Transcriptional regulation in Ebola Virus. The role of VP30

Transcription

1 Transcriptional regulation in Ebola Virus The role of VP30

2 Ebola virus (2018/01/26) First symptoms 2 days after infection (fever, decreased liver- and kidney-function, bleeding...) Death after 6 16 days (~ 50%)

3 Ebola virus VP40 (Matrix) VP24 (Matrix) GP (Glycoprotein) NP (Nucleoprotein) L (Polymerase) VP30 (Transcription factor) (2017/10/05) VP35 (Polymerase cofactor) nonsegmented, filamentous, encapsidated, (-) ssrna Family: Filoviridae (Marburg virus, Lloviu virus,...)

4 Ebola virus 3' leader NP VP35 VP40 L VP35 19 kb genome GP VP30 VP24 L trailer mrna 5' (-)

5 Question 1.) VP30-dependence of transcription 2.) Influence of NP hairpin 3.) Potential binding sequences of VP30?

6 Question 1.) VP30-dependence of transcription 2.) Influence of NP hairpin 3.) Potential binding sequences of VP30?

7 1.) VP30-dependence of transcription NP (+) hairpin, ΔG = kcal/mol N small RNAs < 200 nt poly(a) RNAs max. reads : 24% 57: 25% 58: 43% max. reads NP mrna leader levels mrna/leader transcripts ~9:1 stem loop stem NP (-) hairpin, ΔG = kcal/mol

8 Question 1.) VP30-dependence of transcription 2.) Influence of NP hairpin 3.) Potential binding sequences of VP30?

9 2.) Influence of NP hairpin: Minigenome infected human cells Viral genome 3 NP NP leader GP GP luciferase 3 leader WT leader + VP30 small RNA (sequencing) trailer LL trailer 5 5 WT leader - VP30 poly-(a) RNA (sequencing)

10 2.) Influence of NP hairpin 3' NP VP35 VP40 GP VP30 VP24 L leader WT ΔG = kcal/mol... GC AU GC GC AU AU GU AU UA UA A U A ΔG = kcal/mol Weik et al., J. Virol NheI (hairpin mutant) U A U A AU AU U C CG U AU U C CG CG UA G G A G AG A G A U U A ΔG = kcal/mol C U C CG AUU ΔG = kcal/mol A U C G A A trailer 5'

11 2.) Influence of NP hairpin: Minigenome infected human cells Viral genome 3 NP NP leader luciferase 3 leader WT leader + VP30 small RNA (sequencing) GP GP WT leader - VP LL trailer trailer 5 5 NheI leader + VP30 NheI leader - VP30 poly-(a) RNA (sequencing)

12 2.) Influence of NP hairpin Leader-readthrough Leader WT +VP30 3' leader NP VP35 WT -VP30 VP40 GP NheI +VP30 VP30 VP24 NheI -VP30 L trailer 5'

13 2.) Influence of NP hairpin Leader-readthrough Leader NP WT +VP30 3' leader WT -VP30 NP NheI +VP30 VP35 VP40 WT +VP30 NheI -VP30 GP VP30 VP24 WT -VP30 L NheI +VP30 trailer 5' NheI -VP30

14 2.) Influence of NP hairpin qpcr (mrna, n 4, 2 independent experiments) 120 relative mrna level VP30 - VP wt NheI HEK293 cells 3' leader NP VP35 VP40 GP VP30 VP24 L trailer 5'

15 Question 1.) VP30-dependence of transcription 2.) Influence of NP hairpin 3.) Binding sequences of VP30

16 3.) Binding sequences of VP30 iclip UV UV AAA UV AAA AAA AAA cell lysis AAA AAA immunoprecipitation 32 P RNase cdna synthesis labeling 32 P 32 P sequencing proteinase..aaatttatatga....gagcaagcatc....cattgatttctg....atatgaatttaa....attgattcaatc..

17 3.) Binding sequences of VP30 (in vitro) VP30 WT WT leader 3' leader NP Aeo 6S RNA VP35 VP40 GP VP30 VP24 L 5' trailer

18 3.) Binding sequences of VP30 (in vitro) 450 k (+) 5' Ebola (Leader) hairpin hairpin 3'

19 3.) Binding sequences of VP30 (in vitro) 450 k (+) 5' Ebola (Leader) hairpin hairpin (-) 18 k 32 k 3'

20 3.) Binding sequences of VP30 (in vitro) UCCUAAAAAUUCUUCUUU AGAGGAAAAUU +1 5' +79 Ebola (Leader) hairpin hairpin 3'

21 3.) Binding sequences of VP30 P P VP30 P (in vivo) P P NP P L VP35 VP35 (-) replication (+) VP30 DD mutant phosphorylated Biedenkopf et al. VP30 L VP35 VP35 (-) transcription VP30 AA mutant dephosphorylated

22 3.) Binding sequences of VP30 (in vivo) VP30 WT VP30 AA mutant VP30 DD mutant Viral genome 3 NP NP leader GP GP luciferase 3 leader trailer LL 5 trailer 5

23 3.) Potential binding sequences of VP30 host transcripts C control C C VP30 DD VP30 WT VP30 AA

24 3.) Potential binding sequences of VP30 Motives (enriched pentamers) (in vivo) VP30 AA mutant VP30 WT VP30 DD mutant control

25 Conclusion: Transcription Model VP30 L 3 - VP30 VP35 L VP (-) VP30 NheI L 3 - VP30 5 (-) 5 (-) VP35 NheI L VP (-)

26 Results in brief VP30: 1) Binds to loop region of leader and NP hairpin (in vitro) 2) Has hardly any effect on leader transcripts 3) Is essential for transcription of NP 4) Does hardly bind to viral RNA (in vivo)? 5) Might bind to host RNAs (anion transporter, uncharacterized ncrna)

27 Results in brief NheI (hairpin mutant): 1) Reduces leader transcription in favor of NP transcription 2) Introduces leaky NP transcription in absence of VP30

28 Conclusion VP30 effects transcription transition from leader to NP, effect might mediated via host factors Weakening the NP hairpin (NheI mutant), enhances transcription of NP

29 Acknowledgments Marcus Lechner Roland Hartmann Simone Bach Julia Schlereth Dennis Streng Thorsten Hain Stephan Becker Nadine Biedenkopf

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33 RAW reads FastQ Trimming (cutadapt) Removed: Low quality sequences Poly-A Remaining sequencing adapters Work flow FastQC Quality of reads Piechart Number of reads Barcode assignment NNNXXXNN experiments Mapping (segemehl) Read length Boxplot In vitro: puc19, E. coli genome, Aquifex aeolicus 6SRNA In vivo: Minigenome & Human transcript Differential Expressionanalysis AA vs DD hnrnp vs VP30/AA/DD mock vs VP30/AA/DD Enriched Pentamers Mapping/random data Read separation In vitro: puc19 mapped only In vivo: Minigenome mapped only Visualization (IGB) Start / coverage number of reads

34 2.) Influence of NP hairpin Leader-readthrough Leader WT -VP Leader-readthrough WT +VP30 NheI -VP Leader Leader transcript NheI +VP30 Luc (NP) WT -VP30 Tail qpcr (mrna, n 4, 2 independent experiments) Read start Read end NheI +VP30 NheI -VP VP relative mrna level WT +VP30 NP NP VP wt HEK293 cells NheI

35 3.) Binding sequences of VP30 iclip