- Poliovirus uses host cell proteins like TDP2 for replication. Previous studies showed TDP2 relocalizes from the nucleus to the cytoplasm during peak viral replication at 4 hours post-infection. - The study aimed to determine TDP2 localization in poliovirus-infected cells after peak replication at 5-6 hours. - Results showed that after peak replication, TDP2 can be found in both the cytoplasm and nucleus of infected cells. Further research is needed to understand how TDP2 relocalizes during infection.

1. Lisa Chen
Dr. Bert L. Semler
August 7, 2013
Determining the localization of TDP2
during poliovirus infection

2. History of poliovirus
— Widespread in the early twentieth century
— Tens of thousands were infected
— Poliovirus vaccine was first tested in 1952 by Jonas Salk
— Since then, the number of individuals infected by poliovirus each year
has declined from hundreds of thousands to less than a thousand today
— Although poliovirus is wiped out of most first world countries,
outbreaks remain common in undeveloped areas in SoutheastAsia and
Africa
Image taken from Travelers Disease Information.
N.p., n.d. Web.

3. Poliovirus overview
— Belongs to the Picornaviridae family
— Includes: coxsackievirus B3 and human
rhinovirus
— Is the prototype picornavirus
— Has a small (~7-8 kb) single-stranded,
positive-sense RNA genome
— Completes its entire life cycle in the
cytoplasm of infected cells
— Due to its limited coding capacity, it uses
host proteins for viral translation and
RNA replication
Three dimensional structure
of poliovirus
(Hogle et al., 1985)

4. 5’ tyrosyl-DNA phosphodiesterase
(TDP2)
— Is a cellular DNA repair enzyme
— Repairs topoisomerase-mediated damaged DNA
— Can be found in the cell cytoplasm but is predominantly a
nuclear enzyme
— Cleaves a unique viral protein-RNA covalent linkage in
poliovirus infected cells
— Previous studies show thatTDP2 becomes relocalized
from the nucleus to the cytoplasm during peak times
(4 hours post-infection) of viral replication
— TDP2 remains at the cell periphery, a site distinct from
putative viral replication complexes

5. TDP2 relocalizes to the cytoplasm
during poliovirus infection
— TDP2:
— Is predominantly a
nuclear enzyme
— Begins to relocalize
from the nucleus to
the cytoplasm 2
hours post-infection
— Relocalizes to the
cell periphery
during peak time in
poliovirus
replication
Virgen-Slane et al., 2012

6. Experimental Question
— Where isTDP2 localized in poliovirus-infected cells
following peak times of viral replication?
Hypothesis
— TDP2 will remain in the cell periphery of the poliovirus-
infected cells following peak times of viral replication

7. Specific aim
— Determine the localization of TDP2 in poliovirus-
infected cells following peak times of viral
replication (5 and 6 hours post-infection)
Method
Image taken from Hart and
Shears, 1997
— Use immunofluorescence
to detectTDP2 and
poliovirus protein 3A in
poliovirus-infected cells

8. Figure 1. Determining the poliovirus
titer for immunofluoresence
Mock Infected 10 -7 10 -8 10 -9
Figure 1. Determining the poliovirus titer for immunofluoresence. HeLa cells were grown on 60
x 15 mm plates.They were then infected with decreasing 10-fold dilutions of poliovirus 1 (P3)
stock and incubated for 30 minutes at room temperature to allow for viral adsorption.The cells
were overlaid with 2% agrose-DMEM supplemented with serum and incubated at 37o C for 48
hours.The cells were stained with 0.1% crystal violet and the plaques were counted.The
poliovirus 1 (P3) stock’s viral titer was calculated to be 8 x 10 -9 PFU/ml.

9. Mock5Hours6Hours
TDP2 3A DAPI Merged
Figure 2. TDP2 is found in both the nucleus and
cytoplasm following peak viral replication
Figure 2. TDP2 is
found in both the
nucleus and cytoplasm
following peak viral
infection. HeLa cells
were infected with
poliovirus 1 (P3) at an
MOI 20.The cells were
then fixed at 5 hours or
6 hours post-infection
with 3.7%
formaldehyde.The fixed
cells were blocked with
donkey serum, stained
with αTDP2 (1:150)
and α3A (1:500), and
fluorescently labeled
with goatαrabbit
(1:1000) and
goatαmouse (1:1000)
respectively. Images
were taken on the
confocal microscope.

10. Summary
— Since poliovirus has a limited coding capacity, it uses host
proteins, such asTDP2, for viral translation and RNA
replication
— Previous studies show thatTDP2 becomes relocalized from
the nucleus to the cytoplasm during peak times (4 hours
post-infection) of viral replication
— Conclusion:TDP2 can be found in both the cytoplasm and
nucleus of poliovirus-infected cells following peak times (5
and 6 hours) of viral replication
— Future directions:Determine the mechanismTDP2 employs to
relocalize during poliovirus infection

11. Acknowledgements
— Dr. Bert L. Semler
— Sonia Maciejewski-Flores
— Cancer Research Institute
— Nita Driscoll
— Patty Depetris