1. The Construction of Mutations in the 3 Cyclization Sequence of Dengue Virus Genome, for the Study of Translation Tari Tan Dr. Theo Dreher Dr. Connie Bozarth HHMI, Summer 2005

2. The Global Impact of Dengue Virus Dengue Fever & Dengue Hemorrhagic Fever Endemic in more than 100 countries 50 million cases each year Areas infested with Aedes aegypti Areas with Aedes aegypti and dengue epidemic activity

3. Dengue: Viral Properties Flavivirus Genome Ranges from 10-11 kilobases 5' nucleotide cap +ssRNA Serotype 2 (DEN-2)

4. Translation DEN-2: +ssRNA Viral Proteins are responsible for replication, assembly, maturation, and exit from the cell

5. Conserved Features Dumbbells 1 and 2 (DB1, DB2) Stemloops A and B (SLA, SLB) 5' Cyclization Sequence (cCS1) 3' Cyclization Sequence (CS1)

6. Previous Studies Kunjin Virus, observed effect of cCS1 and CS1 mutations on replication 3' mutant - no replication 5' mutant - no replication Deletion of cCS1 - no replication 5'+3' mutant restoring complementarity - delayed start, but efficient replication DEN-2, effect of cCS1 and CS1 mutations on translation Replace cCS1 with modified CS1 - little impact (efficient translation) Replace CS1 with modified cCS1 - only 19% translation 5'+3' mutant restoring complementarity - could not rescue translation Dr. Alexander Khromykh Wei Wei Chiu

7. Five Mutations 1) IS TRANSLATION SEQUENCE-SPECIFIC FOR CS1? 2) Does the degree of complementarity between cCS1 and CS1 affect translational efficiency?

8. Definitions Vector Insert Mutant/Variant The fragment (or, in general terms, the DEN-2 construct containing the fragment) containing the mutated CS1, which is ligated into the vector The resultant vector+insert construct containing the mutated CS1 The DEN-2 construct into which the mutated sequences are ligated

9. Methodology DCLD WT (insert); DCLD ∆DB1+2 (vector)

10. PCR amplification of mutated sequences, using DCLD WT Methodology (Cont.)

11. Digest Final PCR product with Hind III and Kpn I; isolate 474 bp fragment Insert mutants into DCLD ∆DB1+2 (which has already been digested with Hind III and Kpn I) via a ligation Ultimately, the mutants will be used to assess translation by measuring luciferase luminescence

12. Troubleshooting I.PCR - poor yield II.Digests - not cutting III.Liquid Cultures - not growing / no plasmid First PCR Purify Megaprimer Second PCR Hind III/Kpn I Digest Purify Fragment Ligation with DCLD ∆DB1+2 Grow/Prep Colonies Hind III/Kpn I Digest (Select) Send For Sequencing

13. PCR Modification Annealing Temperature/ Mg 2+ concentration 2nd PCR, A-Rich (DCLD-SnaB1/Not1 template, WW12 + A-Rich megaprimer) 60 o C 2 1.5.75 mM mM mM 65 o C, 1.5 mM MgCl 2 2nd PCR, Khromykh (DCLD-SnaB1/Not1 template, WW12 + Khromykh megaprimer) 137 bp 128 bp D K L S Gel-pure Megaprimers, (3 ul/ 30) 479 bp K Final PCR product, “K” mutant New Oligos

14. Diagnostic Digests Performed by Dr. Bozarth DigestedNot Digested BUFFERS ENZYMES KpnI HindIII Buffer R (HindIII buffer) Buffer KpnI

15. Bacterial Growth NH 2 Ampicillin NH CH 3 H N O O O OH S H N O ß - lactam

16. Liquid Cultures & Loss of Plasmid Plate Investigation Hard-to-clone sequences? Mutants toxic to cells?

17. Vector Reconstruction Phosphatased, phenol-extracted, EtOH precipitated, gel-pure vectors (digested with Hind III and Kpn I) DCLD ∆DB 1+2 - Problem with the vector? - Make ∆DB1+2, as well as DCLD

18. Summary I. PCR - poor yield A.Annealing Temperature: 60 o C B.MgCl 2 Concentration: 1.5 mM C.Re-designed oligos to be shorter: better yield A.Dr. Bozarth’s Trials: bad KpnI or Buffer KpnI (bought new enzyme and buffer) II. Digests - not cutting III. Liquid cultures not growing / Loss of plasmid A.DCLD as control: only controls grew (2 x YT not the problem) B.Streaking: selective growing, but still problems in liquid cultures C.Re-make vector, both ∆DB 1+2 and DCLD: under investigation These issues remain unresolved.

19. Acknowledgements HHMI - Dr. Kevin Ahern URISC Dr. Theo Dreher Dr. Connie Bozarth Everyone else in the Dreher lab for helping me out Thank you very much!