11 Replication of Large DNA Virus

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11 Replication of Large DNA Virus
2012-11-30 01:37:49

Herpesvirus, Poxvirus
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  1. Family
    •“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
  2. Herpes
    Simplex Virus (HSV-1)
    •Infect mucous membranes and skin

    •HSV-1:  mainly oral & facial area

    •Latent in neurons
  3. Herpes
    Simplex Virus -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
  4. Varicella/Zoster Virus (VZV)
    •One virus, two diseases

    •Varicella – chickenpox

    •Latent in neurons

    •Zoster– shingles, uncommon reactivation along nerve trunk in adults
  5. Epstein-Barr
    Virus (EBV)
    •Infectious mononucleosis

    •Infects B lymphocyte, epithelial, fibroblast cells

    •Latent in lymphoid tissue

    •Co-carcinogen – Burkitt’s lymphoma, nasopharyngeal carcinoma
  6. 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 
  7. Human Herpesvirus 6 (HHV-6)
    •Exanthema subitum (roseola)

    •Common rash in young children

    •Infects lymphocytes
  8. HHV-7
    •Isolated from lymphocytes of AIDS patient

    •“orphan” virus

    •No associated disease
  9. HHV-8
    • •Infects lymphocyte, vascular endothelial
    • cells

    • •Viral DNA found in Kaposi’s sarcoma tissue of
    • AIDS patients

    •Co-carcinogen for Kaposi’s sarcoma
  10. HSV-1 (structure)
    •Envelope with surface projections, 200 nm

    • •Tegument (matrix) structure between capsid
    • and envelope

    •Icosahedral capsid, 130 nm

    •Core with virus DNA wound in cylinder
  11. 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)
  12. HSV:  Attachment/
    •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
  13. 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
  14. 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 polII
  15. 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
  16. 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)
  17. HSV:  Assembly
    • •Viral proteins transported into nucleus,
    • assemble into capsid

    •Viral DNA “head-full” insertion into capsid
  18. 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
  19. Latent
    • •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) 
  20. Family
    •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
  21. Human
    •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
  22. Occassional Poxvirus Zoonosis to Humans
    •Localized lesions

    •Transmitted by contact

    • •Orf
    • – sheep, goat

    •Cowpox – rodents, cats, cows

    •Monkeypox – monkeys, squirrels
  23. 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
  24. Vaccinia Virus: 
    •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
  25. 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
  26. DNA
    Replication (vaccinia virus)
    •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)

    •Cleavedinto two genome lengths

    • •Fill in and ligate ends into dsDNA,
    • closed ends
  27. 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
  28. 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
  29.  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
  30. 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)


    •Best defense?