A point mutation in the RBCC domain of PML I that disrupts its self-interaction was investigated. Confocal microscopy showed that the PML I L73E mutant failed to aggregate at ND10 nuclear bodies like wildtype PML I, but could dynamically interact with and decrease the number of endogenous ND10 bodies over time. This suggests the L73E mutation prevents PML I oligomerization necessary for ND10 structure but it can still affect ND10 composition and antiviral ability.

2. Point mutation disrupting PML
I self-interaction interferes with
ND10 formation
Nolan Shoukri, Haidong Gu*
Department of Biological Sciences, Gu Lab, Wayne State University

3. Human herpes simplex virus 1 (HSV-1) is a prevalent virus infecting over 70% of
adults that have evolved to efficiently infect its host. During infection, the antiviral ND10
nuclear bodies are used as host’s efforts to repress the expression of the incoming viral
genome. To counteract, HSV-1 deploys its immediate early protein, ICP0, which
colocalizes to the ND10 nuclear bodies and uses its E3 ubiquitin ligase activity to
degrade important proteins at ND10. Consequently, ND10 nuclear bodies are disrupted,
and the repression imposed on viral DNA is released. Promyelocytic Leukemia (PML)
protein is the important structure and functional component of the cellular ND10
nuclear bodies. PML has seven isoforms that share the same N-terminus and differ only
in their C-termini. The RBCC domain located in the PML N- terminus triggers PML
oligomerization, which helps to build the framework of ND10. Our lab has been
investigating viral and cellular elements that regulate the PML degradation triggered by
ICP0. Here we report that an L73E mutation within the RBCC domain of PML I disrupts
its self-interaction to the endogenous PML isoforms. Confocal microscopy assay showed
that PML I containing L73E mutation fails to aggregate at ND10 nuclear bodies, but it
has the ability to decrease the number of endogenous ND10 nuclear bodies. These
results suggest that PML L73E mutant can dynamically interact with endogenous ND10
components and affect the ND10 restrictive ability on HSV-1 infection.
Abstract

4. Introduction
What are viruses?
• Viruses are obligate parasites; they
cannot complete their life cycle
outside of their host.
• For a virus infection to be successful:
• The virus must be able to enter
the host cell.
• The virus must be able to hide
from or counteract the host
antiviral response.
• The cell must have the
appropriate cellular machinery
to support viral replication.
Virus
Eukaryotic cell

5. Introduction
Herpes Simplex Virus-1
Envelope
Tegument
Capsid
Core
Spikes
a b b’ a’
UL Us
c’ c a
Viral
genome
Diagrams showing the virion structure of Herpes Simplex
Virus-1, general layout of the viral genome, as well as the cellular
transmission of the virus.
• The virus has a linear dsDNA
genome size of 152kb.
• Codes for ≥ 84 viral proteins, many
which have more than one function.
• Generates multiple mRNA through
alternate splicing of mRNA
transcripts.
• Can infect epithelial cells and later
establish infection in sensory nerve
cells.
• Has a latent and lytic phase, the
switch from latent to lytic phase is
usually due to environmental
stressors.
• Stressors include physical or
emotional stress, surgery, or any
illness associated with a
compromised immune system.
Primary
infection
Latent infection
Central
Nervous
System
`
Recurrent infection, virus
spread

6. Introduction
Prevalence of diseases caused by HSV-1
>70% of world
population
20-30 millions recurrent
genital herpes
500,000 recurrent herpes
keratitis
2000+ disseminated
infection of newborn 5,000+ adult
encephalitis

7. Introduction
An overview of the viral replication cycle and essential proteins
HCF
VP16
 (IE) gene products
ICP0,
ICP4,
ICP22,
ICP27,
ICP47
STOP
proteins enhance viral expression
structural proteins
(proteins necessary to form the
infection virion)
 (E) gene products
UL23,
UL30,
UL39,
UL42, etc
Viral DNA replication &
g (L) gene expression
proteins essential for
replication
Oct-1
Cytoplasm
Nucleus
Tegument protein
VP16 prevents
viral repression
ICP0 is necessary
for a successful
infection

8. Introduction
What ICP0 is and why it is necessary for
infection
Why is it necessary for infection?
• It interrupts the interferon response which
is an important host antiviral response.
• It prevents chromatin remodeling and
repression of viral genome.
• It plays a part in regulating the host cell
cycle and DNA damage responses.
What is ICP0?
• ICP0 is an E3 ubiquitin ligase, which
means it can ubiquitinate and mark
important cellular proteins, such as PML
and Sp100, for degradation.
• It is highly regulated via post
translational modifications such as
phosphorylation, ubiquitination, etc.

9. Introduction
A deeper look at ND10
• ND10 is an important protein complex
that is multifunctional depending on
the needs of the cell.
• It can function as an antiviral response,
antioxidant response, or anticancer
response complex.
• It is made up structurally of PML I
oligomers which interact with and
recruit other antiviral proteins such as
Sp100.
• PML I self-oligomerizes to form ND10
• Degradation of PML I leads to
dispersion of ND10 components due to
lack of structure holding the complex
together

10. Introduction
Focus of the research
• We are looking at
components that
contributed to PML I
degradation.
• We know that a L73E
mutation in RBCC domain of
PML I disrupts
oligomerization.
• We hypothesize that PML I
L73E will not aggregate with
ND10 and decrease the
amount of ND10.
Normal PML I
PML I L73E
mutant
PML I oligomer
PML I oligomer

11. Methodology/Experimental
• Generate Hep2 cell line that can be induced.
• Cells were then prepped.
• Fluorescently labelled and stained.
• Confocal imagery.
• Analyzed via Las X.
• Compared cells by expression levels.
• Counted ND10.

12. Methodology/Experimental
Generation of the TetOn Hep2 cell line

13. Methodology/Experimental
Induction of PML I L73E expression
TR repressor
TetOn myc-PML
PML
m
Dox
• Cells contain 2 plasmids.
• One plasmid contains the
Tetracycline Receptor (TR)
repressor gene which is
always active.
• Synthesized TR protein
dimerizes.
• pcDNA4/TO plasmid which
contains the mutant PML
is instantly repressed by
TR repressor dimer.
• Doxycycline can bind to
the dimer and release it,
inducing mycPML I L73E
expression. PML
m
Noncoding
DNA
mycPML I L73E
gene
TR repressor
gene
TR repressor
protein
Doxycycline
mycPML I L73E

14. Methodology/Experimental
Cell prep and fluorescent labeling
• The Hep-2 TetOn cells were induced
with Dox for certain periods of time,
either 6 hours or 25 hours.
• The cells were then fixed,
permeabilized, and blocked to
prepare them for imaging.
• Nucleus stained with DAPI which is
blue.
• mycPML I L73E and Sp100 were
labeled using primary and a
fluorescent secondary antibody.
• mycPML I L73E fluoresces red while
Sp100 fluoresces green.
Antibody labeling of target proteins
Target protein
Sp100 or
mycPML I L73E
Primary
antibody
Target protein and
antibody complex
Secondary antibody
with fluorescent
compound Target protein and antibody
complex with secondary antibody
Energy in the
form of light
Fluorescence

15. Results and Discussion
Example images from confocal microscopy
6 hours
-Dox
25 hours
-Dox
6 hours
+Dox
25 hours
+Dox
Legend:
Sp100 DAPI Myc
• Images on the
right are
examples of
images analyzed.
• -Dox means no
doxycycline was
added.
• +Dox means
doxycycline was
added.
• All images taken
at 100x
magnification
with the same
contrast and
zoom parameters
Saturation increased 400% for better visualization

16. Results and Discussion
DAPI/myc/Sp100 Sp100 and myc myc only
25 hour
induced
cells
Saturation increased 900% for better visualization.
Legend
Sp100
DAPI
Myc
• The scattering
of red
throughout
the cell
signifies a lack
of aggregation
of mycPML I
L73E to ND10
(green dots)
• Occasional red
dots that
overlap with
ND10
represent a
dynamic
interaction
between the
mutant PML I
and ND10
complexes.

17. Results and Discussion
High expression
Low expression
6
hours
induced
25
hours
induced
Legend:
Blue line is DAPI
intensity
Red line is mycPML I
L73E intensity
• Histograms were generated using LasX, ROI lines were drawn border to border of the nucleus
and intensity was measured along the line.
• High expression and low expression is based on mycPML I L73E intensity.
• Average cell intensity of >30 is considered high expression while anything <20 is considered low
expression.

18. Results and Discussion
***
0
1
2
3
4
5
6
7
8
9
10
6 hours induced <20 6 hours induced >30 25 hours induced <20 25 hours induced >30
Average
number
of
ND10
Experimental Condition
Average Number of ND10 vs Experimental Condition
*** = p<0.001, >30 signifies high expression and <20 signifies
low expression. Data was statistically analyzed via T-tests. Bars are
standard error lines.
As shown to the right:
• There is no
significance between
low and high
expression of the 6
hours induced
group.
• There is high
significance at the
p<0.001 level
between the high
and low expression
25 hours induced
group.

19. Conclusions
• Lack of aggregation of mutated PML I with ND10.
• As hypothesized, the lack of the oligomerization abilities of mycPML I L73E
resulted in the inability to regularly colocalize with ND10 complexes
• Decreased levels of ND10 with high levels of PML I
mutant.
• As we saw in the bar graph on slide 18, high expression levels of mycPML I L73E
resulted in the disruption of ND10 and an overall decrease in ND10 levels. This
means that the mutated PML I must disrupt the interactions of the component
proteins.

20. Conclusions
• Time dependent effect on ND10.
• Also related to the bar graph on slide 18, we can see that only the 25-hour
group had a decrease in ND10 levels at high expression levels while the 6-hour
group did not have a significant decrease, so there could be a time dependent
effect caused by mycPML I L73E.
• Dynamic interaction of PML I L73E with ND10.
• The timely decrease of ND10 levels explained in the conclusion above, points
towards the fact that PML I L73E is interacting dynamically with the ND10
complex.

21. Acknowledgments
I thank…
Undergraduate student Fizzah Hussain for generating the TetOn Hep2 cell line used
for this research.
Dr. Haidong Gu for providing confocal microscopy images for analysis and as the
mentor of my project.
National Institute of Allergy and Infectious Diseases, grant number R01AI118992 for
providing the funds for the project.