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DNA VIRUSES. 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.

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DNA VIRUSES

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DNA VIRUSES


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


Replication Challenges for DNAViruses

  • Access to nucleus

  • Competing for nucleotides

  • Cell cycle control in eucaryotes - S phase dependent materials for some

  • Primer removal and replacement (completing ends)


Transcriptional/translational challenges

  • Access to RNA polymerase

  • Monogenic expression in eukaryotes

  • Temporal control of gene expression

  • Competition with host for ribosomes


Bacteriophages: T4

  • Linear dsDNA - ~ 1.2 x 10^8 d (>280 genes)

  • circular permuted

  • terminally redundant


http://www.brunel.ac.uk/depts/bl/blst/emma/molecgen/virus/lytic/lytfrm.htm


http://www.brunel.ac.uk/depts/bl/blst/emma/molecgen/virus/lytic/lytfrm.htm


Concatemer formation and packaging of headful genome


What affect does T4 infection have on macromolecular synthesis in the cell?

  • What MOI would you use?

  • 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?


DNA

protein

Rel

conc

RNA

0

time


RNA production in cell

  • Temporal control of transcription

    • Immediate early: will occur in presence of ps inhibitor What RNA-P is used?

    • Delayed early - needs protein synthesis and before DNA replication

    • Late - after DNA replication begins - structural proteins


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 (ADPr) RNA-P to recognize DE promotors

    • Antitermination

    • Nucleases (host DNA and tRNA)

    • Membrane repair


  • DE further changes to RNA-P

    • Antisigma factor (ASiA)

    • Activator proteins

    • Phage tRNAs

    • Nucleotide metabolism

    • DNA replication

  • Late requires different sigma factors


T4 genome - also 127 ORFs of unknown fucntion


T7 control

  • Linear dsDNA

    • ~ 25 x 10^6d

  • Unique with TR - how is this formed?

  • Genes are in order of entry on chromosome


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


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


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


HPV Transcription using host RNA-P

  • Multiple promotors some with overlapping reading frames

  • Alternative splicing - more genes for your genome


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


Inverted terminal repeats


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


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


DNA replication


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


Families of Herpes viruses


Temporal expression of genes


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


Herpes virus supplies all DNA machinery

  • No need for cell to be in S phase

  • Model for replication

    • Rolling circle leads to concatemers


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?


Packaging of Herpesviruses


Protection from host are early products

  • Prevention of apoptosis

  • Use mutants and see affects

  • Cisplatin is apo inducer (+ control)

apoptosis

wt

cisplatin

ICP-


KSHV v-cyclin/v-FLIP gene gives a single transcript

  • Both cell homologs

    • Cyclin regulates cell cycle

    • FLIP delays apoptosis

  • How are two proteins produced from one message?


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