title: Dicodon monitoring of protein synthesis (DiCoMPS) reveals levels of synthesis of a viral protein in single cells.
abstract: The current report represents a further advancement of our previously reported technology termed Fluorescent transfer RNA (tRNA) for Translation Monitoring (FtTM), for monitoring of active global protein synthesis sites in single live cells. FtTM measures Förster resonance energy transfer (FRET) signals, generated when fluorescent tRNAs (fl-tRNAs), separately labeled as a FRET pair, occupy adjacent sites on the ribosome. The current technology, termed DiCodon Monitoring of Protein Synthesis (DiCoMPS), was developed for monitoring active synthesis of a specific protein. In DiCoMPS, specific fl-tRNA pair combinations are selected for transfection, based on the degree of enrichment of a dicodon sequence to which they bind in the mRNA of interest, relative to the background transcriptome of the cell in which the assay is performed. In this study, we used cells infected with the Epizootic Hemorrhagic Disease Virus 2-Ibaraki and measured, through DiCoMPS, the synthesis of the viral non-structural protein 3 (NS3), which is enriched in the AUA:AUA dicodon. fl-tRNA(Ile)UAU-generated FRET signals were specifically enhanced in infected cells, increased in the course of infection and were diminished on siRNA-mediated knockdown of NS3. Our results establish an experimental approach for the single-cell measurement of the levels of synthesis of a specific viral protein.
authors: Department of Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel, Anima Cell Metrology, Inc., Bernardsville, NJ 07924-2270, USA and Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104-6323, USA.
authors: Sima
authors: S
authors: Barhoom
authors: Ian
authors: I
authors: Farrell
authors: Ben
authors: B
authors: Shai
authors: Dvir
authors: D
authors: Dahary
authors: Barry S
authors: BS
authors: Cooperman
authors: Zeev
authors: Z
authors: Smilansky
authors: Orna
authors: O
authors: Elroy-Stein
authors: Marcelo
authors: M
authors: Ehrlich
mesh_terms: D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D003062:Codon; D006224:Cricetinae; D003412:Cricetulus; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D018164:Hemorrhagic Disease Virus, Epizootic; D014176:Protein Biosynthesis; D034622:RNA Interference; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism; D059010:Single-Cell Analysis; D017361:Viral Nonstructural Proteins / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins / Q000096:biosynthesis*
chemical_list: D003062:Codon; C089247:S10 protein, epizootic hemorrhagic disease virus 2; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; D012343:RNA, Transfer
references: Eur J Biochem. 2002 Jun;269(11):2810-9
references: 12047392
references: J Cell Biol. 2006 Oct 9;175(1):67-76
references: 17030983
references: Nucleic Acids Res. 2011 Oct;39(19):e129
references: 21795382
references: J Cell Biol. 2012 Apr 2;197(1):45-57
references: 22472439
references: Proc Natl Acad Sci U S A. 2012 Jan 10;109(2):413-8
references: 22160674
references: Microbiol Mol Biol Rev. 2006 Dec;70(4):1032-60
references: 17158706
references: Science. 2010 Jul 30;329(5991):533-8
references: 20671182
references: Histochem Cell Biol. 2013 Mar;139(3):501-4
references: 23229864
references: Virol J. 2013 Mar 05;10:73
references: 23497128
references: Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2127-32
references: 17283340
references: Nat Rev Mol Cell Biol. 2005 Apr;6(4):318-27
references: 15803138
references: J Virol. 2009 Jul;83(13):6806-16
references: 19369335
references: Chem Biol. 2004 Jul;11(7):999-1008
references: 15271358
references: Anim Genet. 2006 Apr;37(2):171-4
references: 16573533
references: RNA. 2011 Jul;17(7):1393-400
references: 21628433
references: FEBS Lett. 2009 Dec 17;583(24):3966-73
references: 19850042
references: Biochem Biophys Res Commun. 2010 May 28;396(2):258-64
references: 20399740
references: RNA. 2009 Feb;15(2):346-54
references: 19118261
references: J Virol. 2006 Jan;80(1):460-73
references: 16352570
references: Nat Rev Genet. 2012 Mar 13;13(4):227-32
references: 22411467
references: Nucleic Acids Res. 2010 Apr;38(6):e89
references: 20040572
references: Viruses. 2012 Oct 29;4(11):2598-635
references: 23202496
references: Brain Res Mol Brain Res. 2005 Apr 4;134(2):333-7
references: 15836928
references: FASEB J. 2011 Mar;25(3):1028-39
references: 21148113
references: Nucleic Acids Res. 2013 Feb 1;41(3):1914-21
references: 23254333
references: J Biol Chem. 2004 Oct 8;279(41):43092-7
references: 15292261
references: Nat Methods. 2009 Apr;6(4):275-7
references: 19305406