Reverse Genetics Systems for Nonsegmented Negative Strand RNA Viruses (NNSVs) Mi 13.07.2016
Reverse Genetik Reverse Genetik (reverse genetics) gezielten genetische Veränderung Funktion Klassische Genetik (forward genetics) Veränderung von Merkmalen genetische Basis
Reverse Genetics Systems / Reverse Genetics reverse genetics systems Cloning of Virus genomes in vectors (plasmids, phagmids, bacs) Genetic Modification (Engeneering) reverse genetics approach Recombinant Viruses with Defined Mutations forward genetics Phenotypic Charaterisation Selection of Mutatants Phenotypic Characterisation
From Virus Genome to Infectious Clone DNA viruses DNA/RNA viruses (+) viruses (-) RNA viruses DNA cdna cdna cdna transcription by cellular RNA-Pol II transcription by cellular RNA-Pol II virus own RNA-dependen RNA polymerase (RdRp) virus own RNA-dependen RNA polymerase (RdRp) translation translation translation no translation infectious infectious infectious not infectious
Relaxed (nicht kondensiertes) RNP Ribonukleoprotein (RNP): Nur das RNP, nicht aber die freie genomische RNA kann von der viralen RNP-abhängigen RNA Polymerase als Matrize für die virale RNA-Synthese Verwendet werden. vs kondensiert
Rhabdovirus Transkription und Replikation lea lea und 5 mrnas P L RNP-abhängige RNA Polymerase 3 gp N P M G L (-) RNP N P L N P L 5 agp (+) RNP lea = leader RNA gp = Genompromotor agp = Antigenompromotor
( RNP ) Polymerase-Template: Ribonukleoprotein RNA ist nur als RNP Template für die RNA-Polymerase RNP: RNAse resistente Verpackung der genomischen und ( N antigenomischen Virus RNA durch Nukleoprotein N (9 Basen pro stabile Genomstruktur intermolekulare Rekombinationsereignisse unwahrscheinlich Structure of nucleoprotein of VSV: Green et al., Science 2006
( RNP ) Polymerase-Template: Ribonukleoprotein Während der RNA-Synthese (Transkription + Replikation) muss die RNApartiell entpackt werden, damit ein komplementärer RNA Strang synthetisiert werden kann Konformationsänderungen im N-Protein Structure of RV nucleoprotein with RNA: Albertini et al., Science 2006
Rhabdovirus Transkription und Replikation lea lea und 5 mrnas P L RNP-abhängige RNA Polymerase 3 gp N P M G L (-) RNP N P L N P L 5 agp (+) RNP lea = leader RNA gp = Genompromotor agp = Antigenompromotor
Stop-Start Mechanismus: Termination und Reinitiation an intergenischen Signalsequenzen 1. mrna Transkription bis (A) 7 -Sequenz und stoppt 2. Polymerase gleitet vor und zurück (polymerase slippage) poly(a)-tail und Termination 3. Reinitiation und 5 -Capping an der folgenden Transkriptions- Startsequenz intergenische Sequenz zwischen Stop- und Startsignalen wird nicht transkribiert teilweise Dissoziation der Polymerase vom Template Transkriptionsgradient aus Flint et al. Principles of Virology (2000), ASM Press
Rhabdovirus Transkription und Replikation sequentielle Transkription monocistronischer, polyadenylierter mrnas mit 5 -Cap-Struktur (Stop-Startmechanismus) Transkriptionsgradient Virale RNA-Polymerase (P/L) ist sowohl Transkriptase als auch Replikase Ko-replikative Verpackung der neu synthetisierten genomischen RNA (Ribonukleoprotein-Komplex; RNP) RNP-abhängige Polymerase
The Idea cdna-plasmid transcription of RNA genomes packaging of RNA in RNPs RNA-Synthesis by P, L (and N) cdnaplasmid vrna transcript RNP + mrnas + vrna
1990
1990 1995
1994 Rabies Virus (RABV): First recombinant Negative Strand RNA Virus (NSV) generated from cloned cdna
System Vaccinia T7-Polymerase Driven T7 N-P-M-G-L HdVRb T7T
System Vaccinia T7-Polymerase Driven T7 N-P-M-G-L HdVRb T7T T7 promotor-gggacgct..agcgt-hdvrb-t7t.. transcription GGG cleavage GGG not capped and cleaved RNA
CAT Aktivitäten in Zellkultur Insertion einer zusätzlichen Transkriptionseinheiten N P M G L N P M G CAT L CAT CAT Aktivitäten in vivo (Mäusehirn) STOP START
Insertion einer zusätzlichen Transkriptionseinheiten N P M G L N P M G CAT L keine homologe Rekombination stabile Expression in vitro und in vivo Beispiele für Fremdgenexpression von NNSV: STOP START CAT Reporter: CAT, luciferase, GFP viral: HIV gag, env; HCV E1, E2; Flu HA zellulär: IFN, Interleukine, CD4, CXCR4
1 st Generation Rabies Virus Rescue co-transfection of full length cdna and N, P and L coding support plasmids freeze-thaw cells and mix with supernatant passage on fresh cells over-infection with Vaccina Virus T7 (VVT7) CPE pelleting of VVT7 by centrifugation still containes VVT7 mix of rescued Virus and Vaccina Virus
Development and Use of BSR T7/5 cells: BHK cells Expressing Bacteriophage T7 DNA-dependent RNA-Polymerase T7 N-P-M-G-L HdVRb T7T T7 promotor-gggacgct..agcgt-hdvrb-t7t.. transcription GGG cleavage GGG not capped and cleaved RNA
Development and Use of BSR T7/5 cells: BHK cells Expressing Bacteriophage T7 DNA-dependent RNA-Polymerase T7 N-P-M-G-L HdVRb T7T T7 promotor-gggacgct..agcgt-hdvrb-t7t.. GGG GGG not capped and cleaved RNA Type 1 IFN induction
2 st Generation Rabies Virus Rescue - Get Rid of Vaccinia Virus Helper Virus -
Impact by Method Development - BSR T7/5 cells amoung the 15 most cited articles in Journal of Virology -
2 st Generation Rabies Virus Rescue co-transfection of full length cdna and N, P and L coding support plasmids passage supernatant on fresh cells No Helper Virus!
3 rd Generation Rabies Virus Rescue - Get Rid of T7-Polymerase - RNA Polymerase II driven transcription of full length virus RNA and mrnas for N, P and L CMV promoter ndependent of cell type
Requirements for modern RNA-Pol II driven Rescue systems We need chalk!
4 th Generation Rabies Virus Rescue - Improvement of HdVRb - T7 improvement by nt insertion N-P-M-G-L HdVRb T7T T7 promotor-gggacgct..agcgt-hdvrb-t7t.. transcription GGG cleavage GGG not capped and cleaved RNA
Reverse Genetics Systems / Reverse Genetics reverse genetics systems Cloning of Virus genomes in vectors (plasmids, phagmids, bacs) Genetic Modification (Engeneering) reverse genetics approach Recombinant Viruses with Defined Mutations forward genetics Phenotypic Charaterisation Selection of Mutatants Phenotypic Characterisation
Next Generation Reverse Genetics Vaccinia Helfervirus-freie Systeme Buchholz et al., 1999; Finke et al., 1999 1994 1999 ABER: Geringe Vielfalt klonierter Virusgenome in Reverse Genetics Vektoren T7-RNA Polymerase freie Systeme Inoue et al., 2003; und andere 2003 Moderne Populationsgenetik Researchers Reality Optimierte Ribozymsequenzen Ghanem et al., 2012 2012 Rescue-Effizienz
Der Traum: von deskriptiv zu präskriptiv
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Recombinatorial Cloning of Complete Virus cdnas by Homologous Linear-to-Linear recombination
Der Traum: von deskriptiv zu präskriptiv