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This is better then a cartoon!

Chris Clark Infected with SV40!?!? Nope he’s just passed out again!. This is better then a cartoon!. SV40-encoded microRNAs Regulate Viral Gene Expression and Reduce Susceptibility to Cytotoxic T Cells.

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This is better then a cartoon!

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  1. Chris Clark Infected with SV40!?!? Nope he’s just passed out again! This is better then a cartoon!

  2. SV40-encoded microRNAs Regulate Viral Gene Expression and Reduce Susceptibility to Cytotoxic T Cells Christopher S. Sullivan1, Adam T. Grundhoff1, Satvir Tevethia2, James M. Pipas3 & Don Ganem1 1Howard Hughes Medical Institute and Departments of Microbiology and Medicine, G. W. Hooper Foundation, University of California, San Francisco, California 94143-0552, USA. 2Department of Microbiology and Immunology, The Pennsylvania State College of Medicine, Hershey, Pennsylvania 17033, USA. 3Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.

  3. miRNAs • ~22 nt long • Important for regulating development and gene expression • Transcribed from DNA, but is not translated into a protein From http://www.genosensorcorp.com/images/miRNA-Genesis.jpg

  4. SV40 • Polyomavirus • ds DNA • Small (40-50 nm) • No lipoprotein envelope • Oncogenic • Binds p53 • Binds Rb

  5. SV40 • Figure 1a • The viral genome • Early promoter • Small t antigen • Large T antigen • Late promoter • 3 proteins in viral coat • miRNA found in late-polarity

  6. VirMir Analysis • Supplemental Figure 1 • Computer program scanning genome for likely miRNA precursors • No RNA was found with Northern blotting in early polarity

  7. Northern blot of Proposed miRNA • Figure 1b • Northern blot with radiolabelled antisense oligonucleotides • Detected small 62 nt RNA and multiple 22 nt RNA

  8. RNase Protection Assay • RPA with radioactive probes transcribed in vitro • Probe hybridizes • RNase is added and destroys any ssRNA • Target mRNA is protected From http://edoc.hu-berlin.de/dissertationen/moericke-anja-2002-01-25/HTML/chapter3.html

  9. RPA of Unprocessed and Processed miRNA • Figures 2a, 2b, and 2c • Different sizes in end probes likely due to RNase efficiency • Middle probe has no detectable ambiguity

  10. Proposed miRNA structure • Figure 2d • Northern and RPA results used to determine miRNA location within genome • Hairpin structure

  11. Temporal Expression • Figure 3a • Northern blot • miRNA expression increases as infection progresses • Consistent with late-polarity transcription

  12. Temporal Expression of Proposed Target mRNA • Figure 3b • Northern blot • 3’ probe detects cleaved and uncleaved mRNA • 5’ probe detects only uncleaved mRNA

  13. RPA of mRNA After 70 h • Figure 3c • RPA and schematic demonstrating both miRNA clusters associate with the same mRNA

  14. SV40 Mutant Strain (SM) • Supplemental Figure 2 • Created with wobble pairing to conserve the integrity of the Large T antigen sequence

  15. Wild-type vs. SM Mutant • Figure 4a and 4b • Northern blot • miRNA deficient strain has no detectable cleaved target mRNA • Expression of LTAg and stAg downregulated in functional miRNA strain

  16. Wild-type vs. SM Mutant • Figures 4c and 4d • Regulation of LTAg has no effect on replicative activity • Cells with less LTAg have less CTL mediated lysis

  17. miRNA Effects on Cell Lysis • Supplemental Figure 3 • Enhanced CTL mediated lysis data • Cytokine data

  18. VirMir Analysis of Other Primate Polyomaviruses

  19. Conclusions • An miRNA identified in the SV40 genome • miRNA mediates mRNA cleavage • miRNA is also involved in downregulation of Large T antigen • Cells with downregulation due to miRNA experience less CTL attack • “The existence of these autoregulatory miRNAs indicates that viruses can use the host RNAi machinery, which is often speculated to have evolved as an antiviral mechanism, to generate small RNAs that serve their own purposes” -C. S. Sullivan

  20. References • Ekenburg, S. and G. Hudson. 1994. RNase protection assay system: a versatile technique of the analysis of RNA. Promega Notes Mag. 46:14. • Peden, K. W. C., Pipas, J. M., Pearson-White, S., and D. Nathans. 1980. Isolation of mutants of an animal virus in bacteria. Science. 209:1392-1396. • Sullivan, C.S., Grundhoff, A. T., Tevethia, S., Pipas, J. M., and D. Ganem. 2005. SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature. 435:682-686.

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