Transcript Poxvirus - Rutgers

Cryo EM of
purified Poxvirus
particles
30 nm-thick surface
domain (S)b is noted
Poxviruses
From Principles of Virology,
Academic Press 2004
Poxvirus particle is large and complex
100 nm
Poxviridae
• Large family of large, complex viruses
• Infect vertebrate and invertebrate hosts
• Two subfamilies:
– Chordopoxvirus, 8 genera (vertebrate hosts)
– Entomopoxvirus, 3 genera (invertebrate hosts)
• All replication steps in cytoplasm, but probably
requires host factors late in infection process
• Particles contain enzymes associated with replication,
transcription, protein and nucleic acid modification
• One of the few DNA viruses that make their own RNA
polymerase; therefore, promoter specificity achieved
• Substantial cross hybridization and cross reactivity
within genera
• Relatively narrow host cell specificity
Poxvirus particle composition
• Nucleic acid: Single large segment of dsDNA, ~
3% of particle weight (130-375 kbp); 20
completely sequenced genomes
• Proteins: ~ 100 total virion proteins, ~ 90% of
particle weight
• Carbohydrate: ~ 3% of particle weight, most as
N- or C-linked glycans or glycolypids
• Lipids: ~ 4% of particle weight, most as
modified cellular lipids
Biology of poxviruses
• Cause disease in many vertebrates, including fowl
• May cause mild lesion disease or systemic lethal
disease
• Different strains cause different diseases; different
animal species react differently to a given strain
• Myxoma virus of hare used to control feral European
rabbit population in Australia
• Transmission
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Aerosol (common for smallpox)
Direct contact
Arthropods - biting vectors, no virus replication
Indirect contact
Biology of poxviruses
• After primary infection is successfully fought,
virus is cleared - does not remain in animal
• Early in infection, host immune system may be
severely compromised:
– “virokines” - secreted, virus-coded proteins that
mimic host cytokines and cellular growth factors,
thus interfering with normal cell growth, causing cell
proliferation
– “viroceptors” - secreted, virus-coded proteins that
mimic host cytokine receptors that are involved with
host immune response
Poxvirus genome organization
• Linear dsDNA 130-375 kbp; covalently closed termini.
• Large hairpin structure at each terminus - up to 10 kb
total at each end is repeat sequence (replicationassociated).
• Encode 150-300 proteins.
• Coding regions are closely spaced, no introns.
• Coding regions are on both strands of genome, and
are not tightly clustered with respect to time of
expression or function.
Poxvirus genome expression
• Early - polyA+, capped mRNAs representing ~ 50% of
genome synthesized from both strands by virus-coded
enzymes within the core
• Unspliced transcripts extruded from core for
translation by host ribosomes.
• Host macromolecular synthesis inhibited.
• Early genes expressed prior to DNA replication
encode enzymes for replication, intermediate gene
transcription, neutralization of host response.
• Intermediate genes required for replication, DNA
modification, transcription of late genes.
• Late genes required for structural proteins, early
transcription factors.
Poxvirus infection cycle
1. Entry and release of core
(mechanism unknown).
2-5. Early mRNA synthesis
products release core,
cause cell proliferation, and
local immune suppression.
6-9. DNA synthesized for
packaging and as template
for intermediate gene
expression – products
include transcription
specificity factors for late
gene expression.
10-18. Transcription and
translation of late (structural)
protein genes; particles
assembled at Golgi;
particles released on cell
lysis or directly infect
adjoining cell.
Vaccinia virus exocytosis
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•
•
•
Immature virion (IV) formed from golgi, not by budding
IV matures to form infectious intracellular mature virion (IMV)
IMV acquires another membrane to form intracellular enveloped virion (IEV)
IEV transported to, fuses with plasma membrane
Vaccinia virus movement on actin tails
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•
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Actin in uninfected
cell (A) is reduced
and dramatically
reorganized by
Vaccinia virus
infection (B)
Intracellular
enveloped virions
bind to actin tails
(one virion per tail)
and move through
cytoplasm (C)
Actin tails push
virus into adjoining
cell
Smallpox
• Epidemic for more than 2 centuries.
• Two main strains:
– Variola major - general lethality >20%
– Variola minor - lethality < 5%
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“Variolation” first used for protection.
Subject of first vaccine with related cowpox.
Survives for years in desiccated state.
Human-to-human transmission the only normal
infection route.
• Declared eradicated in 1980 by WHO.
• Duration of vaccine unclear – some level of
protection appears long-lived.
Sources of poxvirus gene acquisition
• Extensive sequence divergence
• Recombination
• Horizontal transfer
• AMV-EPB_034 – inhibitor of apoptosis from Amsacta
moorei entomopoxvirus (AMV-EPB)
• GenBank sequence – inhibitor of apoptosis from Bombyx
mori (silkworm) BLAST e-value 9e-81
• Bombyx and Amsacta both Order Lepidoptera
• 62% of best non-viral GenBank hits are from same
taxonomic Class as viral host
Poxvirus Phylogeny
Poxvirus conserved gene order and spacing
Monkeypox 2003
• Spread by rodents and monkeys
• Infects, but rarely fatal in humans
– (1-10% mortality in Africa, <1% in developed countries)
• Infected ~30 people in the Midwest in
spring, 2003
• Rarely found outside of Africa
• Likely will not become a major problem in
humans
• Smallpox vaccine partially protects against
monkeypox
Vaccinia virus vectors
• 187 kbp linear DNA genome of Vaccinia virus has more than 12
sites where additional DNA can be inserted.
• Insertion of genes in these sites is by recombination at flanking
homologies
• Insert sizes up to 25 kbp accepted
• The major use of vaccinia virus vectors is as antigen delivery
vehicles for immunization.
– First successful trials against rabies in foxes
– Problems:
• Delivery to those already immune
• Very cytotoxic – not good for long-term treatment
• As antitumor therapy
– For replicative "oncolysis" or intratumoral expression of toxic or
immunostimulatory genes
Poxvirus in experimental systems
• For experimental gene expression studies –
poxviruses make their own RNA polymerases
• For capping studies – capping enzyme purified
early
• Vaccinia virus methyltransferase subunit shown
to substitute in yeast
– (Saha et al., J Virol. 2003 77: 7300–7307)