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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

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slide1

Cryo EM of purified Poxvirus particles

30 nm-thick surface domain (S)b is noted

Poxviruses

From Principles of Virology, Academic Press 2004

poxviridae
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
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
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
    • Aerosol (common for smallpox)
    • Direct contact
    • Arthropods - biting vectors, no virus replication
    • Indirect contact
biology of poxviruses1
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
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 (replication-associated).
  • 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
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.
slide10

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
Vaccinia virus exocytosis
  • 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
Vaccinia virus movement on actin tails
  • 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
Smallpox
  • Epidemic for more than 2 centuries.
  • Two main strains:
    • Variola major - general lethality >20%
    • Variola minor - lethality < 5%
  • “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
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 OrderLepidoptera
  • 62% of best non-viral GenBank hits are from same taxonomic Class as viral host
monkeypox 2003
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
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
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)