Vorlesung Molekulare Virologie und Onkologie Picornaviren und Hepatitis A Virus

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Vorlesung Molekulare Virologie und
OnkologiePicornaviren und Hepatitis A Virus

• Prototyp der Virusfamilie Picornaviridae
• Poliovirus

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Iron lungs used during 1950s poliovirus
epidemics to counter effects of respiratory
muscles paralysis.Fig 26.17
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Poliomyelitis in individual with atrophy of the
left leg due tolack of muscle and bone
development. Results from selective destruction
of motor nerves in spinal cord.Fig. 26.15
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Poliovirus epidemiology and control

• Worldwide infection
• Most frequent in young children, subclinical
• 21,000 polio cases/yr in USA before vaccine
• WHO- USA free in 1994, world free-2000
• Effective vaccine- permanent immunity
• 2 types (4 times over 1-2 yrs)
• Salk- inactivated (early use in USA)
• Sabin- live attenuated virus (global use)

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Fig. 26.18
USA incidence of polio infection (1951-1998)
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Sizes of icosahedric viruses
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Mol Virol HAV
Negative stain of picornavirus particles
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Picornaviridae

• Non-enveloped virion
• Single-stranded linear RNA genome of positive
  polarity
• Lacks 5 cap structure translated from internal
  ribosome entry site (IRES)
• Named as pico (very small), RNA (genome)
• Family includes many important human animal
  pathogens
• Poliovirus
• Hepatitis A virus (HAV)
• Foot-and-mouth disease virus (FMDV)
• Rhinovirus
• Poliovirus FMDV are the best-characterized

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Classification

• Family Picornaviridae has 6 genera
• Aphthovirus
• e.g., FMDV
• Cardiovirus
• e.g., EMCV
• Enterovirus
• Hepatovirus
• Parechovirus
• Rhinovirus
• All contain viruses that infect vertebrates

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Classification

• Aphthoviruses (e.g., FMDV)
• Infect cloven-hoofed animals
• Rarely infect humans
• 7 serotypes, each with subtypes
• Cardioviruses (e.g., EMCV Theilers virus)
• Two clusters
• Encephalomyocarditis-like viruses (e.g., EMCV
  mengovirus)
• Murine viruses
• Also infect humans, monkeys, pigs, elephants
  squirrels
• Sehr wirksame IRES
• Theilers murine encephalomyelitis viruses (e.g.,
  Theilers virus
• Some strains cause polio-like disease in mice
• Others cause chronic demyelinating disease like
  MS
• Vilyuisk virus causes human encephalomyelitis

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FAMILY PICORNAVIRIDAE

• Taxonomic Structure of the Family
• Genus Enterovirus
• Genus Rhinovirus
• Genus Cardiovirus
• Genus Aphtovirus
• Genus Hepatovirus
• Genus Parechovirus

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Species in the Genus
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Classification

• Enteroviruses (e.g., Poliovirus)
• Replicate in alimentary canal
• Resistant to low pH
• Genus includes
• Poliovirus 3 serotypes
• Coxsackie virus 23 serotypes
• Echovirus 28 serotypes
• Human enterovirus 4 serotypes
• Plus many non-human enteric viruses
• Hepatovirus (HAV)
• HAV reclassified from Enterovirus to Hepatovirus
  genus
• Nucleotide amino acid sequ.dissimiliarity from
  other picornaviruses
• No cpe in cultured cells
• Only one serotype

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Classification

• Parechoviruses
• 2 serotypes of human parechovirus
• Reclassified from Enterovirus genus
• Previously echovirus type 22 type 23
• Only 30 amino acid identity with other
  picornaviruses
• Capsid has 3, not 4, proteins
• Rhinoviruses
• 103 serotypes
• Replicate in nasopharynx
• Important agents of common cold
• Acid labile

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Picornavirus Taxonomy
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Physical properties of the virion

• Spherical - 30 nm
• Protein shell surrounding naked RNA genome
• Lack lipid envelope
• Infectivity insensitive to solvents
• Differentially pH stable/labile related to
  replication sites
• Cardio-, entero-, hepato-, parenchoviruses
  resist low pH
• Pass through stomach to infect intestine
• Rhino- Aphthoviruses labile at pH lt6.0
• Replicate in respiratory tract
• Acid lability plays role in genome uncoating
• Differential buoyant densities on cesium
  gradients
• If pores are present in viral capsid permeable
  to cesium
• Poliovirus not cesium permeable
• Aphthovirus cesium permeable
• Rhinovirus intermediate permeable, limited by
  polyamines in capsid

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Poliovirus plaques on a cell culture monolayer.
Each plaque results from cells destroyed by
spread of one infectious virus in the original
inoculum.Fig. 26.16
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ParticlePFU-Ratio

• Ratio of particles to plaque-forming units
• Determined by number of particles (e.m.) divided
  by number of infectious units (plaque assay)
• Fraction of virus particles that are infectious
• N.B. specific for cell-type plaqued on
• For poliovirus ranges from 1 in 30 to 1 in 1,000
• Other picornavirus in same range
• Why so few infectious particles?
• Non-infectious particles have lethal mutations in
  genome?
• All are potentially infectious, but most dont
  complete infectious cycle?
• Fail to attach, enter, replicate or assemble?
• Infectivity of aphthoviruses approaching 11
  all infectious

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High-resolution structure of the virion
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Space models of picornavirus proteins VP1 VP3
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High-resolution structure of the virion

• Basic building block of capsid is protomer
• One copy each of VP1, VP2, VP3 VP4
• Shell formed by VP1 to VP3 interaction, with VP4
  internal
• VP1, VP2 VP3 have no sequence homology, but
  same topology
• Each forms 8-stranded antiparallel ß-barrel
• Swiss-roll or jelly-roll ß-barrel
• Antiparallel ß-sheets make wedge-shape
• One sheet forms wall of wedge
• Other, which has kink, forms wall floor
• Wedges packed together to form dense rigid
  protein shell
• Strengthened by protein-protein interactions
  inside capsid
• Particularly around 5-fold axis

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High-resolution structure of the virion

• Picornavirus capsids have 4 proteins
• VP1, VP2, VP3 VP4
• Exception Parechoviruses have 3
• VP1, VP2 VP0 (uncleaved precursor to VP2 VP4)
• Capsid is icosahedral arrangement of 60
  structural proteins
• Best way to build shell with nonidentical
  subunits icosahedron
• Icosahedron 20 triangular faces 12 vertices
• Minimum numbers of subunits 60
• Caspar Klug, 1960s
• High-resolution structures for many
  picornaviruses determined by X-ray
  crystallography
• First structures for poliovirus type 1 human
  rhinovirus 14

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High-resolution structure of the virion

• VP4 has extended conformation
• Structural differences among VP1, VP2 VP3
• In loops connecting ß-barrels
• In N- C- terminal extending from ß-barrel
• These sequences give each picornavirus its
  distinct morphology antigenicity
• C-termini on surface of virion
• N-termini on interior
• Capsid proteins of many viruses have similar
  topology
• Plant, invertebrate vertebrate viruses
• No sequence homology
• Either, evolved from common ancestor
• Or, ß-barrel domain is best way to pack proteins
  into sphere

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Rhinovirus Rhinovirus 14 X-Ray
Rhinovirus Rhinovirus 14 X-Ray
Enterovirus Poliovirus type 1 X-Ray
AphthovirusFMDV X-Ray
Cardiovirus Mengovirus X-Ray
Rhinovirus Rhinovirus 16 X-Ray - interior
Enterovirus Poliovirus type 1 X-Ray
Rhinovirus Rhinovirus 14 CryoEM
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Surface of the virion

• Surface has corrugated topography
• Prominent star-shaped plateau mesa at 5-fold
  axis of symmetry
• Surrounded by deep depression canyon
• Another protrusion at 3-fold axis
• For poliovirus rhinovirus, canyon is receptor
  binding site
• But, not all picornaviruses have canyons
• Surfaces of aphthoviruses cardioviruses have no
  canyon much smoother receptor binding site
  elsewhere

5-fold axis
3-fold axis
Canyon
Entero
Cardio
Aphtho
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The Hydrophobic Pocket

• Hydrophobic tunnel or pocket
• Within core of VP1
• Usually under canyon floor
• In poliovirus types 1 3 believed to contain
  sphingosine
• In human rhinovirus types 1A 16 contains
  unidentified lipid
• In coxsackie B contains C16 fatty acid
• But, in human rhinovirus type 16 its empty
• Binding site for antipicornavirus drugs
• WIN compounds (Sterling-Winthrop)
• Similar cpds made by Schlering-Plough Janssen
  Pharmaceutica
• Hydrophobic, sausage-shaped
• Bind tightly in hydrophobic pocket/tunnel
• Displace lipid molecule
• Inhibit binding or uncoating

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Picornavirus genome organization
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Genome organization

• Common genome organization
• 7,209 to 8,450 bases long
• Single-stranded, positive-sense RNA genome
• Single ORF, translated as polyprotein
• Cleaved during translation never see
  full-length product
• Long (624-1,199 nt) highly structured 5
  non-coding region
• Contains internal ribosome entry site IRES
• Short (47-125 nt) 3 non-coding region
• Contains 2 structure
• Pseudoknot role in controlling RNA synthesis
• Poly(A) tract (35-100 nt)
• 3 ncr not required for infectivity, but important

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Genome organization

• Polyprotein divided into 3 regions
• P1 Viral capsid proteins
• P2 Proteins involved in protein processing
  genome replication
• P3 ditto

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Polyproteinprocessing of picornaviruses
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Polyprotein processing of different picornavirus
genera
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VPg protein

• VPg covalently linked to 5 end of all picorna
  genomes
• Viral Protein genome-linked
• 22-24 amino acids
• 5uridylylate on RNA joined to tyrosine in VPg
  (3rd aa)
• VPg encoded by single gene
• Except FMDV has 3 VPgs
• VPg not required for infectivity
• VPg not found on viral RNAs associated with
  ribosomes
• Removed by host protein unlinking enzyme
• Prerequisite for or result of association with
  ribosome?
• VPg found on nascent RNA chains of replicative
  intermediates negative-stranded RNAs
• Used as primer for RNA synthesis?
• VPg plays major role in recent models for
  poliovirus RNA replication

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Life cycle of picornaviruses
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Life cycle of picornaviruses
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Life cycle of picornaviruses

• Replication entirely cytoplasmic
• Virus attaches to cell surface receptor
• Structural changes occur in capsid
• Genome uncoated released into cytoplasm
• Translated into polyprotein precursor
• Nascently cleaved into proteins for genome
  replication virion assembly
• 2 viral proteinases (2Apro and 3Cpro/3CDpro)
• Genome replication
• RdRp accessory proteins copy genome to
  negative-sense intermediate
• Used as template to synthesize positive-sense
  progeny
• Occurs on small, semi-membranous vesicles induced
  by virus proteins
• Encapsidation begins when pool of capsid proteins
  is sufficient
• Coat protein precursor P1 cleaved into immature
  protomer
• Assemble into pentamers
• Assemble with newly-synthesized genomes
• Released as cell loses integrity
• Except hepatoviruses released without cpe
• Entire cycle takes 5-10 hours

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Picornavirus receptors
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Attachment of picornaviruses to receptors
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Rhinovirus ICAM-1
Poliovirus CD155 (Pvr)
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Attachment (2)

• How do picornaviruses attach to cells?
• All icosahedral, but surface architecture varies
• Accounts for different serotypes different
  virus-receptor interactions
• Some have canyon (or groove), others dont
• For polio- rhinoviruses, receptor binds in
  canyon
• Mutate canyon aas alter receptor binding
  affinity
• Interactions shown by cryo-EM X-ray
  crystallography
• For FMDV coxsackievirus A9, receptor binds
  through surface loops
• RGD in VP1 loop of FMDV binds integrins
• Blocked by RGD-binding peptides
• Mutating RGD interferes with receptor binding
• HS binds in shallow depression on virion surface
  where VP1, VP2 VP3 meet
• Dependent on His to Arg mutation in VP3
• RGD in C-terminus of Coxsackievirus A9 binds
  integrins
• But, mutating RGD doesnt abolish binding has
  another receptor

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Entry into cells

• After binding, capsid must dissociate to release
  genome
• For some, binding concentrates virions on surface
• Low pH or coreceptor interaction effect genome
  release
• For others receptor causes conformational changes
  to release genome
• For poliovirus, binding to CD155 causes major
  structural changes
• Forms altered or A particles
• VP4 lost hydrophobic N-terminus of VP1 ends up
  on surface
• Inserts into membrane forms pore through which
  RNA enters?

• Lipids in hydrophobic pocket play a role in
  uncoating
• Lock virion in stable conformation prevent
  uncoating
• Lipid likely removed to allow RNA release
• Not known how

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Translation of viral RNA (1)

• Ribosomes bind to IRES
• Dont scan through 5 end of picornavirus RNA
• Structures conserved within type I type II
  IRESs
• Have conserved sequence motif GNRA (Nany nt)
• In stem-loop IV of type I IRES, stem-loop I of
  type II IRES
• Have conserved Yn-Xm-AUG motif
• Yn pyrimidine-rich region
• Xm 15-25 nt spacer, followed by AUG
• In type II IRES, this is the initiator codon
  bound by 40S ribosomal subunit
• In type I IRES, 40S subunit binds scans to an
  AUG 30-150 nt downstream
• IRES-initiated translation requires initiation
  proteins
• Same set as 5-end dependent translation
• eIF4G interacts directly with IRES or through
  cellular IRES binding protein
• eIF4G cleaved in picornavirus-infected cells
• Still able to initiate translation
• La autoantigen polypyrimidine tract binding
  protein bind the IRES
• Recruits eIF3 40S ribosomal subunit

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Genome organization

• Polyprotein divided into 3 regions
• P1 Viral capsid proteins
• P2 Proteins involved in protein processing
  genome replication
• P3 ditto

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Translation of viral RNA (2)

• Polyprotein cleaved by viral proteases
• Multiple proteins from single mRNA
• Primary cleavages
• Cleaved co-translationally (in cis) as proteases
  are formed
• Full-length polyprotein not found in cells
• Secondary cleavages
• Cleaved in cis trans to form proteins
• 3Cpro, 3CDpro 2Apro proteases active in nascent
  polyprotein
• Autocleave themselves from growing chain by
  acting in cis
• Once released, act in trans on polyprotein
• Multiple activities performed by single protein
  efficiency!
• Processing cascade varies for different
  picornaviruses
• Viral protein expression controlled by rate
  extent of proteolytic processing

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Translation of viral RNA (3)

• Encode 3 proteinases
• Lpro thiol protease-like
• Aphthoviruses only
• Cleaves itself from VP4 cleaves eIF4G
• Cardioviruses encode L protein, but not a
  protease cleaved by 3Cpro
• 2Apro chymotrypsin-like protease
• Protease in entero-, rhinoviruses
• Cleaves P1 from P2 (between VP1 2A) cleaves
• Autocleaves 2A from 2B in aphtho- cardioviruses
• Not in hepato- or parechoviruses (function
  unknown)
• 3Cpro chymotrypsin-like serine protease
• All picornaviruses
• Primary cleavage of P2 from P3 (between 2C 3A)
• In hepato- parechoviruses also cleaves between
  2A 2B
• Secondary cleavage of P1 P2 polyproteins

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Genome replication mRNA synthesis (1)

• RNA replication studies mostly with poliovirus
• Produce 50,000 progeny genomes per infected cell
• 3 forms of RNA in infected cell
• Single-stranded RNAs
• Most abundant
• Exclusively positive-sense
• Replicative intermediates (RIs)
• Mostly full-length RNA (ve) with 6 to 8 nascent
  strands (-ve) attached
• Opposite has been detected
• Replicative forms (RFs)
• Full-length, dsRNA
• Viral RNA synthesis is asymmetric
• 25 to 65x more positive-sense made than
  negative-sense

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Picorna RNA replication
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Genome replication mRNA synthesis (2)

• Picornavirus RdRp is 3Dpol
• Found in RNA Replication Complexes
• Associated with smooth membranes of cell
• Requires oligo(U) primer to copy poliovirus RNA
• Template- primer-dependent polymerase
• Looks like a hand
• Palm active site of enzyme
• May oligeromize
• Through interface I to form long fibers
• Through interface II to form networks of fibers
• Has additional activities
• Cooperative ssRNA-binding protein
• Unwinds duplex RNA without ATP hydrolysis
• Keeps RNA single-stranded, unwound continuously
  replicating?

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Genome replication mRNA synthesis (3)

• RNA replication uses viral accessory proteins
• Most proteins in P2 P3 involved in RNA
  replication
• 2A
• Protease cleaves polyprotein
• Plays unknown additional role in replication
• 2B
• Plays unknown role in RNA synthesis
• Induces cell membrane permeability
• Partly responsible for proliferation of
  membranous vesicles (with 3A)
• 2C
• Binds RNA has NTPase activity
• Shares homology with known helicases, but
  purified protein not active
• Mutations in 2C block antiviral activity of
  guanidine hydrochloride
• Specifically inhibits initiation of
  negative-strand synthesis
• 3AB
• Anchors VPg in membranes to prime RNA synthesis
• Stimulatory cofactor for 3Dpol

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Genome replication mRNA synthesis (4)

• RNA replication uses cellular accessory proteins
• Cytoplasmic component(s) required for initiation
  of RNA synthesis
• If purified poliovirus RNA incubated in vitro
  with cytoplasmic extract
• Viral RNA translated
• Polyprotein processed
• Genome replicated
• Virions assembled
• If cytoplasmic extract removed after
  preinitiation complex forms
• No RNA synthesized
• Cellular poly r(C)-binding proteins required
• Bind cloverleaf in 5 end of positive-sense RNA
• Mediate attachment of 3CD to cloverleaf
• Essential to initiate viral RNA synthesis
• Cellular Sam68 (Src-associated in mitosis, 68kd)
  may play role
• Normally nuclear, but redistributed by poliovirus
  infection
• Associated with 3Dpol /or 2C

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Cellular site for RNA synthesis

• Replication occurs entirely in cytoplasm
• Poliovirus-infected cells accumulate small
  membranous vesicles
• Because membrane vesicle transport from ER to
  cell surface inhibited
• Blocked by 2B and/or 3A
• Proteins 2BC 2C cause membrane proliferation
• Vesicles important for RNA synthesis
• Lipid synthesis inhibitor (cerulenin) brefeldin
  A (blocks accumulation of vesicles) inhibit
  poliovirus replication
• Because poliovirus is non-enveloped doesnt need
  functional ER or Golgi
• Replication complex brought to membrane vesicles
  by 2C 3AB
• 2C binds RNA anchors RNA to membranes in
  replication complex?
• 3AB anchors VPg in membrane for RNA synthesis

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Life cycle of picornaviruses
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Recombination

• Exchange of nucleotide sequences among different
  genome RNA molecules
• First discovered for poliovirus replication
• Now shown for most RNA viruses
• Frequency of recombination is high
• Occurs mainly between nt sequs of RNAs with high
  identity
• Called base-pairing dependent or copy choice
  recombination
• Occurs by template switching during
  negative-strand synthesis
• RdRp copies 3 end of one parental genome
• Switches templates
• Continues at corresponding position on 2nd
  parental genome
• 100x more frequent among type 1 polioviruses
  than between type 1 type 2
• Cause of template switching? Happens when RdRp
  pauses?

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Assembly of Picornaviruses
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Assembly of virus particles

• Studied extensively for picornaviruses SIMPLE!!

• Capsid protein precursor P1 folds nascently
• Cleaved from P2 (1), into VP0, VP3 VP1 by
  3CDpro
• 5S protomers self-assemble into 14S pentamers
• Pentamers assemble into 80S empty capsids
• Either, RNA threaded into capsid making 150S
  provirion (with uncleaved V0)
• But, no opening has been seen
• Or, pentamers assemble around RNA empty capsids
  for pentamer storage?
• Final step in morphogenesis is cleavage of VP0
  into VP2 VP4 producing 160S infectious virion
• Protease unknown inside virion autocatalysis?

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Effects of viral multiplication on the host cell

• Poliovirus inhibits 5 end-dependent mRNA
  translation
• By 2 h p.i. polyribosomes disrupted, translation
  of cellular RNA stops
• Replaced by viral mRNA translation
• eIF4GI/II inactivated by cleavage - so cant bind
  eIF4E
• eIF4E cant bind 5 cap of mRNAs - cant recruit
  40S ribosomal subunits
• Picornaviruses inhibit host cell RNA synthesis
• DdRps from infected cells are active
• Accessory proteins may be targeted
• Transcription factors required by each DdRp
  inhibited
• Poliovirus induces cytopathic effect (cpe)
• Leakage of lysosomal contents?
• Poliovirus-encoded inhibitor blocks apoptosis in
  vitro
• But, poliovirus causes apoptosis in vivo

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Perspectives

• Picornaviruses, especially poliovirus, have been
  researched extensively
• But many questions remain
• What determined receptor selection?
• Does receptor-mediated signaling help/hinder
  virus?
• How does genome come out of particle?
• Etc
• But the eradication of poliovirus is planned by
  2005
• Poliovirus is truly a virus with a brilliant
  past, but with no future
• Vincent Racaniello
• Fortunately many of these issues can be studied
  with other picornaviruses

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Phylogenetic tree of picornaviridae
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HAV infection

• Infection by the fecal oral route
• Rarely by the blood route
• 4 weeks incubation period
• Liver inflammation due to cytotoxic T cell
  response
• Often inapparent in young people
• Occasionally very severe or lethal in elderly
  with pre-existing HBV or HCV infection
• Complete resolution within 1 -2 months
• Some cases may be protracted up to 12 months
• No chronic cases known
• Complete immunity due to neutralising antibody

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HAV properties

• Very heat stable
• Survives for years in water or shell fish
• Transmitted also by fecal smears
• Only one serotype
• Difficult to grow in cell culture
• Highly infectious
• Rare in Germany, mostly imported

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Course of HAV infection
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Infectivity of HAV in body excretions
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Hepatitis immunisation

• Passive with immunoglobulin
• Protects even up to 1 week post exposure
• But only for 2 months
• Active with cell culture-produced HAV
• Killed with formalin
• Purified 2 x 25 nanograms HAV doses
• Protects before exposure
• Combination with HBV vaccine (Twinrix) available
  and recommended

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Antibody titers after HAV immunization
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Efficacy of HAV vaccine