The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation

The Role of the 3’ UTR of Dulcamara mottle virus RNAin Translation Alma Laney Dr. Yannis Tzanetakis Dr. Theo Dreher

mRNA Translation An mRNA structure 5’ cap 3’ UTR 60s Enzyme 40s An mRNA translation scheme

An Positive Strand RNA Viruses mRNA, Flexiviruses, Togaviruses TLS Tymo-, Tobamoviruses Flaviviruses, Closteroviruses Picornaviruses, Potyviruses An VPg Barley yellow dwarf virus 5´cap: m7G(5´)ppp(5´)-N

RNA virus translation • How do the untranslated regions (UTR) of RNA viruses enhance translation?

TYMV translation • The TLS of TYMV mimics a tRNA.

M T R TYMV and DuMV genomes TYMV RNAi suppressor P r o t e o l y t i c M a t u r a t i o n MP (69 kD) CP (20 kD) C l e a v a g e * P R O H E L P O L TLS (Val) 109 nt 3´-UTR 87 nt 5´-UTR p141 p66 (206 kD) RP DuMV RNAi suppressor MP (62 kD) CP (20 kD) * M T R P R O H E L P O L 248 nt 3´-UTR 129 nt 5´-UTR (196 kD) RP

CUCU U A C C A A U G CCCG CCC UCGGA A CC(A) UUC CGUC GAGC GCGA T A A GGGC GGG AGCCU CUCG GCAG CGCU U C C C G U U UCA UUAAA U C G U C A A U U Dulcamara mottle virus 3´- pseudoknot D A C 5´ C A U G-C C-G TYMV TLS G-C A-U G-C 5´ G U-A C-G U-A G-C U-A C-G C C A/C C C A C A TYMV and DuMV 3’ termini

U A C A G UUC CGUC GAGC GCGA A CUCG GCAG CGCU C C U U UUAAA C C U C 5´ AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAAAAAAAAAAAAAACAAAACAAAACAAA The 3’ UTR of DuMV 44 nt 59 nt 145 nt DuMV 248 nt Dulcamara mottle virus 3´- pseudoknot

The Question • Does the 3’ UTR of DuMV enhance translation? 44 nt 59 nt 145 nt

The Hypothesis • The combination of the 59 nt and 145 nt sequences are responsible for translational enhancement. 59 nt 145 nt ( Poly A Tail) (Pseudoknot)

Methodology • Six plasmid constructs were created with the luciferase gene. • The plasmids will be tested to see luciferase expression. 3’ UTR luciferase reaction luciferase Light generated Luciferin + ATP

Luciferase Spacer Luciferase Spacer A Track Luciferase A Track Luciferase Pseudoknot Luciferase A Track Pseudoknot Luciferase Spacer A Track Pseudoknot Plasmid constructs

Creating the plasmid constructs 1. PCR of UTR section 2. Ligation of UTR fragment to pLUC

Creating the plasmid constructs cont. 3. Transformation 4. Restriction Enzyme Digest

Creating the plasmid constructs cont. 5. Gel Electrophoresis 6. DNA sequencing

Luciferase assay to test for translational enhancement 1. Linearize plasmid 2. In vitro transcription LUC * 3. RNA transfection +/- DuMV UTR fragments Cowpea protoplasts 4. Incubation under light 5. Cell lysis 6. Luciferase reaction

Cloned Sequenced Assayed Results Luc + Spacer X X Luc + Spacer + A Tail X X Luc + A-tail X X Luc + Pseudoknot X X Luc + A-tail + Pseudoknot x Luc + complete X X

Luc Spacer A Track Pseudoknot Example of Luciferase Assay Luc Luc Luc Luc

Future Work • The next step is to create several genome chimeras to test infectivity in plants. TYMV Complete Genome DuMV 3’ UTR DuMV Complete Genome TYMV TLS

Acknowledgements • Thanks to the Howard Hughes Medical Institute. • Dr. Yannis Tzanetakis • The Theo Dreher Lab

The Role of the 3’ UTR of Dulcamara mottle virus RNA in Translation