
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Replication
1 Attachment.
Capsid
RNA
Nucleus
Host cell
Cytoplasm
2 Entry
and uncoating.
4 Translation and syn-
thesis of viral proteins.
3 RNA replication by viral RNA-
dependent RNA polymerase.
– strand is transcribed
from + viral genome.
Capsid
protein
+ strand
mRNA is transcribed
from the – strand.
Uncoating releases
viral RNA and proteins.
Viral
genome
(RNA)
ssRNA;
+ or sense strand;
Picornaviridae
Viral
protein
Replication
1 Attachment.
Capsid
RNA
Nucleus
Host cell
Cytoplasm
2 Entry
5 Maturation
and uncoating.
and release.
4 Translation and syn-
thesis of viral proteins.
3 RNA replication by viral RNA-
dependent RNA polymerase.
– strand is transcribed
from + viral genome.
Capsid
protein
Uncoating releases
viral RNA and proteins.
Viral
genome
(RNA)
+ strand
mRNA is transcribed
from the – strand.
ssRNA;
+ or sense strand;
Picornaviridae
Viral
protein
Picornavirus Translation:
1. Picornavirus translation is cap independent
2. Picornavirus mRNAs have no cap (pUp) at their 5’
terminus and no VPg.
3. The genome has a (743 nt) long UTR untranslated
leader sequence that contains 8 upstream AUGs
preceding the translational initiation site.
4. Internal ribosomal binding occurs at an Internal
Ribosomal Entry Sequence (IRES).
5. The IRES consists of a high level of secondary
structure in the UTR leader sequence that mediates
ribosome 40 S subunit binding and initiation.
6.
eIF-3, eIF-4G and eIF-4a promote IRES
assembly. A host factor X is also required.
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A Host Protein is Required for Synthesis of Polio Virus RNA
A highly based paired
structure formed from the
108 nucleotides at the 5’ end
of the (+) strand RNA of
poliovirus, which forms a
cloverleaf like structure.
The viral protein 3CD and a PCBP a host Cellular protein, (PolyrC Binding
Protein) interact with different loops of the cloverleaf.
This interaction brings the 5’ end close to the 3’ end.
At this point VPg priming can occur.
Formation of this ribonucleoprotein complex is required for synthesis of
both plus and minus strand RNA.
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Steps in the Assembly of Poliovirus
 After Translation and release
by the 2A protease, P1 folds
and is cleaved by the 3CD
protease to form the 5S
protomers.
 assemble into a 14S complex.
 The 14S complex may then
form a 75S empty capsid
that assembles with the RNA
to form a noninfectious 150S
provirion.
 Some researchers argue that
the 14S subunits assemble in a
series of steps with the
viral RNA to form provirions.
 The provirion is converted to
an infectous form by internal
cleavage of VP0 molecules to
VP4 plus VP2.
P1 Peptide
Assembly steps are not reversible
Folded P1
5S Unit
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Poliovirus Life Cycle
1) The capsid binds to
host receptors and
releases Viral RNA
into the cell.
2) Translation of the
released RNA
produces a polyprotein
that is processed by
the 2A and 3CD
proteinases to form
the viral gene
products.
3) RNA synthesis is
initiated by the 3AB
proteins and elongated
by the 3D protein.
4) As proteins viral
capsid assembly
begins in the
cytoplasm from P1
products.
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Laboratory Diagnosis of
Infections
• Most common method: isolate virus from stool
samples or throat samples from the mouth
– Rarely isolated from CSF
• Grows well in any human or monkey kidney cell
lines (causes good CPEs).
• Identify serotype with neutralization assays
• Nucleic acid methods: genomic sequencing to
determine if the infection is caused by
vaccine or wild-type virus.
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