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
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
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
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
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
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?
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?