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

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dna viruses1
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.
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
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)
  • Terminally redundant ends enable circularization
what affect does t4 infection have on macromolecular synthesis in the cell
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?
slide8

DNA

protein

Rel

conc

RNA

0

time

rna production in cell
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
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 (ADP ribosylation) RNA-P to recognize DE promotors
    • Antitermination
    • Nucleases (host DNA and tRNA)
    • Membrane repair
slide11
DE further changes to RNA-P
    • Antisigma factor (ASiA)
    • Activator proteins
    • Phage tRNAs
    • Nucleotide metabolism
    • DNA replication
  • Late requires different sigma factors
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

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
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
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?
host cell defense
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.
hsv 1 icp
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.
protection from host are early products
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-