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

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. 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. Poxvirus infection cycle

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. 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 Vaccinia virus movement on actin tails

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 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 Extensive sequence divergence Recombination Horizontal transfer

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 homologiesrecombination 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)