Exploring the role of TGF-β
signaling in Mouse Papillomavirus
induced pathogenesis
Max Golden
Abstract
Human Papillomavirus (HPV) is a double stranded DNA virus that is implicated in genital and head and
neck cancers in humans. Papillomaviruses are strictly species tropic and currently there is no tractable
infection model to study these viruses in a laboratory setting. In this light, the recent discovery of
Mouse Papillomavirus (MmuPV1), as the first ever papillomavirus to naturally infect laboratory mice
provides us the unique opportunity to understand the biology of papillomaviruses in a genetically
manipulatable host. Preliminary studies by our collaborators at Dr. Karl Munger’s lab (Harvard University)
have shown that the viral protein E6 of MmuPV1 inhibits transforming growth factor beta (TGF-β) signaling
potentially by interacting with the cellular SMAD3 protein, which is a downstream regulator of the TGFβ
signaling pathway. We want to understand whether inactivation of TGFβ signaling by MmuPV1 E6 protein
plays a critical role in MmuPV1 pathogenesis. The Munger lab is currently in the process of identifying
MmuPV1 E6 mutants that are selectively altered in their ability to bind to SMAD3. However, structural
analysis of E6 proteins in both HPV16 and BPV (Bovine Papillomavirus) has led to the hypothesis that it may
be difficult to generate a functional E6 mutant deficient in the ability to interact with SMAD3. If this were
true, then it will be difficult to use this approach for studying the biology of the E6 protein. Therefore we
are trying to use an alternative approach by identifying a SMAD3 mutant which is deficient in its ability to
bind E6, but retains other biological properties of SMAD3. A panel of SMAD3 mutants were generated in
Dr. Michael Hoffman’s lab which were disrupted in their binding to some but not all Smad binding
partners. We intend to test these mutants for their ability to bind E6 using a high-throughput
screening luminescence based interactome mapping (LUMIER) assay. We have been successful in
optimizing the LUMIER assay in the context of MmuPV-1 E6. optimized and performed several
successful LUMIER assays. Now that we have a working assay, we want to identify mutants of
SMAD3 that are selectively defective for binding E6. Our eventual goal is to be able to use the
identified mutant to generate a transgenic mouse strain which can be used to understand MmuPV-1
biology.
Hypothesis
Inactivation of TGFβ signaling by MmuPV1 E6
protein plays a role in pathogenesis
Problem
Mutating E6 in papillomavirus proteins has pleiotropic effects on the overall
function of E6
Potential Solution
Identify a SMAD3 mutant that does not bind MmuPV-1 E6 but retains other
biological functions
Approach
Using a high throughput screening luminescence based interactome mapping
(LUMIER) assay to identify SMAD3 mutants
Experimental Design
Generation of flag tagged MmuPV-E6 plasmids
Expansion of flag tagged MmuPV-E6 plasmids
Optimization of LUMIER assay using flag tagged E6 and known SMAD3 binding partners
Using optimized assay for identifying SMAD3 mutants that do not bind E6
Testing identified mutants in tissue culture for their ability to rescue TGF-beta signaling
Description of LUMIER
After completing a maxiprep with our plasmid DNA, we began the LUMIER
assay. In this assay, renilla luciferase-tagged partners, ALK5, and FLAG-tagged
partners are transfected into HEK 293 cells using TransIT-LT1 transfection
reagent (from Mirus). The next day, a high throughput co-immunoprecipitation
is done. The wells of a plate are pre-coated with G-protein antibody, which
pulls down the FLAG-tagged partner. If the E6 has binded to the renilla-tagged
partner, the entire protein complex will be pulled down and the luciferase will
give off a signal. We also used Alk5, a TGF-beta type 1 receptor which
phosphorylates SMAD3 and increases binding. A LUMIER assay is split into
three steps and is done across three days. The first step involves seeding cells
for transfection. To do this, we seed 10^5 cells/well of a 12 well plate.
The next step was to perform a LUMIER transfection. In order to perform the
LUMIER assay, we begin by preparing an anti-flag coated 96-well plate by
coating each well with 0.008 µg anti-flag antibody. 15µl of the sample is
collected for analysis of total luminescence activity and the remaining 185 µl is
incubated on the anti-flag coated plate and incubated at 4 oC on plate shaker
for 2 hours. The assay was developed using the dual-glo Luciferase assay
reagents (Promega) as per manufacturer’s instructions and luciferase counts
were read using a plate reader.
Methods: LUMIER Workflow
Flag tagged
partner
Renila tagged
partner
1. Read total renilla luciferase
activity to evaluate transfection
efficiency
Lyse Cells
2. Read activity of CO-IP
HEK293 Cells
Flag tagged
partner
Renila tagged
partner
Anti-flag coated
G-protein plate
Results
Fig. 1 Verifying presence of plasmid
DNA using agarose gel electrophoresis
Fig. 1
Flag tagged E6 plasmids were provided to us by the Munger lab. Our first task was to
verify the presence of plasmid DNA shared with us by our collaborators in the Munger
lab via agarose gel electrophoresis. The ladders were resolved well in lanes 1 and 5.
Lanes 2, 3, and 4 show the presence of supercoiled, relaxed, and nicked forms of
plasmid DNAs.
Fig. 2 Bacterial transformation using
flag-tagged E6 plasmids
Results of bacterial transformation (Table 1)
Plate Number
Label/Type
Colonies (Number)
1.
Positive Control (pUC 19 plasmid)
>20
2.
Negative Control (No DNA)
0
3.
CMV-N-mE6
>100
4.
CMV-C-mE6
>100
Flag tagged E6 plasmids (CMV-N-mE6 Transformation of E. Coli with CMV-N-E6 (Fig. 2)
and CMV-C-mE6) were transformed
into E.Coli cells, and plated on LB-amp
plates. Number of colonies that grew
after 24 hours are shown in
Table 1. Fig.2 shows an example of a
transformed plate with bacterial
colonies
Fig. 3 Small scale preparation of
plasmid DNA from transformed
bacteria
Plasmid DNA isolation by miniprep (Fig. 3)
I then performed a miniprep using a QIAGEN kit to isolate and purify our plasmid DNA and verified
presence of plasmid DNA via agarose gel electrophoresis. I used the plasmid vector pUC19 as a size
reference since it is known that pUC19 is about 3000 base pairs. Lanes 2 through 7 showed the
presence of supercoiled, relaxed, and nicked forms of plasmid DNAs. Lanes 8 and 9 showed the
presence of pUC19, which was our positive control. From our results, we were able to verify that
plasmid DNA was present and that our miniprep had been successful.
Fig. 4: Large scale preparation of plasmid DNA
UV Spectrophotometer Readings (Table 2)
S. No.
Sample Type
OD260
260/280
(from spec)
(from spec)
Dil. Factor
Conc.
(µg/ml)
Conc.
(µg/µl)
OD*DF*50
Conc.(µg/ml)
/1000
1
CMV-C-mE6
.1682
1.8156
200
1682
1.682
2
CMV-N-mE6
.1878
1.8107
200
1878
1.878
Presence of linear DNA confirmed by restriction analysis (Figure 4)
After conducting a successful miniprep, a maxiprep was then done to isolate and purify large amounts of plasmid DNA.
We assessed the purity and quantity of DNA by UV spectrophotometry (Table 2). The DNA isolated had a 260/280 ratio
of 1.8, which meant that the DNA was pure and not contaminated by RNA or proteins. We also set up a restriction
enzyme digestion using BamH1 (which linearizes the DNA) and ran an agarose gel to confirm the correct size (Fig. 4).
Fig. 5: COIP Luciferase expressed as a percentage of total luciferase of lysate
COIP Luciferase/total Luciferase expressed as percentage
2.5
2
1.5
1
0.5
0
Percentage
TrxGA
Smad4
N-E6
C-E6
0.01979315
1.912229939
0.714163611
0.457239453
The amount of luciferase activity in cells transfected with each of the indicated
proteins that coimmunoprecipitated with Smad3 is shown. TrxGA is the negative
control vector, whereas the Smad 4 serves as the positive control since it interacts
strongly with Smad 3. Both the n-terminal and c-terminal tagged viral E6 proteins
interacted with Smad3.
Fig. 6: Titrating the amount of Flag
tagged E6 keeping Smad3 constant
Conclusion
We have verified that E6 binds to Smad3 via LUMIER assay
Future Directions
• We want to identify mutants of SMAD3 that are selectively defective for
binding E6 and to then perform follow-up studies in tissue culture to confirm
that the ability of E6 to bind to SMAD3 is required for E6 to inactivate the
TGF-beta signaling pathway.