Transcript Gene Delivery Course - University of Pittsburgh

Tumor Virology Course: MSMVM 3435, Dec 2, 2009.
Who Am I?
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: [email protected]
Web: http://labs.mmg.pitt.edu/sarkar/
What Am I going to talk about? And Why?
• 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
Evolution of the “central dogma” for Cancer.
Weinbarg, 2000
Examples:
• Helicobacter pylori (Marshall & Warren, 2005)
• Human papillomavirus (zur Hausen, 2008)
• Hepatitis B and C virus
Karin, Mantovani, 2006..
Mechanisms of Inflammation mediated Cancer.
Montavani (2008) Nature. 454, 436
Karin (2006) Cell. 124, 823
Kinetics of immune response after virus infection
Biron & Sen (2007) Fields Virology
Interferons
Type I (a1, a2.., b, d, e, t, k..)
Size
Type II (g)
~ 18 kDa
21 kDa
Cell Source
Most Nucleated cells
Activated T Cells, NK Cells
Target Cells
All
Monocytic Cells and others
Mainly Antiviral
Antiviral and Antitumor
IFNAR
IFNGR
Effect
Receptor
Transcription Factors: NF-kB, STAT and IRF
NF-kB
IRF
STAT
Family
P50, p52, p65 (RelA),
RelB, c-Rel
IRF1 to IRF9
STAT1 to STAT6; 5A & 5B
Activation
IkB Degradation, pS, pT
pS, pT, Ub
pY, pS, pT
Activators
Everything!!
TLR, RLR
IFN, Hormone
Repressor
IkB
Not Clear
SOCS
Kinases
IKK family to IkB
TBK1, IKKe
JAK
Genes
Cytokines, Inflammation,
Growth Promoting
IFN, Innate Immunity,
Growth Inhibitory
ISGs, Growth
Promoting/Inhibitory.
IFN Signaling
Protein Kinase RNA activated (PKR)
Enzyme: Ser/Thr kinase, Activator dsRNA, PACT (cofactor)
Substrate: eIF2a, PKR, other substrates in vitro.
Function: Translation inhibition, other
PKR Structure
PKR Activation
2’-5’ Oligoadenylate Synthetases (OAS)
INTERFERON
2’-5’ OAS
ATP
dsRNA
O
O
O
P
O 3
N
O
5
3
N
N
2
N
N
O
O
O
P
O
O
N
O
5
3
N
N
2
N
O
O
Inactive RNase L
Active RNase L
RNA Degradation
N
Structure of OAS1
Hartmann, Sarkar et al. (2003) Mol Cell. 19, 1173
Other ISGs
ISG56 Family:
Human ISG 56, 54, 60, aka IFIT
Interaction with eIF3, translation inhibition
ADAR:
RNA-Specific Adenosine Deaminase
A
||
U
Mx/GBP: small GTP binding proteins
ISG15: Similar to Ubiquitin
I
||
U
I
||
C
Virus - dsRNA – Interferon – anti-viral state
Interferon b Enhansosome
PRD IV
Escalante et al, Mol. Cell 26, 703-16
PRD III-I
PRD II
Toll-like Receptors
TLR Structures
Jin et al, Immunity 29, 182-91
NLRs, RLRs Inflammosomes
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.
Meylan et al, Nature 442, 39-44
Cellular Sensors of DNA and DNA viruses
• DNA transfection induces IFNb independent of TLRs (Stetson, 2005)
• Cytoplasmic DNA sensor – DAI/ZBP1 (Takaoka, 2007)
o
o
Transient Expression allows DNA dependent IRF
activation, siRNA.
DAI interaction with DNA
• NALP3/ASC Inflammosome (Muruve, 2008)
o
o
Adenoviral particles, capsid, DNA induced IL-1 secretion in a NALP3, ASC
dependent manner.
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
PRR Signaling Pathways
TLR Signaling Pathways: Gene Induction
RLR Signaling Pathways: Gene Induction
Takeuchi and Akira Curr. Opin. Immun. 20, 17-22
NLR Signaling Pathways: IL-1 Production & Gene Induction
Meylan et al, Nature 442, 39-44
Viral Evasion
Strategies to evade host response
Finlay et al, Cell 124, 767
Examples of Viral Evasion
TLR/RLR
IFN
ISG
Pox V
IFNARs
IFNGRs
TNFR
Flu NS1
Paramyxo
V, W
HCV NS3/4A
Adeno VAI RNA
Mu V
Nipah W
Adeno E1A
EBNA1
HHV8 K9
HPV E6
Adeno VAI RNA
EBV EBER RNA
HIV TAR RNA
Vaccinia E3L
Multifunctional viral proteins with multiple targets!!
Paramyxo V and W, Flu NS1, Adeno E1A, SV40 T……
Putting it together
Natural
Selection
References
• Innate Immunity, TLR, NLR
o
o
o
Immunity's Early-Warning System, Luke A. J. O'Neill, Scientific American, Jan 2005. (@ sarkarlab/research)
Recognition of microorganisms and activation of the immune response, Ruslan Medzhitov, Nature, Oct 18, 2007
(PMID: 17943118)
Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell. 124, 783-801.
(PMID: 16497588)
• Infalammation Cancer
o
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)
Cancer-related inflammation. Mantovani A, Allavena P, Sica A, Balkwill F. Nature. 454: 436 (PMID: 18650914)
• Viral Evasion
o
o
Pathogen subversion of cell-intrinsic innate immunity. Roy CR, Mocarski ES. Nat Immunol. 8:1179 (PMID:
17952043)
Viral evasion and subversion of pattern-recognition receptor signalling. Bowie AG, Unterholzner L. Nat Rev Immunol.
8: 911(PMID: 18989317)
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.