Transcript Investigation of the Roles of Viral Proteinases

Investigation of the Roles
of Viral Proteinases in the
Vaccinia Virus Life Cycle
Jessica Page
Mentor: Dr. Dennis Hruby
Department of Microbiology
Background
Vaccinia Virus: A close relative to variola
virus, the causative agent
of smallpox.
Vaccinia
The Disease
Smallpox (variola major)
Highly infectious
30-40% mortality
Effective vaccine (VV)
Global eradication in 1977
Remaining variola stocks
to be destroyed in 2002
Biological threat gone
……or is it?
Significance



Smallpox has
become a possible
weapon of
bioterrorism
U.S. population has
become
immunologically
naïve
Vaccine not totally
effective in attack
situation
Scientific Approach
Selection of appropriate antiviral target
Proteinase inhibitor paradigm (HIV, HCV, etc.)
Poxvirus proteinases
I7L
G1L
VV Replication Cycle
Morphogenic
Proteolysis
VV Core Protein Cleavage Sites
Amino Acids
30
90
150
210
240
300
360
420
480
540
600
AGS
660
720
780
840
AGT
P4a (97 kDa)
892
617
697
4a (64 kDa)
23K
AGA
P4b (72 kDa)
645
60
4b (62 kDa)
AGS
AGA
P25K (28 kDa)
18 32
25K (25 kDa)
252
Virion core
formation
Tetracycline
Operator/Repressor
Viral DNA
vvTetO:G1L/I7L
TetO
G1L
TetO
TRex 293 Cell
TetO
G1L
TetO
I7L
“OFF” No
expression
of G1L &
I7L
I7L
TetR
Tet Repressor
Tet
TRex 293 Cell
TetO
G1L
TetO
Tet Repressor
I7L
Tetracycline
“ON” Expression
of G1L &
I7L
Viral Phenotype +/- Tetracycline
A
B
C
D
E
F
+ tet
- tet
My Goals
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

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
Characterize the vvTetO:G1L/I7L modified
virus
Growth curve +/- tet vs. WR to demonstrate
conditional lethality and fitness
Western blots of I7L and late core proteins
p4a, p4b, p25K +/- tet vs. WR and
transfected with G1L/I7L plasmids
Rescue with I7L and G1L alone and in
tandem to demonstrate activity of each
proteinase independently
Analysis of core protein processing
Genomic Sequencing
Tetracycline Operator = TCCCTATCAGTGATAGAGA
I7L
TGATAATCCCTATCAGTGATAGAGAATGGAAAGATA
Promoter
(upstream)
G1L
TetO
Methionine
AAATGATCCCTATCAGTGATAGAGAATGATTGTCTT
Promoter
(upstream)
TetO
Methionine
MOI Optimization
1.00E+09
Titer (pfu/ml)
+ tet
1.00E+08
+ tet
+ tet
1.00E+07
- tet
- tet
- tet
1.00E+06
0.1
0.5
MOI (Multiplicity of Infection)
1
Growth Curve
Titer (pfu/ml)
1.00E+08
1.00E+07
w/out tet
tet
WR
1.00E+06
1.00E+05
0
2
4
6
8
10
Hours Post Infection (hpi)
12
Immunoblot Detection of
p4b Protein Cleavage
+ tet -tet WR
p4b
precursor
72 kDa
4b
cleavage
product
62 kDa
VV Core Protein Cleavage Sites
Amino Acids
30
90
150
210
240
300
360
420
480
540
600
AGS
660
720
780
840
AGT
P4a (97 kDa)
892
617
697
4a (64 kDa)
23K
AGA
P4b (72 kDa)
645
60
4b (62 kDa)
AGS
AGA
P25K (28 kDa)
18 32
25K (25 kDa)
252
Virion core
formation
Immunoblot Detection of
p4b Protein Cleavage
+ tet -tet WR
p4b
precursor
72 kDa
4b
cleavage
product
62 kDa
Rescue Experiments
TRex 293 Cell
TetO
G1L
TetO
TetR
I7L
Tet Repressor
G1L
plasmid
I7L
plasmid
Virion core
formation
and
continuation
of virus life
cycle
Expression of
G1L and/or I7L
Future Progress


Continue to optimize experimental
conditions to achieve successful rescue
and analysis of late core proteins
Try rescue with mutant I7L and G1L
plasmids to determine the functional
areas of the proteins
THANKS!!



Dr. Dennis Hruby
Megan, Jen, Cliff, Chelsea,
Kady, Su-Jung, Dina, Eric,
and all the family
Howard Hughes Medical
Institute