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 10-11 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
Dr. Alexander Khromykh
• 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
Wei Wei Chiu
• 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
Five Mutations
1) IS TRANSLATION
SEQUENCE-SPECIFIC
FOR CS1?
2) Does the degree of
complementarity between
cCS1 and CS1 affect
translational efficiency?
Vector
Definitions
The DEN-2 construct into which
the mutated sequences are
ligated
Insert
The fragment (or, in general terms,
the DEN-2 construct containing the
fragment) containing the mutated
CS1, which is ligated into the vector
Mutant/Variant
The resultant vector+insert
construct containing the
mutated CS1
Methodology
• DCLD WT (insert); DCLD ∆DB1+2 (vector)
Methodology (Cont.)
•
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
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)
I. PCR - poor yield
II. Digests - not cutting
III. Liquid Cultures - not growing / no plasmid
Send For
Sequencing
PCR Modification
•Annealing Temperature/
Mg2+ concentration
60o C
•New Oligos
D K
L S
2 1.5 .75
mM mM mM
128 bp
137 bp
Gel-pure Megaprimers, (3 ul/ 30)
2nd PCR, A-Rich
(DCLD-SnaB1/Not1
template, WW12 + A-Rich
megaprimer)
K
65o C,
1.5 mM MgCl2
2nd PCR, Khromykh
(DCLD-SnaB1/Not1 template,
WW12 + Khromykh
megaprimer)
479 bp
Final PCR product,
“K” mutant
Diagnostic Digests
•Performed by Dr. Bozarth
BUFFERS
Buffer R
Buffer KpnI
(HindIII buffer)
HindIII
Digested
Not Digested
Not Digested
Not Digested
ENZYMES
KpnI
Bacterial Growth
NH2
Ampicillin
NH
H
S
CH3
O
CH3
N
O
OH
ß - lactam
O
Liquid Cultures & Loss of Plasmid
Plate Investigation
•Hard-to-clone sequences?
•Mutants toxic to cells?
∆DB 1+2
DCLD
Vector Reconstruction
- Problem with the vector?
- Make ∆DB1+2, as well as DCLD
Phosphatased, phenol-extracted,
EtOH precipitated, gel-pure vectors
(digested with Hind III and Kpn I)
Summary
I. PCR - poor yield
A. Annealing Temperature: 60o C
B. MgCl2 Concentration: 1.5 mM
C. Re-designed oligos to be shorter: better yield
II. Digests - not cutting
A. Dr. Bozarth’s Trials: bad KpnI or Buffer KpnI
(bought new enzyme and buffer)
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!