1. Tumor Virology Course: MSMVM 3435, Dec 2, 2009. Who Am I? What Am I going to talk about? And Why? Saumendra N. Sarkar, Ph.D. (Saumen) University of Pittsburgh Cancer Institute, HCC 1.8 5117 Centre Avenue, Pittsburgh, PA 15213 Phone: (412) 623-7720, Fax: (412) 623-7715 E-mail: saumen@pitt.edusaumen@pitt.edu Web: http://labs.mmg.pitt.edu/sarkar/http://labs.mmg.pitt.edu/sarkar/ Innate Immunity, Inflammation and tumorigenesis. Host responses to virus infection Innate immune response to virus o Interferons (IFN) o Viral components and their Sensors o TLR, RLR, NLRs Innate Immune evasion strategies of viruses

2. Evolution of the “central dogma” for Cancer. Weinbarg, 2000Karin, Mantovani, 2006.. Examples: Helicobacter pylori (Marshall & Warren, 2005) Human papillomavirus (zur Hausen, 2008) Hepatitis B and C virus

3. Mechanisms of Inflammation mediated Cancer. Montavani (2008) Nature. 454, 436 Karin (2006) Cell. 124, 823

4. Biron & Sen (2007) Fields Virology Kinetics of immune response after virus infection

5. Interferons Size ~ 18 kDa21 kDa Cell Source Most Nucleated cellsActivated T Cells, NK Cells Target Cells AllMonocytic Cells and others Effect Mainly AntiviralAntiviral and Antitumor Receptor IFNARIFNGR Type I (     ..)Type II (  )

6. Transcription Factors: NF-  B, STAT and IRF Family P50, p52, p65 (RelA), RelB, c-Rel IRF1 to IRF9STAT1 to STAT6; 5A & 5B Activation I  B Degradation, pS, pT pS, pT, Ub pY, pS, pT Activators Everything!!TLR, RLRIFN, Hormone Repressor IBIB Not ClearSOCS Kinases IKK family to I  BTBK1, IKK  JAK Genes Cytokines, Inflammation, Growth Promoting IFN, Innate Immunity, Growth Inhibitory ISGs, Growth Promoting/Inhibitory. NF-  B IRFSTAT

7. IFN Signaling

8. Enzyme: Ser/Thr kinase, Activator dsRNA, PACT (cofactor) Substrate: eIF2 , PKR, other substrates in vitro. Function: Translation inhibition, other Protein Kinase RNA activated (PKR) PKR StructurePKR Activation

9. INTERFERON 2’-5’ OAS 2’-5’ Oligoadenylate Synthetases (OAS) Inactive RNase L Active RNase L RNA Degradation ATP dsRNA

10. Structure of OAS1 Hartmann, Sarkar et al. (2003) Mol Cell. 19, 1173

11. ISG56 Family: Human ISG 56, 54, 60, aka IFIT Interaction with eIF3, translation inhibition ADAR: RNA-Specific Adenosine Deaminase Mx/GBP: small GTP binding proteins ISG15: Similar to Ubiquitin Other ISGs A || U I || U I || C

12. Virus - dsRNA – Interferon – anti-viral state

13. Interferon  Enhansosome Escalante et al, Mol. Cell 26, 703-16 PRD IVPRD III-IPRD II

14. Toll-like Receptors

15. TLR Structures Jin et al, Immunity 29, 182-91

16. NLRs, RLRs Inflammosomes Meylan et al, Nature 442, 39-44 PYD: Pyrin NACHT: used to be called NOD NOD: Nucleotide Oligomerization NAD: NACHT Associated CARD: Caspase Recruitment LRR: Leucine Rich Repeat BIR: Baculovirus IAP Repeat.

17. Cellular Sensors of DNA and DNA viruses DNA transfection induces IFN  independent of TLRs (Stetson, 2005) Cytoplasmic DNA sensor – DAI/ZBP1 (Takaoka, 2007) o Transient Expression allows DNA dependent IRF activation, siRNA. o DAI interaction with DNA NALP3/ASC Inflammosome (Muruve, 2008) o Adenoviral particles, capsid, DNA induced IL-1 secretion in a NALP3, ASC dependent manner. o No IRF activation. AIM2, new protein which interacts with ASC (Hornung, 2009) o Binding, localization with ASC o Direct DNA binding o IRF activation? RNA polymerase III, transcribes dA.dT to RNA for RIG-I recognition (Hornung, 2009/Chen, 2009) o 5’pppN is important

18. PRR Signaling PathwaysPRR Signaling Pathways

19. TLR Signaling Pathways: Gene Induction

20. RLR Signaling Pathways: Gene Induction Takeuchi and Akira Curr. Opin. Immun. 20, 17-22

21. NLR Signaling Pathways: IL-1 Production & Gene Induction Meylan et al, Nature 442, 39-44

22. Viral Evasion

23. Strategies to evade host response Finlay et al, Cell 124, 767

24. TLR/RLR IFNISG Examples of Viral Evasion Nipah W HCV NS3/4A Mu V Flu NS1 Pox V IFNARs IFNGRs TNFR Paramyxo V, W Adeno E1A EBNA1 HHV8 K9 HPV E6 Adeno VAI RNA EBV EBER RNA HIV TAR RNA Vaccinia E3L Adeno VAI RNA Multifunctional viral proteins with multiple targets!! Paramyxo V and W, Flu NS1, Adeno E1A, SV40 T……

25. Putting it together Natural Selection

26. References Innate Immunity, TLR, NLR o Immunity's Early-Warning System, Luke A. J. O'Neill, Scientific American, Jan 2005. (@ sarkarlab/research) o Recognition of microorganisms and activation of the immune response, Ruslan Medzhitov, Nature, Oct 18, 2007 (PMID: 17943118) o Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell. 124, 783-801. (PMID: 16497588) Infalammation Cancer o Innate immunity gone awry: linking microbial infections to chronic inflammation and cancer. Michael Karin, Toby Lawrence, and Victor Nizet Cell 124 (4), 823-35 (PMID: 16497591) o Cancer-related inflammation. Mantovani A, Allavena P, Sica A, Balkwill F. Nature. 454: 436 (PMID: 18650914) Viral Evasion o Pathogen subversion of cell-intrinsic innate immunity. Roy CR, Mocarski ES. Nat Immunol. 8:1179 (PMID: 17952043) o Viral evasion and subversion of pattern-recognition receptor signalling. Bowie AG, Unterholzner L. Nat Rev Immunol. 8: 911(PMID: 18989317)

27. My Assessment I think it was pretty good. How to keep people falling off to sleep? Make people more interactive during the class Incorporate more stories and experiments describing how some of these were discovered.