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.

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DNA VIRUSES

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Dna viruses

DNA VIRUSES


Dna genome replication strategies similar in all and similar to host

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

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

Transcriptional/translational challenges

  • Access to RNA polymerase

  • Monogenic expression in eukaryotes

  • Temporal control of gene expression

  • Competition with host for ribosomes


Bacteriophages t4

Bacteriophages: T4

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

  • circular permuted

  • terminally redundant


Dna viruses

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


Dna viruses

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


Concatemer formation and packaging of headful genome

Concatemer formation and packaging of headful genome


What affect does t4 infection have on macromolecular synthesis in the cell

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 viruses

DNA

protein

Rel

conc

RNA

0

time


Rna production in cell

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

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


Dna viruses

  • 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

T4 genome - also 127 ORFs of unknown fucntion


T7 control

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

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

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

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

HPV Transcription using host RNA-P

  • Multiple promotors some with overlapping reading frames

  • Alternative splicing - more genes for your genome


Adenovirus 5 protein primer

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

Inverted terminal repeats


Adenovirus transcription

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

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

DNA replication


Herpes simplex virus

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

Families of Herpes viruses


Temporal expression of genes

Temporal expression of genes


Alpha and beta proteins

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

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

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

Packaging of Herpesviruses


Protection from host are early products

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

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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