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
Differential Transcription of Fathead
Minnow Immune-Related Genes
Following Infection with Frog virus 3
Kwang Cheng, B. Lynn Escalon,
Natalia Garcia-Reyero, and V. Gregory
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
Cellular vs Viral Responses Determine
• 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.
• Identify cellular immune-related
molecules that are differentially
expressed in FHM cells after FV3
• Monitor the expression of cellular
immune-related genes using a 60K
feature fathead minnow (FHM)
Q. When are immune-related genes
M 4h 8h 16h 24h 48h
• 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
• Validate microarray results by qRT-PCR
Confirmation of a Productive Viral
M FV3 ∆18K
M WT Δ18K
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
• Observation: Numerous immune-related
genes, affecting both innate and acquired
immunity, were upregulated in FV3-infected
• Hypothesis: Immune-related molecules impair
virus replication and lead to long term
• Transcripts induced following infection with
wt FV3 and the 18K KO are qualitatively
• However, quantitative differences may reflect
the role that the 18K protein plays in viral
replication and the modulation of cellular
• 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
• 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