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Replication of Large DNA Virus

Replication of Large DNA Virus. Herpesvirus, Poxvirus. Family Herpesviridae. “creeping” spread of rash & vesicle lesions Widely found in nature – plants, fungi, animals, humans Highly infectious Infections – acute, persistent, transform Eight Human herpesvirus (HHV 1-8)

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Replication of Large DNA Virus

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  1. Replication of Large DNA Virus Herpesvirus, Poxvirus

  2. Family Herpesviridae • “creeping” spread of rash & vesicle lesions • Widely found in nature – plants, fungi, animals, humans • Highly infectious • Infections – acute, persistent, transform • Eight Human herpesvirus (HHV 1-8) • Also primate, bovine, equine, swine, murine, avian herpesvirus

  3. Herpes Simplex Virus (HSV-1) • Infect mucous membranes and skin • HSV-1: mainly oral & facial area • Latent in neurons

  4. Herpes Simplex Virus -2(HSV-2) • Mainly genital area • Most infections are asymptomatic • Symptoms of genital lesions soon after exposure, last ~10 days • Latent in neurons, most have recurrent episodes within first year • Mother with active infection may transmit to newborn during delivery

  5. Genital Herpes: USA Epidemic • Estimate >20 million infections • Sexually Transmitted Diseases (STD’s) • Increasing since 1960’s • Social change (sexual freedom, changing moral standards, sex outside of marriage) • Birth control pill developed (non-barrier contraception) • Difficult problem for Public Health

  6. Varicella/Zoster Virus (VZV) • One virus, two diseases • Varicella – chickenpox • Latent in neurons • Zoster – shingles, uncommon reactivation along nerve trunk in adults

  7. Epstein-Barr Virus (EBV) • Infectious mononucleosis • Infects B lymphocyte, epithelial, fibroblast cells • Latent in lymphoid tissue • Co-carcinogen – Burkitt’s lymphoma, nasopharyngeal carcinoma

  8. Human Cytomegalovirus (HCMV) • “giant cells” in culture • syncytia forms multinucleated cell • Infects monocyte, lymphocyte, epithelial cell • Latent in lymphoreticular cells • USA – leading viral infection of fetus/newborns

  9. Human Herpesvirus 6 (HHV-6) • Exanthema subitum (roseola) • Common rash in young children • Infects lymphocytes

  10. HHV-7 • Isolated from lymphocytes of AIDS patient • “orphan” virus • No associated disease

  11. HHV-8 • Infects lymphocyte, vascular endothelial cells • Viral DNA found in Kaposi’s sarcoma tissue of AIDS patients • Co-carcinogen for Kaposi’s sarcoma

  12. HSV-1 • Envelope with surface projections, 200 nm • Tegument (matrix) structure between capsid and envelope • Icosahedral capsid, 130 nm • Core with virus DNA wound in cylinder

  13. HSV Genome: dsDNA • Linear, one strand has nicks, 150 kbp • Two unique components (UL, US) • Terminal and internal repeat sequences • Highly conserved “a” sequence at both ends (used for genome recognition and insertion into capsid)

  14. HSV Genetic & Transcription Map

  15. HSV: Attachment/Entry • Viral surface glycoproteins • Host cell heparan sulphate proteoglycans • Viral attachment blocked by polycations (polylysine, neomycin) • Fusion of viral envelope with cell plasma membrane • Capsid into cytoplasm • Release of VHS (virion host shut-off) tegument protein that degrades cell mRNA in cytoplasm

  16. HSV: Uncoating • Viral capsid transported to nuclear membrane • Release of DNA into nucleus • Viral tegument protein αTIF (trans-inducing factor) transported into nucleus activates virus transcription

  17. HSV: mRNA Transcription • DNA genome circularizes • Promoter/enhancer sites activated by viral αTIF and cell DNA-binding proteins (Oct-1, SP1) • Transcription from both DNA strands, bidirectional (clockwise, counterclockwise) • Uses cell RNA pol II

  18. HSV: Regulated Gene Expression • Immediate-Early – α gene products, mainly regulatory • Early – β gene products, mainly viral enzymes and proteins for DNA synthesis • Late – γ gene products, some regulatory, mainly structual proteins

  19. Cascade of HSV Transcription

  20. DNA Replication (Rolling Circle) • Synthesis of DNA in a long strand (head-to-tail concatemers) • Viral enzymes • Nick DNA strand, ssDNA rolls off • Continous and discontinous (Okazaki fragments) DNA replication • Concatemers later cleaved into genome size (recognition of “a” terminii)

  21. HSV: Assembly • Viral proteins transported into nucleus, assemble into capsid • Viral DNA “head-full” insertion into capsid

  22. HSV: Release • Viral “primary” tegument protein associate with viral glycoprotein, buds through inner & outer nuclear membrane, releasing capsid into cytoplasm • Capsid migrates to tegument proteins and picks up envelope by budding into exocytic vesicle • Virus inside vesicles of cytoplasm; either remain cell associated or “secreted” to outside

  23. Latent Infection • Virus ascend up sensory nerve to neuron • Viral DNA with cell histones and established in host cell as “episome” • Expresses LAT (latency-associated transcripts) • No infectious virus replication • May be reactivated (immune suppression, stress, injury, UV light, hormone)

  24. HSVInfection:ProductivevsLatent

  25. Reading & Questions • Chapter 17: Replication of Some Nuclear-Replicating DNA Viruses

  26. Class Discussion – Lecture 11a • 1. How does HSV upon release of its DNA genome insure that it will be transcribed? • 2. Like a good friend, HSV and its host cell have a lifetime relationship. How is this possible?

  27. Family Poxviridae • Viruses of vertebrates and insects • Large “brick” shape, 200x300 nm • External, inner envelope • Lateral bodies • Complex coat of tubular structures • Replication occurs in cytoplasm • Benign tumors in experimental hosts

  28. Human Poxviruses • Characteristic rash and “pocks” • Variola – smallpox • Transmitted by inhalation and infects respiratory tract, systemic infection • eradicated by WHO vaccination (1977) • Vaccinia – “cowpox” lab recombinant used for vaccine • Molluscum contagiosum – localized lesions, transmitted by contact

  29. Occassional Poxvirus Zoonosis to Humans • Localized lesions • Transmitted by contact • Orf – sheep, goat • Cowpox – rodents, cats, cows • Monkeypox – monkeys, squirrels

  30. Vaccinia Virus Genome: dsDNA • Linear, 186 kbp • Covalently closed ends (“hairpin” loops) • Inverted terminal repeats (10 kbp) • Conserved central region • Genes code for enzymes needed for RNA/DNA synthesis

  31. Vaccinia Virus: Entry/Uncoating • Fusion of virus with plasma membrane or entry by endocytosis • Release of viral core into cytoplasm • Viral proteins shut off host functions • Further uncoating leads to “early” viral transcription/proteins in cytoplasm

  32. Vaccinia Virus: Expression of “Early” Genes • Virus core brings in enzymes required for viral transcription • Half of genome is expressed from “early” gene promoters (activated by viral DNA binding proteins) • Express enzymes needed for DNA replication

  33. DNA Replication • Occurs in cytoplasm • Nick at end creates a free 3’ OH, self-priming • DNA synthesis displaces parent strand, two genome concatemer (tail-to-tail) • Continued DNA synthesis displaces two genome strand concatemer (tail-to-tail, head-to-head) • Cleaved into two genome lengths • Fill in and ligate ends into dsDNA, closed ends

  34. Vaccinia Virus: Expression of “Late” Genes • Switch due to viral regulatory proteins and configuration of newly replicated viral DNA • Use of “late” promoters • Expression of some enzymes, mainly structual proteins

  35. Vaccinia Virus: Assembly and Release • Sequential developmental stages in cytoplasm • Viral membrane form crescent and circular structures • Nucleoprotein mass forms with immature envelope and buds through golgi membrane for envelope • Release by budding through plasma membrane

  36. Vaccinia Virus Replication Cycle

  37. Recombinant Vaccine • Poxviruses have high recombination rate • Dual infection of vaccinia virus + recombinant plasmid cloning vector with foreign virus gene • Use of recombinant vaccinia virus + foreign gene for possible protective vaccine

  38. Smallpox Virus: Potential Terrororist Weapon? • Susceptible population • Easily transmitted by inhalation • Highly virulent strains (up to 40% mortality) • Smallpox virus stored in two Public Health Labs (USA, former Soviet Union) • Fear? • Best defense?

  39. Reading • Chapter 18: Replication of Cytoplasmic DNA Viruses

  40. Class Discussion – Lecture 11b • 1. What would you postulate for the origin of Poxviruses? • 2. If a terrorist ask your suggestion for a biological agent, would you tell him to go buy a herpesvirus or a smallpox virus from an underpaid government research microbiologist ? • 3. Since smallpox has now been eradicated, would it be a good or bad ideal to destroy the remaining virus samples in the U.S.?

  41. MICR 401 Final Exam • Tuesday, Dec. 4, 2012 • 1:30 – 3:00pm • Papovavirus thru Hepadnavirus • Case Study and Questions #9-15 • Lecture & Class Discussion Questions, Reading & Chapter Questions • Exam: • Objective Questions (MC, T/F, ID) • Short Essay Questions

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