Differential Transcription of Fathead
Minnow Immune-Related Genes
Following Infection with Frog virus 3
(FV3)
Kwang Cheng,...
Immune Competence Determines
Survival in Xenopus
• FV3 infection of Xenopus laevis tadpoles and
immunocompromised adults t...
Cellular vs Viral Responses Determine
Survival
• Cellular genes contribute to anti-viral
immunity and viral clearance.
– I...
Goal
• Identify cellular immune-related
molecules that are differentially
expressed in FHM cells after FV3
infection.
Approach
• Monitor the expression of cellular
immune-related genes using a 60K
feature fathead minnow (FHM)
microarray.
Q. When are immune-related genes
expressed?
M 4h 8h 16h 24h 48h
MCPMCP
Mx
actin
Methodology
• Sham infect or infect replicate (6) cultures of
FHM cells with wt FV3 or the 18K KO mutant,
that was shown t...
Confirmation of a Productive Viral
Infection
M FV3 ∆18K
MCP
18K
M WT Δ18K
MCP
18K
18K-1
18K-2
18K-3
18K-5
WT-2
WT-4
WT-5
WT-1
WT-3
M-1
M-3
M-2.1
M-2.2
M-5
Heat map
analysis of
replicate
cultures of
mock-i...
Gene Name (abbreviation WT ∆18K
TNFRSF14 151 51
Interleukin 8 (IL8) 145 70
EIF2S2 96 24
NFIL3 66 13
ICLP2 62 11
NFKBIA 52 ...
Additional Upregulated Genes
Gene Name WT ∆18K
Interleukin 1β (IL1B) 9 7
Mx 9 ND
IRF3 5 2
MHC class I 5 4
RIGI 3 3
IRF1 3 ...
Genes down regulated after FV3 infection
Gene Name WT ∆18K
HSPB8 -3 -5
G6PD -3 -3
POLB -2 ND
POLA2 -3 -2
C9 -3 ND
ATG7 -4 ...
Pathways Affected by FV3 Infection
• IFN induction and regulation
– IFN, IRF-1, IRF-2, IRF-3, IRF-7, RIGI
• Pro-Inflammato...
qRT-PCR Validation of Microarray
Results
0
50
100
150
200
250
Mock WT 18K
RelativeGeneExpression
IL-8
0
10
20
30
40
50
60
...
Q. Do qualitative/quantitative
differences in gene expression
explain why 18K KO mutants were
attenuated in vivo?
Pathway – Apoptosis – WT vs. Mock
Red = UP
Green = Down
Pathway – Apoptosis – 18K vs. Mock
Red = UP
Green = Down
Conclusions
• Observation: Numerous immune-related
genes, affecting both innate and acquired
immunity, were upregulated in...
Conclusions
• Transcripts induced following infection with
wt FV3 and the 18K KO are qualitatively
similar.
• However, qua...
Future Plans
• Use FHM and Xenopus microarrays to analyze
global host expression following infection with
wt and KO mutant...
Acknowledgements
• Kwang Cheng, UMMC (Jackson, MS)
• Lynn Escalon, ERDC (Vicksburg, MS)
• Natalia Garcia-Reyero, Mississip...
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Immune response in FHM cells following infection with frog virus 3

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2013 International Symposium on Ranaviruses
by Greg Chinchar

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  • - What constitutes innate virus response
  • MOI ~ 10 PFU/cell, radiolabled from ___ - ___ hr p.i.
  • NFKBIA = This gene encodes a member of the NF-kappa-B inhibitor family, which contain multiple ankrin repeat domains. The encoded protein interacts with REL dimers to inhibit NF-kappa-B/REL complexes which are involved in inflammatory responses. The encoded protein moves between the cytoplasm and the nucleus via a nuclear localization signal and CRM1-mediated nuclear export. Mutations in this gene have been found in ectodermal dysplasia anhidrotic with T-cell immunodeficiency autosomal dominant disease.NFIL3 = Expression of interleukin-3 (IL3; MIM 147740) is restricted to activated T cells, natural killer (NK) cells, and mast cell lines. Transcription initiation depends on the activating capacity of specific protein factors, such as NFIL3, that bind to regulatory regions of the gene, usually upstream of the transcription start site.TNFRSF14 = The protein encoded by this gene is a member of the TNF-receptor superfamily. This receptor was identified as a cellular mediator of herpes simplex virus (HSV) entry. Binding of HSV viral envelope glycoprotein D (gD) to this receptor protein has been shown to be part of the viral entry mechanism. The cytoplasmic region of this receptor was found to bind to several TRAF family members, which may mediate the signal transduction pathways that activate the immune response.ICLP2 = Putative homolog of mammalianiI (the invariant protein) and thought to play a role in MHC class II antigen presentation analogous to that in mammalian cells. IL3 = IL-3; MCGF; MULTI-CSF …is a potent growth promoting cytokine. This cytokine is capable of supporting the proliferation of a broad range of hematopoietic cell types. It is involved in a variety of cell activities such as cell growth, differentiation and apoptosis. This cytokine has been shown to also possess neurotrophic activity, and it may be associated with neurologic disorders.IL11 = This cytokine is shown to stimulate the T-cell-dependent development of immunoglobulin-producing B cells. It is also found to support the proliferation of hematopoietic stem cells and megakaryocyte progenitor cells.Cyclin T2a = Cyclin T2a was recently identified as one of the regulatory subunits of the cdk-cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation.
  • Calpain - calcium-dependent, non-lysosomal cysteine proteases Initiator caspases (e.g., CASP2,CASP8, CASP9, and CASP10) cleave inactive pro-forms of effector caspases, thereby activating them. Effector caspases (e.g., CASP3, CASP6, CASP7) in turn cleave other protein substrates within the cell, to trigger the apoptotic process. granzyme B (released by cytotoxic T lymphocytes and NK cells), which is known to activate caspase-3 and -7death receptors (like Fas, TRAIL receptors and TNF receptor), which can activate caspase-8 and -10the apoptosome (regulated by cytochrome c and the Bcl-2 family), which activates caspase-9.nuclear laminsICAD/DFF45 (inhibitor of caspase activated DNase or DNA fragmentation factor 45)PARP (poly-ADP ribose polymerase)PAK2 (P 21-activated kinase 2).Ras – signal transducersAPAF1 - Upon binding cytochrome c and dATP, this protein forms an oligomeric apoptosome. The apoptosome binds and cleaves caspase 9 preproprotein, releasing its mature, activated form.
  • Immune response in FHM cells following infection with frog virus 3

    1. 1. Differential Transcription of Fathead Minnow Immune-Related Genes Following Infection with Frog virus 3 (FV3) Kwang Cheng, B. Lynn Escalon, Natalia Garcia-Reyero, and V. Gregory Chinchar
    2. 2. Immune Competence Determines Survival in Xenopus • FV3 infection of Xenopus laevis tadpoles and immunocompromised adults triggers severe systemic disease and high mortality. • In contrast, FV3 infection of immunocompetent adults is confined to the kidney and associated with high levels of survival.
    3. 3. Cellular vs Viral Responses Determine Survival • Cellular genes contribute to anti-viral immunity and viral clearance. – Immunocompetent adults display protective innate (IFN, pro-inflammatory) and acquired (Antibody, CTLs) responses that resolve infection. • In contrast, viral immune evasion genes antagonize cellular immune responses. – For example ranavirus vIF2α is thought to block PKR-mediated translational shut-off.
    4. 4. Goal • Identify cellular immune-related molecules that are differentially expressed in FHM cells after FV3 infection.
    5. 5. Approach • Monitor the expression of cellular immune-related genes using a 60K feature fathead minnow (FHM) microarray.
    6. 6. Q. When are immune-related genes expressed? M 4h 8h 16h 24h 48h MCPMCP Mx actin
    7. 7. Methodology • Sham infect or infect replicate (6) cultures of FHM cells with wt FV3 or the 18K KO mutant, that was shown to be attenuated in vivo. • Prepare total RNA at 8 hr after infection. • Monitor cellular gene expression by microarray analysis • Validate microarray results by qRT-PCR
    8. 8. Confirmation of a Productive Viral Infection M FV3 ∆18K MCP 18K M WT Δ18K MCP 18K
    9. 9. 18K-1 18K-2 18K-3 18K-5 WT-2 WT-4 WT-5 WT-1 WT-3 M-1 M-3 M-2.1 M-2.2 M-5 Heat map analysis of replicate cultures of mock-infected, wt FV3- infected, and ∆18K knock mutant-infected FHM cells
    10. 10. Gene Name (abbreviation WT ∆18K TNFRSF14 151 51 Interleukin 8 (IL8) 145 70 EIF2S2 96 24 NFIL3 66 13 ICLP2 62 11 NFKBIA 52 23 GILT 30 9 TRAF3 20 12 CAV1 14 8 POLR2A 14 2 Interferon (IFNα) 11 4 Differential Gene Expression: WT FV3 and ∆18K versus Mock- infected
    11. 11. Additional Upregulated Genes Gene Name WT ∆18K Interleukin 1β (IL1B) 9 7 Mx 9 ND IRF3 5 2 MHC class I 5 4 RIGI 3 3 IRF1 3 2 CCNT2 3 -2 ADAR 1* 4
    12. 12. Genes down regulated after FV3 infection Gene Name WT ∆18K HSPB8 -3 -5 G6PD -3 -3 POLB -2 ND POLA2 -3 -2 C9 -3 ND ATG7 -4 -3
    13. 13. Pathways Affected by FV3 Infection • IFN induction and regulation – IFN, IRF-1, IRF-2, IRF-3, IRF-7, RIGI • Pro-Inflammatory immune response – IL8, IL3, IL1β • Immune activation/ anti-viral activity – NFKBIA, TNFRSF14, MxD, ADAR • Antigen Presentation – MHC class I, ICLP2 • Metabolism – EIF2S2 (eIF-2β), cyclin T2a, HSP70 family members, POLR2A, CAV1
    14. 14. qRT-PCR Validation of Microarray Results 0 50 100 150 200 250 Mock WT 18K RelativeGeneExpression IL-8 0 10 20 30 40 50 60 70 80 Mock WT 18K RelativeGeneExpression INF -2 -1.5 -1 -0.5 0 0.5 1 1.5 2 2.5 3 Mock WT 18K RelativeGeneExpression Cyt2a 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 Mock WT 18K RelativeGeneExpression IRF3 -5 -4.5 -4 -3.5 -3 -2.5 -2 -1.5 -1 -0.5 0 Mock WT 18K RelativeGeneExpression HSPB8
    15. 15. Q. Do qualitative/quantitative differences in gene expression explain why 18K KO mutants were attenuated in vivo?
    16. 16. Pathway – Apoptosis – WT vs. Mock Red = UP Green = Down
    17. 17. Pathway – Apoptosis – 18K vs. Mock Red = UP Green = Down
    18. 18. Conclusions • Observation: Numerous immune-related genes, affecting both innate and acquired immunity, were upregulated in FV3-infected cells. • Hypothesis: Immune-related molecules impair virus replication and lead to long term survival.
    19. 19. Conclusions • Transcripts induced following infection with wt FV3 and the 18K KO are qualitatively similar. • However, quantitative differences may reflect the role that the 18K protein plays in viral replication and the modulation of cellular gene expression.
    20. 20. Future Plans • Use FHM and Xenopus microarrays to analyze global host expression following infection with wt and KO mutants in vivo and in vitro. • Determine whether specific cellular gene products are critical for a protective immune response.
    21. 21. Acknowledgements • Kwang Cheng, UMMC (Jackson, MS) • Lynn Escalon, ERDC (Vicksburg, MS) • Natalia Garcia-Reyero, Mississippi State University (Starkville, MS) • Jacques Robert/Guangchun Chen (U. Rochester School of Medicine) • US ARMY Corps of Engineers • National Science Foundation

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