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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 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 similar to host

  • 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 similar to host

  • Access to RNA polymerase

  • Monogenic expression in eukaryotes

  • Temporal control of gene expression

  • Competition with host for ribosomes


Bacteriophages t4
Bacteriophages: T4 similar to host

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


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 synthesis in the cell?

protein

Rel

conc

RNA

0

time


Rna production in cell
RNA production in cell synthesis in the 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 synthesis in the cell?

  • 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 synthesis in the cell?

    • Antisigma factor (ASiA)

    • Activator proteins

    • Phage tRNAs

    • Nucleotide metabolism

    • DNA replication

  • Late requires different sigma factors



T7 control
T7 control synthesis in the cell?

  • 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 synthesis in the cell?

  • 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 synthesis in the cell?

  • 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 synthesis in the cell?

  • 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 synthesis in the cell?

  • Multiple promotors some with overlapping reading frames

  • Alternative splicing - more genes for your genome


Adenovirus 5 protein primer
Adenovirus - 5’protein primer synthesis in the cell?

  • 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 synthesis in the cell?


Adenovirus transcription
Adenovirus - transcription synthesis in the cell?

  • 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 synthesis in the cell?

  • 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 synthesis in the cell?


Herpes simplex virus
Herpes Simplex Virus synthesis in the cell?

  • 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 synthesis in the cell?


Temporal expression of genes
Temporal expression of genes synthesis in the cell?


Alpha and beta proteins

Beta synthesis in the cell?

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 synthesis in the cell?

  • 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?



Protection from host are early products
Protection from host are early products proteins

  • 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 proteins

  • Both cell homologs

    • Cyclin regulates cell cycle

    • FLIP delays apoptosis

  • How are two proteins produced from one message?


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