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