1. DNA VIRUSES

2. 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

3. 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 )

4. Transcriptional/translational challenges Access to RNA polymerase Monogenic expression in eukaryotes Temporal control of gene expression Competition with host for ribosomes

5. Bacteriophages: T4 Linear dsDNA - ~ 1.2 x 10^8 d (>280 genes) circular permuted terminally redundant

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

8. Concatemer formation and packaging of headful genome

9. 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?

10. RNA protein DNA Rel conc time 0

11. 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

12. 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

13. DE further changes to RNA-P –Antisigma factor (ASiA) –Activator proteins –Phage tRNAs –Nucleotide metabolism –DNA replication Late requires different sigma factors

15. T4 genome - also 127 ORFs of unknown fucntion Gene function% of known genome functions Metabolic, essential (22) 15 Metabolic, unessential (60) 39 Structural (34)27 Assembly, nonstructural (19) 10

16. T7 control Linear dsDNA – ~ 25 x 10^6d Unique with TR - how is this formed? Genes are in order of entry on chromosome

17. 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

18. Papovaviruses Papilloma/Polyoma/Vacuo lating 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

19. 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

20. HPV Transcription using host RNA-P Multiple promotors some with overlapping reading frames Alternative splicing - more genes for your genome

21. 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

22. Inverted terminal repeats

23. 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

25. 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

26. DNA replication

27. 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

28. Families of Herpes viruses

29. Temporal expression of genes

30. 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

31. Herpes virus supplies all DNA machinery No need for cell to be in S phase Model for replication –Rolling circle leads to concatemers

32. 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?

33. Packaging of Herpesviruses

34. Protection from host are early products Prevention of apoptosis Use mutants and see affects Cisplatin is apo inducer (+ control) apoptosis wtcisplatinICP-

35. What do the results show? Infect cell with virus in presence of Actinomycin D (inhibits transcription from DNA) Add S-35 methionine and measure ps after 1 hour

36. 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?