Date post: | 20-Dec-2015 |
Category: |
Documents |
View: | 214 times |
Download: | 1 times |
Investigation of the Investigation of the Roles of Viral Roles of Viral Proteinases in the Proteinases in the Vaccinia Virus Life CycleVaccinia Virus Life Cycle
Jessica PageJessica Page
Mentor: Dr. Dennis HrubyMentor: Dr. Dennis Hruby
Department of MicrobiologyDepartment of Microbiology
Vaccinia Virus: A close relative to variola virus, the causative agent
of smallpox.
Vaccinia
Background
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?
SignificanceSignificance
Smallpox has Smallpox has become a possible become a possible weapon of weapon of bioterrorismbioterrorism
U.S. population has U.S. population has become become immunologically immunologically naïvenaïve
Vaccine not totally Vaccine not totally effective in attack effective in attack situationsituation
Selection of appropriate antiviral target
Proteinase inhibitor paradigm (HIV, HCV, etc.)
Poxvirus proteinases
Scientific Approach
I7L G1L
Morphogenic Proteolysis
VV Replication CycleVV Replication Cycle
30 36090 420150 480210 240 540300 600 660 720 780 840
P4b (72 kDa)
Amino Acids
AGS AGA
252P25K (28 kDa)18 32
P4a (97 kDa)617
AGS
697
AGT
AGA
60
VV Core Protein Cleavage VV Core Protein Cleavage SitesSites
645
892
25K (25 kDa)
4b (62 kDa)
4a (64 kDa) 23K
Virion core formation
I7LG1LTetO TetO
TRex293 Cell
TetRepressor
I7LG1LTetO TetO
TRex293 Cell
TetRepressor
Tetracycline Tetracycline Operator/RepressorOperator/Repressor
I7LG1LViral DNA
vvTetO:G1L/I7L
TetO TetO
No expression of G1L & I7L
Tetracycline
Expression of G1L & I7L
TetR
Tet
“OFF”
“ON”
A B C
D E F
Viral Phenotype +/- Tetracycline
+ tet
- tet
My GoalsMy Goals
Characterize the vvTetO:G1L/I7L Characterize the vvTetO:G1L/I7L modified virusmodified virus
Growth curve +/- tet vs. WR to Growth curve +/- tet vs. WR to demonstrate conditional lethality and demonstrate conditional lethality and fitnessfitness
Western blots of I7L and late core Western blots of I7L and late core proteins p4a, p4b, p25K +/- tet vs. WR proteins p4a, p4b, p25K +/- tet vs. WR and transfected with G1L/I7L plasmidsand transfected with G1L/I7L plasmids
Rescue with I7L and G1L alone and in Rescue with I7L and G1L alone and in tandem to demonstrate activity of each tandem to demonstrate activity of each proteinase independently proteinase independently
Analysis of core protein processingAnalysis of core protein processing
Genomic SequencingGenomic Sequencing
Tetracycline Operator = TCCCTATCAGTGATAGAGA
I7L TGATAATCCCTATCAGTGATAGAGAATGGAAAGATA
G1L
AAATGATCCCTATCAGTGATAGAGAATGATTGTCTT
TetO
TetO
Methionine
Methionine
Promoter (upstream
)
Promoter (upstream
)
MOI OptimizationMOI Optimization
1.00E+06
1.00E+07
1.00E+08
1.00E+09
0.1 0.5 1
MOI (Multiplicity of Infection)
Tit
er
(pfu
/ml)
+ tet
+ tet
+ tet
- tet- tet
- tet
Growth CurveGrowth Curve
1.00E+05
1.00E+06
1.00E+07
1.00E+08
0 2 4 6 8 10 12
Hours Post Infection (hpi)
Tit
er (
pfu
/ml)
w/out tet
tet
WR
Immunoblot Detection Immunoblot Detection of p4b Protein of p4b Protein CleavageCleavage
p4b precursor
72 kDa
4b cleavag
e product 62 kDa
+ tet -tet WR
30 36090 420150 480210 240 540300 600 660 720 780 840
P4b (72 kDa)
Amino Acids
AGS AGA
252P25K (28 kDa)18 32
P4a (97 kDa)617
AGS
697
AGT
AGA
60
VV Core Protein Cleavage VV Core Protein Cleavage SitesSites
645
892
25K (25 kDa)
4b (62 kDa)
4a (64 kDa) 23K
Virion core formation
Immunoblot Detection Immunoblot Detection of p4b Protein of p4b Protein CleavageCleavage
p4b precursor
72 kDa
4b cleavag
e product 62 kDa
+ tet -tet WR
I7LG1LTetO TetO
TRex293 Cell
TetRepressor
Rescue ExperimentsRescue Experiments
TetR
G1L plasmid I7L
plasmid Expression of G1L and/or I7L
Virion core formation
and continuation of virus life cycle
Future ProgressFuture Progress
Continue to optimize Continue to optimize experimental conditions to experimental conditions to achieve successful rescue and achieve successful rescue and analysis of late core proteinsanalysis of late core proteins
Try rescue with mutant I7L and Try rescue with mutant I7L and G1L plasmids to determine the G1L plasmids to determine the functional areas of the proteinsfunctional areas of the proteins
THANKS!!THANKS!! Dr. Dennis HrubyDr. Dennis Hruby Megan, Jen, Cliff, Megan, Jen, Cliff,
Chelsea, Kady, Su-Jung, Chelsea, Kady, Su-Jung, Dina, Eric, and all the Dina, Eric, and all the familyfamily
Howard Hughes Medical Howard Hughes Medical InstituteInstitute