DNA VIRUSESDNA VIRUSES

DNA (genome) replication strategies DNA (genome) replication strategies similar in all and similar to host 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 Replication Challenges for DNAVirusesDNAViruses

• 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 Transcriptional/translational challengeschallenges

• Access to RNA polymerase

• Monogenic expression in eukaryotes

• Temporal control of gene expression

• Competition with host for ribosomes

Bacteriophages: T4Bacteriophages: 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 Concatemer formation and packaging of headful genomeof headful genome

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

RNA

protein

DNA

Rel

conc

time0

RNA production in cellRNA 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-PT4 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 T4 genome - also 127 ORFs of unknown fucntionfucntion

Gene function % of known genome functions

Metabolic, essential (22)

15

Metabolic, unessential (60)

39

Structural (34) 27

Assembly, nonstructural (19)

10

T7 controlT7 control

• Linear dsDNA– ~ 25 x 10^6d

• Unique with TR - how is this formed?

• Genes are in order of entry on chromosome

T7 promotors differT7 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

PapovavirusesPapovaviruses• 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 How do DNA Viruses Get cells out of G1 and into S phaseand 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-PHPV Transcription using host RNA-P

• Multiple promotors some with overlapping reading frames

• Alternative splicing - more genes for your genome

Adenovirus - 5’protein primerAdenovirus - 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 repeatsInverted terminal repeats

Adenovirus - transcriptionAdenovirus - 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 morphologyPoxvirus: 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 replicationDNA replication

Herpes Simplex VirusHerpes 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 virusesFamilies of Herpes viruses

Temporal expression of genesTemporal expression of genes

Alpha and Beta proteinsAlpha 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

Herpes virus supplies all DNA Herpes virus supplies all DNA machinery machinery

• No need for cell to be in S phase

• Model for replication

– Rolling circle leads to concatemers

Thymidine kinase and Ribonucleotide Thymidine kinase and Ribonucleotide reductase are early proteinsreductase 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 HerpesvirusesPackaging of Herpesviruses

Protection from host are early productsProtection 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 KSHV v-cyclin/v-FLIP gene gives a single transcripttranscript

• Both cell homologs– Cyclin regulates

cell cycle– FLIP delays

apoptosis• How are two proteins

produced from one message?