Knockdown of the Bovine Leukemia Virus Tax Gene by RNAi and Its Effects on Host Gene Expression

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Knock-down of the Bovine Leukemia Virus Tax Gene
by RNAi and Its Effects on Host Gene Expression

• Rosane Oliveira, Allison Sommers, Robin Everts,
  Harris Lewin
• ESALQ-UIUC Genomics Workshop
• December 17th 2007 Piracicaba, Brazil

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(Doolittle et al, 1990)
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Genetic Map
(Retroviruses, 1997)
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Bovine Leukemia Virus (BLV) as a Model for
Systems Biology of Retroviral Transformation of
Mammalian Cells

• Features of the BLV Model System
• BLV is a chronic transforming deltaretrovirus in
  same family as the HTLVs
• BLV infects CD5 B cells and causes persistent
  lymphocytosis (pre-leukemic state) in 30 of
  infected animals (dependent on host class II DRB3
  genotype)
• 1 develop lymphosarcoma (100 mortality)
• Costs to US dairy industry at least 100
  million/year
• Two-hit model of cell transformation
• First Hit genome instability
• Provirus integration (random)
• Trans-activation/repression of host cell genes by
  BLV tax
• Second Hit
• Deletion and/or mutation of tumor suppressor
  genes
• Chromosome rearrangements
• Goal
• To understand the role of BLV Tax in driving
  B-cell proliferation and leukemogenesis

Tumors
Persistent Lymphocytosis
Tumor
Seropositive (asymptomatic)
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What Are the Host Cell Target Genes and
Biological Pathways that Are Affected by BLV Tax?

• RNA interference (RNAi)
• siRNA targeting Tax gene
• Amaxa Nucleofector II
• 2 cell lines (BL3º and BL3)
• 6 biological replicates
• 5-6 technical replicates per experiment
• Tax- and Scrambled-siRNA 2 controls
• Microarray
• 13,245-element cattle-specific oligo array
• (Loor et al., 2007)
• Quantitative PCR
• BLV Tax mRNA
• Host cellular genes (validation of microarray
  results)
• Data Mining
• Ingenuity Pathway Analysis Metacore by GeneGO
  Inc.

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What Are the Host Cell Target Genes and
Biological Pathways that Are Affected by BLV Tax?

• RNA interference (RNAi)
• siRNA targeting Tax gene
• 2 cell lines (BL3º and BL3)
• 6 Biological Replicates
• 5-6 technical replicates
• Tax- and Scrambled-siRNA
• 2 controls
• 4 Comparisons
• BL3 scrambled-siRNA/BL3º scrambled-siRNA
• BL3 tax-siRNA/BL3º tax-siRNA
• BL3º tax-siRNA/BL3º scrambled-siRNA
• BL3 tax-siRNA/BL3 scrambled-siRNA

Amaxa Nucleofector II
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What Are the Host Cell Target Genes and
Biological Pathways that Are Affected by BLV Tax?

• Transcription Profiling of Tax Knock-down
• 13,245-element cattle-specific oligonucleotide
  array (Loor et al., 2007)
• Direct comparison, 4 different comparisons
• Loess normalization and data analysis with
  Genespring
• Tested for differences in ratios using T-test
  Benjamini and Hochberg false discovery rate (FDR)
  for multiple testing correction (P-value lt 0.05)
• Confirmation of DEG by Quantitative PCR
• BLV Tax mRNA
• Host cellular genes
• CDK6, CLU, DNAJB6, DUSP1 (MPK1), NDUFA2, STAT1,
  BHLHB2 (DEC1), IL4R, MARCKS, PRKCD
• Pathway Analysis and Modeling (Data Mining)
• Ingenuity Pathway Analysis
• GO and canonical pathways distributions
• Metacore by GeneGO Inc.
• Networks and pathways

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Rationale of RNAi Knock-down of BLV Tax
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Experimental Approach
Transcription Profile
BL3
Comparison I
Tax-siRNA
Scrambled-siRNA/ºScrambled-siRNA
RNA Isolation qPCR for Tax
Comparison II
Scrambled-siRNA
Tax-siRNA/Scrambled-siRNA
BL3º
Comparison III
Tax-siRNA
ºTax-siRNA/ºScrambled-siRNA
RNA Isolation qPCR for Tax
Scrambled-siRNA
Comparison IV
Tax-siRNA/ºTax-siRNA
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Tax knock-down Assessed by qPCR
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Results
Comparison I (Scrambled-Off Target) 3
Scrambled/3º Scrambled
Cell line-specific
4,057
1,694
908
Comparison IV (3 Tax-Off Target) 3 Tax/3º Tax
Benjamini Hochberg false discovery rate P lt0.20
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Genome-wide Effects of RNAi Knock-down of BLV Tax
mRNA

• Microarray analysis
• 184 differentially expressed genes in BL3 cells
  treated with tax-siRNA

Benjamini Hochberg false discovery rate, P lt
0.2
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Quantitative PCR Validation of 10 Host Genes
Affected by BLV Tax mRNA Knock-down








t-test, FDR Plt0.05 t-test, FDR Plt0.01
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Canonical Pathways Affected by Knock-down of BLV
Tax mRNA in BL3 cells
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Biological Functions Affected by Knock-down of
Tax mRNA in BL3 cells
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Cell Death Gene Network
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Model for Role of BLV in B-Cell Transformation
Apoptosis
Cell Cycle
BLV Tax
mRNA processing
Transcription
Viral replication
Protein biosynthesis, ribosome
Cellular Growth and Proliferation
Cell-Cell Signaling
New virions
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Conclusions Future Directions

• Critical pathways of cell growth and
  proliferation (e.g. cell signaling, cell cycle
  and cell death/apoptosis) are affected directly
  by BLV-tax (likely through STAT1).
• The gene networks affected by tax knock-down
  provide strong evidence that BLV-tax creates
  mitotic instability in BLV-infected cells by
  disrupting normal processes of cell division.
• We hypothesize that these tax-mediated effects on
  host gene transcription lead to the oligoclonal
  expansion of B cell precursor(s) and the onset of
  persistent lymphocytosis (PL).
• Future studies will focus on direct
  identification of BLV-tax binding sites in the
  cattle genome (ChIP-PET). These studies will
  permit molecular dissection of the upstream
  transcription pathways that lead to mitotic
  instability and cell transformation in BLV
  infected cattle.
• Understanding the molecular pathways leading to
  PL and lymphosarcoma may provide novel vaccine
  and therapeutic strategies for disease caused by
  BLV and HTLVs.

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Acknowledgments
Prof. Harris A. Lewin
Dr. Robin E. Everts
Allison M. Sommers