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

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

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

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

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

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

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

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

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

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

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

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

PCR Modification Annealing Temperature/ Mg 2+ concentration 2nd PCR, A-Rich (DCLD-SnaB1/Not1 template, WW12 + A-Rich megaprimer) 60 o C 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

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

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

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

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

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.

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!