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DNA VIRUSES

DNA VIRUSES. DNA Viruses. Except for poxviruses, transcription occurs in the nucleus and translation in the cytoplasm.

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DNA VIRUSES

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  1. DNA VIRUSES

  2. DNA Viruses • Except for poxviruses, transcription occurs in the nucleus and translation in the cytoplasm. • Generally, the primary transcripts, generated by RNA polymerase II, are larger than the mRNAs found on ribosomes, and in some cases, as much as 30% of the transcribed RNA remains untranslated in the nucleus. • The viral messengers, however, like those of animal cells, are monocistronic. • Transcription has a temporal organization, with most DNA viruses only a small fraction of the genome is transcribed into early messengers. • The synthesis of early proteins is the key initial step in viral DNA replication. • After DNA synthesis, the remainder of the genome is transcribed into late messengers. • The complex viruses have immediate early genes, which are expressed in the presence of inhibitors of protein synthesis, and delayed early genes, which require protein synthesis for expression. • Regulation is carried out by proteins present in the virions, or specified by viral or cellular genes, interacting with regulatory sequences at the 5' end of the genes.

  3. DNA (genome) replication strategies similar in all and similar to host • ssDNA becomes dsDNA • 5’ to 3’ synthesis; need for primer • Variety of enzymes of host or viral origin : DNA polymerase (proofreading), helicases, ss binding proteins, ligases • In nucleus except for poxviruses Phage T4 replisome

  4. Replication Challenges for DNAViruses • Access to nucleus • Competing for nucleotides • Cell cycle control in eucaryotes - S phase dependent materials

  5. Transcriptional/translational challenges • Access to RNA polymerase • Monogenic expression in eukaryotes • Temporal control of gene expression • Competition with host for ribosomes

  6. Bacteriophages: T4 • Linear dsDNA - ~ 1.2 x 10^8 d (>280 genes) • Terminally redundant ends enable circularization

  7. What affect does T4 infection have on macromolecular synthesis in the cell? • How would you measure DNA synthesis? RNA synthesis? Protein synthesis? • How can you distinguish between phage and host DNA synthesis? • How can you distinguish between phage and host RNA synthesis?

  8. DNA protein Rel conc RNA 0 time

  9. RNA production in cell • Temporal control of transcription • Immediate early (IE): will occur in presence of ps inhibitor What RNA-P is used? • Delayed early(DE) - needs protein synthesis and before DNA replication • Late - after DNA replication begins - structural proteins

  10. T4 changes host RNA-P • RNA-P - 4 subunits plus sigma factor • IE uses host enzyme but at promotors that differ from E. coli (high affinity) • IE gene products • modifies (ADP ribosylation) RNA-P to recognize DE promotors • Antitermination • Nucleases (host DNA and tRNA) • Membrane repair

  11. DE further changes to RNA-P • Antisigma factor (ASiA) • Activator proteins • Phage tRNAs • Nucleotide metabolism • DNA replication • Late requires different sigma factors

  12. T4 genome - also 127 ORFs of unknown fucntion

  13. T7 control • Linear dsDNA • ~ 25 x 10^6d • Unique with TR - how is this formed? • Genes are in order of entry on chromosome

  14. T7 promotors differ • IE - host polymerase • Creation of a new polymerase/inactivation of host polymerase • T7 polymerase promoter often used in gene cloning for control of expression

  15. Papovaviruses • Papilloma/Polyoma/Vacuolating agent • Bidirectional replication from single ori (similar to Bacteria) • Early to late strategies • T ags in SV40 enhance first and then suppresses early; • E ag in BPV is an enhancer for late genes • Mutations in T or Eag/transition lead to tumors

  16. How do DNA Viruses Get cells out of G1 and into S phase • Inactivate Rb/p53 - cell cycle regulators • SV40 uses T ag against p53 • p53 inactivation probably stops apoptosis • Multiple functions for T ag increases genome potential

  17. HPV Transcription using host RNA-P • Multiple promotors some with overlapping reading frames • Alternative splicing - more genes for your genome

  18. Adenovirus - 5’protein primer • Linear dsDNA • 20-30 x 10^6 d • Terminal protein linked to 5’nucleotide • Sequential replication from linear DNA • No Okazaki fragments This is now a template

  19. Inverted terminal repeats

  20. Adenovirus - transcription • Monogenic proteins with individual promotors • Uses host RNA-P • Multliple splicing of mRNA yields different proteins • E1A is IE gene- activates at other E promotors

  21. DNA replication

  22. Poxvirus: DNA with a complex morphology • Large genomes - 130 n- 240 x 10^6d • Denatured genome is ss circle • Replicates in cytoplasm • Brings in RNA-P; mRNA is capped • Makes all replicating enzymes

  23. Herpes Simplex Virus • Tegument - ~ 18 proteins • Access to nucleus • TIF (VP16 /UL48 ) trans inducing factor • binds with host factors to begin transcription • 500 - 1000 copies/virion • Determines tissue tropism • VHS (UL41) degrades preexisting mRNA but is stopped so virus can work

  24. Temporal expression of genes

  25. Beta DNA replication (polymerae,binding proteins, helicase/primase) Thymidine kinase DNA repair proteins Turn on Gamma/off Alpha Gamma Structural proteins Tegument proteins Alpha ICP27 - blocks host RNA splicing Immune escape (MHC1 downregulation) Turn on Beta genes Alpha and Beta proteins

  26. Herpes virus supplies all DNA machinery • No need for cell to be in S phase • Model for replication • Rolling circle leads to concatemers

  27. Thymidine kinase and Ribonucleotide reductase are early proteins • Needed for virulence but not in cell culture WHY? • TK needed to activate acyclovir • DNA polymerase - target of acyclovir • Many proteins have some cellular homolog - stolen genes? • Stress response gene - counter stress of viral infection?

  28. Packaging of Herpesviruses

  29. Host cell defense? • Prevention of translational initiation is a commonly utilized mechanism of cellular anti-viral defence. • Strategies have been adopted by viruses to overcome host cell attempts to terminate protein translation in the face of infection. • The mechanisms utilized by viruses such as HSV, are known in some detail and the host cell enzyme PKR is a central mediator in these mechanisms. • PKR is normally present in cells in a latent form. • It is induced byinterferon following virus infection, PKR is activated via autophosphorylation. • Activated PKR can phosphorylate the a subunit of the initiation factor eIF-2 (eIF2a), which generally results in prevention of translational initiation and thus halts protein synthesis.

  30. HSV -1 ICP • Herpes simplex virus type 1 (HSV-1) infected cell protein • (ICP) 34.5 gene null mutants exhibit severely attenuated • replication in animal models of HSV pathogenesis, but replicate as well as wild-type HSV in many malignant cells in vitro and in vivo • Capitalizing on this selective lytic replication, it has been used to successfully treat brain tumours, including melanoma, intraperitoneal human mesotheliomaand subcutaneous human melanoma, in various immunodeficientand immunocompetent mouse models.

  31. Protection from host are early products • Prevention of apoptosis • Use mutants and see affects (ICP = infected cell protein) • Cisplatin is apo inducer (+ control) apoptosis wt cisplatin ICP-

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