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Chris Clark Infected with SV40!?!? Nope he’s just passed out again!

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.

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

SV40

Polyomavirus ds DNA Small (40-50 nm) No lipoprotein

envelope

Oncogenic Binds p53 Binds Rb

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

VirMir Analysis

Supplemental Figure 1 Computer program

scanning genome for likely miRNA precursors

No RNA was found with Northern blotting in early polarity

Northern blot of Proposed miRNA

Figure 1b Northern blot with

radiolabelled antisense oligonucleotides

Detected small 62 nt RNA and multiple 22 nt RNA

RNase Protection Assay

RPA with radioactive probes transcribed in vitro

Probe hybridizes RNase is added and

destroys any ssRNA Target mRNA is

protectedFrom http://edoc.hu-berlin.de/dissertationen/moericke-anja-2002-01-25/HTML/chapter3.html

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

Proposed miRNA structure

Figure 2d Northern and RPA results used to determine

miRNA location within genome Hairpin structure

Temporal Expression

Figure 3a Northern blot miRNA expression

increases as infection progresses

Consistent with late-polarity transcription

Temporal Expression of Proposed Target mRNA

Figure 3b Northern blot 3’ probe detects

cleaved and uncleaved mRNA

5’ probe detects only uncleaved mRNA

RPA of mRNA After 70 h

Figure 3c RPA and schematic

demonstrating both miRNA clusters associate with the same mRNA

SV40 Mutant Strain (SM)

Supplemental Figure 2 Created with wobble pairing to conserve the

integrity of the Large T antigen sequence

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

Figures 4c and 4d Regulation of LTAg

has no effect on replicative activity

Cells with less LTAg have less CTL mediated lysis

Wild-type vs. SM Mutant

miRNA Effects on Cell Lysis

Supplemental Figure 3

Enhanced CTL mediated lysis data

Cytokine data

VirMir Analysis of Other Primate Polyomaviruses

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

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.