From Principles of Virology , Academic Press 2004

1. Cryo EM of purified Poxvirus particles 30 nm-thick surface domain (S)b is noted Poxviruses From Principles of Virology, Academic Press 2004

2. Poxvirus particle is large and complex 100 nm

3. 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

4. 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

5. 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

6. 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

7. 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.

9. 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.

10. 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.

11. 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

12. 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

13. 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.

14. 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

15. Poxvirus Phylogeny

16. Poxvirus conserved gene order and spacing

17. 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

18. 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

19. 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)