Poster design workshop for CUROP students

1. Cardiff Undergraduate Research Opportunities
Programme (CUROP):
Poster Design Workshop
4th September 2014
Dr Nathan Roberts
Dr Iain Mossman
CUROP Co-ordinators

2. CUROP Poster Session
• 17 October 2014
• Main Building, VJ Gallery (foyer)
• Two sessions, either side of lunch

3. Poster Design Tips and Principles
Why posters?
• Help you get your work across in a very
concise way
• Common dissemination tool in many
disciplines – a good skill to have
• A research output that we can use

4. Poster Design Tips and Principles
The poster session
• A busy, noisy room with up to 50 other posters
• Specialist and non-specialist viewers
• People look at posters for an average of 90
seconds. May decide in 10 seconds how much they
want to engage at all
• Make it visually interesting and easy to understand
at a glance

5. Poster Design Tips and Principles
• Powerpoint is often the easiest option
– Design Tab, Page Setup – customise the size of
the poster
• A2 = 59.4cm x 42cm
• A1 = 84.1cm x 59.4cm
• A0 = 118.9cm x 84.1cm
– In most instances landscape is better than
portrait.
• Other options for design, include Adobe
Illustrator/Photoshop and InDesign – but require
software and know-how

6. Poster Design Tips and Principles
• No matter what size you choose – no more
than 1000 words!
• Fonts:
– Sans Serif fonts (Arial, Helvetica, Calibri)
are easier to read, and should be used for
headings
– Serif fonts (Times New Roman etc.) are
fine for body text.
– 22 point font size is a minimum.
• Instead of using words, think of ways in
which you can use photographs, cartoons or
illustrations to illustrate your concept.

7. Poster Design Tips and Principles
• Where to print?
– University: Graphic Services (Bute),
Media Resources (Heath Park)
– Print Centre, Students Union. Use code
CUROP2014 for discount

8. Poster Design Tips and Principles
Elements of the poster
Title

9. Poster Design Tips and Principles
Elements of the poster
Title
Introduction
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Conclusion
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah

10. Poster Design Tips and Principles
Elements of the poster
Title
Introduction
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Conclusion
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Methods
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Findings
Images
Graphs
Acknowledgements, references, contact details

11. Poster Design Tips and Principles
Elements of the poster
Title
Introduction
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Conclusion
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Methods
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Findings
Images
Graphs
Acknowledgements, references, contact details
Include CUROP!

12. Poster Design Tips and Principles
Colours and backgrounds
Title
Introduction
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Conclusion
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Methods
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Findings
Images
Graphs
Acknowledgements, references, contact details
Include CUROP!

13. Poster Design Tips and Principles
Colours and backgrounds
Title
Introduction
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Conclusion
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Methods
Blah blah Blah blah
Blah blah Blah
blahBlah blah Blah
blah Blah blah Blah
Findings
Images
Graphs
Acknowledgements, references, contact details
Include CUROP!

14. Example Posters

17. Exercise
• In groups, think about what is good
and bad about the design of some
example posters.
• Be ready to share an example of
one good thing and one bad thing
about the poster!

18. Introduction Method
• 44 sites across upland Wales (Fig. 3)
sampled
• Water-PAM (Walz, Germany) used to take
approx. 60 readings at each site
• Readings calibrated using a light curve
that was obtained from each site
• Water samples collected for DNA and
nitrate analysis.
• Habitat survey conducted
• Analysis of water chemistry
(pH,temperature, conductivity and % O2)
using a portable multiparameter meter
(HANNA instruments, USA) for further
analysis of nitrates concentration in
stream water.
Results, conclusion and Future Directions
• Establish nitrate concentrations in rivers and streams across upland Wales.
• Practical implications include evaluating the effect of pesticide run off from farm land
• Future work would involve continued assessment on a biannual basis. Developing biofilm as a important early bio-indicator of water
quality. Providing vital information to reduce and predict pesticide run off.
• Thus reducing the harmful effect of pesticide on a river biofilm(4).
• Reducing this negative effect suggest that that biodiversity and the economically valuable ecosystem services provided by rivers and
streams , which are dependent on biofilm and correct nitrate levels can be sustainably maintained.
Biofilms are made up from a
cluster of bacteria, algae,
cyanobacteria and protozoa,
which are held in a
polysaccharide matrix(1).
Biofilms , as well as having
medical implications are a
essential component to the
river ecosystem. The levels of
biofilm have been shown to be
affected by water
temperature, , nutrient levels
and pesticides(2). Through
analysis of biofilm levels we
are able to predict the
corresponding levels of algae
biomass, functional
chlorophyll A and nitrates.
Such factors are key to
British river biodiversity and to
the maintenance of ecosystem
services, a industry which is
currently valued at £200
billion (3).
References
1) Diaz Villanueva, V., Font, J., Schwartz, T., Romani, A.M. (2011). Biofilm formation at warming temperature: accelerationof microbialcolonization and microbialinteractive effects. Biofouling 27:59-71 •2)Proia, L., Osorio, V., Soley, S., Köck-Schulmeyer, M., Pérez, S., Barceló, D., Romaní,
A.M., Sabater, S.(2013). Effects of pesticides and pharmaceuticalson biofilms in a highly impacted river. EnvironmentalPollution 178:220-8• 3) http://nerc-duress.org/?page_id=40 [Accessed, August 2013.]•4) Hayashi, S., Jang, J.E., Itoh, K., Suyama, K., Yamamoto, H. 2011.
Construction of river model biofilm for assessing pesticide effects. Arch Environ Contam Toxicol. 60:44-56.
Fig. 1) The water –PAM used that took one of
5,500 readings taken during this study.
Assessing the role of biofilm in nitrate regulation in
streams and rivers in upland Wales using fluorescence
analysis
Fig. 3) Map of Wales with the 44 sites (black points)
sampled.
Fig 4) Looking downstream
form our site on the Honddu
river, Powys
Fig. 2) Rhys Luckwell carrying out
water chemistry analysis on the
Conway river, near Snowden in
upland Wales.

19. ANALYSIS OF CD112 CELL SURFACE EXPRESSION MODULATION BY HUMAN
CYTOMEGALOVIRUS
STEALTHOGEN
Department of Infection, Immunity and Biochemistry
Cardiff University School of Medicine
Cardiff University
Health Park
Cardiff
CF14 4XN
Year 4 MBBCh
ABSTRACT
IMMUNE EVASION BY MODULATION METHOD
CONCLUSIONS & FURTHER STUDYΔUS1-11 / ΔUL13-20 ENCODE GENE
PRODUCTS MODULATING CD112
EXPRESSION
Human Cytomegalovirus (Human Herpesvirus 5, HHV5, HCMV) is a clinicallyrelevant pathogen [1]. As the leading cause of infectious congenital birth defects and responsible for significantmorbidityin immunocompromised patients (HIV/AIDS, post-transplant), it has been designated a target of highest priority for vaccine
research by the US Institute of Medicine [2]. HCMV encodes multipleimmune evasion functions and relies heavily on these for virulence., including downregulationof cell surface membraneligands for NK Cell receptors [3]. Recently, UL141, an HCMV gene putativelyinvolved in immune evasion function, was shown to
downregulate NK cell ligands CD155 and CD112 from cell surface membranes, attenuating NK Cell mediated responses. However, UL141 alone was unable to reproduce functional downregulation of CD112, suggesting co-operationwith other viral functions is required [4]. This investigationsought to identifycandidate genes
for co-operativefunction through screening a gene block deletion mutant library for restoring downregulationfunction [5, 6]. Gene loci UL13-20 and US1-11 were identifiedas encoding functions involved in cell surface CD112 modulation. Moreover, US2 and US11 showed trend for specific role in this capacity.This study
highlights these genes for further focussed research.
The Human Cytomegalovirus genome (~236kbps) encodes an estimated 164 genes with protein products. Significantly, only 45 are essential for replication in
fibroblast culture, suggesting the remainderhave alternativefunction. Indeed, many have been associated with immune evasion [3].
HCMV UL141, a gene located in the ULb/b’ cassette has been shown to downregulate cell surface expression of CD155 (Polio Virus Receptor, Nectin-Like
Molecule 5) and CD112 (Herpes Entry MediatorB, Nectin-2). Both are ligands for the activatingNK cell receptor CD226 (DNAM-1). Reduction in CD155 /
CD112 cell surface expression results in fewer interactions with CD226, thereby inhibitingNK cell primingand reducing NK cell mediatedcytotoxicity. This
promotes virus survival and facilitates further replication [2, 3, 4].
Experimentation showed HCMV ΔUL141 (derived from strain Merlin, Cardiff)was associated with enhanced susceptibilityto NK cell cytolysis. However, when
UL141 was re-introducedas a transgene, a resistant phenotype was rescued, suggesting an immune evasion function for this gene. Subsequently, UL141 was
found to be associated with downregulationof CD155 and CD112, the former targeted for proteasomal degradation. Interestingly,when expressed alone in
cells, neither UL141 or the protein product (gpUL141) were able to downregulate CD112 cell surface expression, although downregulation of CD155 was
preserved. This suggested UL141 required co-operationwith further HCMV genes (present in the HCMV ΔUL141 clone) to modify the expression of CD112 [4].
The aim of this investigationwas to use a library of HCMV block deletion mutants (knockout for specific gene loci), to screen for modulation of CD112 cell
surface expression. Once identified, the individualgenes involved would be determined by rescuing their function in cells currently infected with block
deletion mutants. Using a model of co-infection, transgenes were deliveredby replicationdeficientadenovirus vectors and CD112 expression analysed.
Human Cytomegalovirus Block Deletion Mutant Library Screen
HCMV block deletion mutants were obtained from a pre-constructedlibrary. Human Fibroblasts (HF) were infected with block deletion mutants at multiplicity
of infection= 10 (MOI=10)for 2 hours, then incubated at standard conditions for 48 hours. Cells were then harvested and analysed by flow cytometry.
Immunohistochemistrystaining was performedas described previously [1]. 1o Antibodies: Control Ig (mIgG),Mouse IgG 1:500 (Santa Cruz, SC-2025); Anti-CD155 Mouse D171 IgG 1:500 (Abcam,AB3142);Anti-CD112
Mouse IgG2b 1:50 (Santa Cruz, SC65333); Anti-MHC Class I W632 IgG2a 1:10000(Serotec, MCA81EL). 2o Antibody: Goat Anti-Mouse Fab fragments AF647 (Molecular Probes, A21237).
Flow Cytometry
Flow cytometrywas performed using a BD Accuri C6 flow cytometer [1].
Optimisation of Co-Infection Protocol
Recombinantadenovirus vectors expressing green fluorescent protein (RAd-GFP)and RAd-Control were prepared as previously described. For the timecourse
assay, Human Foteal Foreskin Fibroblasts preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd’s (MOI=5)for 2
hours and incubated under standard conditions for 24 hours and 48 hours, then analysed using fluorescence microscopy (Lecia DMIRBE microscope) and
western blotting. For virus titration,HFFF-CARs were infected with RAd’s at MOI=5 and MOI=10 for 2 hours and incubated for 48 hours under standard
conditions, then analysed by spectrophotometry(BMG Omega FluStar)and western blotting. For co-infectionoptimisation,HF’s were co-infectedat the same
time with HCMV (MOI=50)and RAd’s (MOI=10)for 2 hours, then incubated under standard conditions for 48 hours. Cells were analysed by flow cytometry [1,
3]. WB antibodies: Control Anti-Actin Rabbit IgG + HPO 1:10000 (Sigma, A2066);1o Anti-GFP Rabbit IgG 1:5000(Santa Cruz, SC-8334);2o GoatAnti-Rabbit IgG + HPO 1:10000(BioRad,170-6515).
US1 – US11 Screen
Human Fibroblasts were co-infectedwith HCMV Deletion Mutant (MOI=10)and RAds-US1-US11 (MOI=50)for 2 hours, then incubated at standard conditions
for 48 hours. Cells were harvested and analysed by flow cytometryas per Block Deletion Mutant Library Screen.
CONFIRMATION OF TRANSGENE EXPRESSION IN MODEL
OF CO-INFECTION
US2 AND US11 MODULATE CELL SURFACE
CD112 EXPRESSION IN HUMAN FIBROBLASTS
Previous study showed HCMV UL141 was unable to modulate cell
surface expression of CD112 when expressed alone in cell culture. We
hypothesised a co-operativeinteractionby UL141 with other viral
protein(s) was required for functional downregulationof CD112. To
screen for putative interactingfunction, we analysed cell surface CD112
expression in Human Fibroblasts infected with HCMV containing block
deletions of known gene loci (excludingUL141). Positive results were
considered a greater than 50% increase in CD112 expression relative to
control.
Two gene loci were associated with significantlyincreased CD112 cell
surface expression, corresponding to US1-11 and UL13-20 (relative
staining signal increase of 502% and 168% vs. control respectively)
(Figure.1.). This suggested HCMV encoded at least two further products
involved in cell surface CD112 modulation.
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
SSC
FSC
A
FSC
FL1
D
CELLCOUNT
B
CD155
FL4
cIg
HCMV
Mock
C
CD112
FL4
cIg
HCMV
Mock
CD155
CELLCOUNT
FL4
H
cIg
HCMV
ΔUL13-20
ΔUS1-11
CD112
FL4
I
cIg HCMV
ΔUL13-20
ΔUS1-11
MHC Class I
FL4
J
cIg
HCMV
ΔUL13-20
ΔUS1-11
Figure.1. ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface expression relative to HCMV control. Human Fibroblasts (HF’s) were
infected with HCMV block deletion mutants (MOI=10) and analysed by flow cytometry 48h p.i. A: Cells were gated for HCMV infection. B, C: HCMV
control infected cells downregulated CD155 and CD112 relative to mock infected cells. D: GFP fluorescence was used to select cells infected with
HCMV block deletion mutants, since these viruses encode a gene for GFP. E-J: ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface
staining relative to HCMV control. Modulation of CD155 did not reach the 150% threshold for any HCMV block deletion mutant. Isotype matched
Mouse IgG (cIg) was used for staining control.
RELATIVEEXPRESSION(%)
CD155 CD112cIg
E F G
CELLCOUNTCELLCOUNT
FSC
SSCFL1
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg cIg cIg
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
K L M N
O P Q R
24h48h
Phase
Contrast
FL1 Merge
A B C
D E F
CELLCOUNT
FL1
H
MFI(LOG10)
RAdGFP
RAdCtrl.
MOI=10
MOI=10
MOI=5
MOI=5
IMock
HCMV
RAd GFP (5)
RAd GFP
(10)
G
J
GFP
Figure.3. HF’s were co-infected with RAds-US1-US11 (MOI=50) and HCMV deletion mutant ΔUS1-11 (MOI=10) for 2h and analysed by flow
cytometry 48h p.i. A: Live cells were selected and B-C: HCMV infection validated by downregulation of CD155 and CD112. D: Cells co-infected with
RAd-US1-US11 were selected by gating for GFP expression. E-J: Two RAds, corresponding to US2 and US11 showed trend towards recovering
CD112 downregulation function of HCMV, reaching significance threshold of 80% relative staining vs. control HCMV. CD155 expression showed no
similar trend. Isotype matched Mouse IgG (cIg) was used as staining control.
CD155 CD112 MHC Class I
CD155 CD112
CD155 CD112cIg
RELATIVEEXPRESSION(%)
DB C
H I J
A
FSC
SSC
CELLCOUNT
FL4 FL4
FL1
FSC
CELLCOUNT
FL4 FL4 FL4
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
80%
100%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
E F G
cIg cIg
cIg cIg cIg
Mock Mock
HCMV HCMV HCMV
RAd + HCMV
HCMV
RAd + HCMV
HCMV
US2
US11
US2
US11
US2
US11
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
SSC
FSC
A
FSC
FL1
D
CELLCOUNT
B
CD155
FL4
cIg
HCMV
Mock
C
CD112
FL4
cIg
HCMV
Mock
CD155
CELLCOUNT
FL4
H
cIg
HCMV
ΔUL13-20
ΔUS1-11
CD112
FL4
I
cIg HCMV
ΔUL13-20
ΔUS1-11
MHC Class I
FL4
J
cIg
HCMV
ΔUL13-20
ΔUS1-11
Figure.1. ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface expression relative to HCMV control. Human Fibroblasts (HF’s) were
infected with HCMV block deletion mutants (MOI=10) and analysed by flow cytometry 48h p.i. A: Cells were gated for HCMV infection. B, C: HCMV
control infected cells downregulated CD155 and CD112 relative to mock infected cells. D: GFP fluorescence was used to select cells infected with
HCMV block deletion mutants, since these viruses encode a gene for GFP. E-J: ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface
staining relative to HCMV control. Modulation of CD155 did not reach the 150% threshold for any HCMV block deletion mutant. Isotype matched
Mouse IgG (cIg) was used for staining control.
RELATIVEEXPRESSION(%)
CD155 CD112cIg
E F G
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
SSC
FSC
A
FSC
FL1
D
CELLCOUNT
B
CD155
FL4
cIg
HCMV
Mock
C
CD112
FL4
cIg
HCMV
Mock
CD155
CELLCOUNT
FL4
H
cIg
HCMV
ΔUL13-20
ΔUS1-11
CD112
FL4
I
cIg HCMV
ΔUL13-20
ΔUS1-11
MHC Class I
FL4
J
cIg
HCMV
ΔUL13-20
ΔUS1-11
Figure.1. ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface expression relative to HCMV control. Human Fibroblasts (HF’s) were
infected with HCMV block deletion mutants (MOI=10) and analysed by flow cytometry 48h p.i. A: Cells were gated for HCMV infection. B, C: HCMV
control infected cells downregulated CD155 and CD112 relative to mock infected cells. D: GFP fluorescence was used to select cells infected with
HCMV block deletion mutants, since these viruses encode a gene for GFP. E-J: ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface
staining relative to HCMV control. Modulation of CD155 did not reach the 150% threshold for any HCMV block deletion mutant. Isotype matched
Mouse IgG (cIg) was used for staining control.
RELATIVEEXPRESSION(%)
CD155 CD112cIg
E F G
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
HCMV
UL2-11
UL13-20
UL22A-25
US12-17
US27-28
US29-34A
RL1-6
RL10-UL1
US1-11
150%
100%
SSC
FSC
A
FSC
FL1
D
CELLCOUNT
B
CD155
FL4
cIg
HCMV
Mock
C
CD112
FL4
cIg
HCMV
Mock
CD155
CELLCOUNT
FL4
H
cIg
HCMV
ΔUL13-20
ΔUS1-11
CD112
FL4
I
cIg HCMV
ΔUL13-20
ΔUS1-11
MHC Class I
FL4
J
cIg
HCMV
ΔUL13-20
ΔUS1-11
Figure.1. ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface expression relative to HCMV control. Human Fibroblasts (HF’s) were
infected with HCMV block deletion mutants (MOI=10) and analysed by flow cytometry 48h p.i. A: Cells were gated for HCMV infection. B, C: HCMV
control infected cells downregulated CD155 and CD112 relative to mock infected cells. D: GFP fluorescence was used to select cells infected with
HCMV block deletion mutants, since these viruses encode a gene for GFP. E-J: ΔUL13-20 and ΔUS1-11 mediated recovery of CD112 cell surface
staining relative to HCMV control. Modulation of CD155 did not reach the 150% threshold for any HCMV block deletion mutant. Isotype matched
Mouse IgG (cIg) was used for staining control.
RELATIVEEXPRESSION(%)
CD155 CD112cIg
E F G
Figure.2. RAd and HCMV co-infection proves viable technique for assessment of transgene expression and function. Human Fibroblasts
preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd-GFP for 2h. RAd-GFP mediated GFP
expression visualised by fluorescence microscopy at A-C: 24h p.i. and D-F: 48h p.i. G: Total GFP expression was similar at both 24h and 48h p.i.
as assessed by western blot. H-J: Similar GFP expression and fluorescence intensity was detected at RAd-GFP MOI=5 and MOI=10. HF’s were co-
infected with RAd-GFP (MOI=25, MOI=50) and HCMV (strain Merlin) for 2h and analysed by flow cytometry 48h p.i. K: Cells were gated and L-N:
HCMV infection validated by downregulation of CD155, CD112 and MHC Class I. O: Cells were gated for GFP expression and P-R: importantly,
RAd-GFP infection did not appear to substantially alter expression of stained cell surface proteins. S-U: Confirmation of RAd-GFP and HCMV
infection was achieved by observation that GFP expressing cells has simultaneously downregulated cell surface markers of HCMV infection.
CD155 CD112 MHC Class I
CELLCOUNTCELLCOUNT
FL4 FL4 FL4
FSC
SSC
FSC
FL1
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg cIg cIg
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
K L M N
O P Q R
24h48h
Phase
Contrast
FL1 Merge
A B C
D E F
CELLCOUNT
FL1
H
MFI(LOG10)
RAdGFP
RAdCtrl.
MOI=10
MOI=10
MOI=5
MOI=5
IMock
HCMV
RAd GFP (5)
RAd GFP
(10)
G
J
GFP
FL1
FL4
GFP
CD155
FL1
FL4
GFP
CD112
FL4
FL1
GFP
MHCClassI
S T UMock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Figure.2. RAd and HCMV co-infection proves viable technique for assessment of transgene expression and function. Human Fibroblasts
preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd-GFP for 2h. RAd-GFP mediated GFP
expression visualised by fluorescence microscopy at A-C: 24h p.i. and D-F: 48h p.i. G: Total GFP expression was similar at both 24h and 48h p.i.
as assessed by western blot. H-J: Similar GFP expression and fluorescence intensity was detected at RAd-GFP MOI=5 and MOI=10. HF’s were co-
infected with RAd-GFP (MOI=25, MOI=50) and HCMV (strain Merlin) for 2h and analysed by flow cytometry 48h p.i. K: Cells were gated and L-N:
HCMV infection validated by downregulation of CD155, CD112 and MHC Class I. O: Cells were gated for GFP expression and P-R: importantly,
RAd-GFP infection did not appear to substantially alter expression of stained cell surface proteins. S-U: Confirmation of RAd-GFP and HCMV
infection was achieved by observation that GFP expressing cells has simultaneously downregulated cell surface markers of HCMV infection.
CD155 CD112 MHC Class I
CELLCOUNTCELLCOUNT
FL4 FL4 FL4
FSC
SSC
FSC
FL1
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg cIg cIg
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
K L M N
O P Q R
24h48h
Phase
Contrast
FL1 Merge
A B C
D E F
CELLCOUNT
FL1
H
MFI(LOG10)
RAdGFP
RAdCtrl.
MOI=10
MOI=10
MOI=5
MOI=5
IMock
HCMV
RAd GFP (5)
RAd GFP
(10)
G
J
GFP
FL1
FL4
GFP
CD155
FL1
FL4
GFP
CD112
FL4
FL1
GFP
MHCClassI
S T UMock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Figure.2. RAd and HCMV co-infection proves viable technique for assessment of transgene expression and function. Human Fibroblasts
preferentially expressing the Coxsackie and Adenovirus Receptor (HFFF-CAR) were infected with RAd-GFP for 2h. RAd-GFP mediated GFP
expression visualised by fluorescence microscopy at A-C: 24h p.i. and D-F: 48h p.i. G: Total GFP expression was similar at both 24h and 48h p.i.
as assessed by western blot. H-J: Similar GFP expression and fluorescence intensity was detected at RAd-GFP MOI=5 and MOI=10. HF’s were co-
infected with RAd-GFP (MOI=25, MOI=50) and HCMV (strain Merlin) for 2h and analysed by flow cytometry 48h p.i. K: Cells were gated and L-N:
HCMV infection validated by downregulation of CD155, CD112 and MHC Class I. O: Cells were gated for GFP expression and P-R: importantly,
RAd-GFP infection did not appear to substantially alter expression of stained cell surface proteins. S-U: Confirmation of RAd-GFP and HCMV
infection was achieved by observation that GFP expressing cells has simultaneously downregulated cell surface markers of HCMV infection.
CD155 CD112 MHC Class I
CELLCOUNTCELLCOUNT
FL4 FL4 FL4
FSC
SSC
FSC
FL1
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg
HCMV
Mock
cIg cIg cIg
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
RAd-GFP
(25)
RAd-GFP
(50)
K L M N
O P Q R
24h48h
Phase
Contrast
FL1 Merge
A B C
D E F
CELLCOUNT
FL1
H
MFI(LOG10)
RAdGFP
RAdCtrl.
MOI=10
MOI=10
MOI=5
MOI=5
IMock
HCMV
RAd GFP (5)
RAd GFP
(10)
G
J
GFP
FL1
FL4
GFP
CD155
FL1
FL4
GFP
CD112
FL4
FL1
GFP
MHCClassI
S T UMock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Mock
HCMV
RAd-GFP
(25)
RAd-GFP
(50)
Figure.3. HF’s were co-infected with RAds-US1-US11 (MOI=50) and HCMV deletion mutant ΔUS1-11 (MOI=10) for 2h and analysed by flow
cytometry 48h p.i. A: Live cells were selected and B-C: HCMV infection validated by downregulation of CD155 and CD112. D: Cells co-infected with
RAd-US1-US11 were selected by gating for GFP expression. E-J: Two RAds, corresponding to US2 and US11 showed trend towards recovering
CD112 downregulation function of HCMV, reaching significance threshold of 80% relative staining vs. control HCMV. CD155 expression showed no
similar trend. Isotype matched Mouse IgG (cIg) was used as staining control.
CD155 CD112 MHC Class I
CD155 CD112
CD155 CD112cIg
RELATIVEEXPRESSION(%)
DB C
H I J
A
FSC
SSC
CELLCOUNT
FL4 FL4
FL1
FSC
CELLCOUNT
FL4 FL4 FL4
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
80%
100%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
E F G
cIg cIg
cIg cIg cIg
Mock Mock
HCMV HCMV HCMV
RAd + HCMV
HCMV
RAd + HCMV
HCMV
US2
US11
US2
US11
US2
US11
Figure.3. HF’s were co-infected with RAds-US1-US11 (MOI=50) and HCMV deletion mutant ΔUS1-11 (MOI=10) for 2h and analysed by flow
cytometry 48h p.i. A: Live cells were selected and B-C: HCMV infection validated by downregulation of CD155 and CD112. D: Cells co-infected with
RAd-US1-US11 were selected by gating for GFP expression. E-J: Two RAds, corresponding to US2 and US11 showed trend towards recovering
CD112 downregulation function of HCMV, reaching significance threshold of 80% relative staining vs. control HCMV. CD155 expression showed no
similar trend. Isotype matched Mouse IgG (cIg) was used as staining control.
CD155 CD112 MHC Class I
CD155 CD112
CD155 CD112cIg
RELATIVEEXPRESSION(%)
DB C
H I J
A
FSC
SSC
CELLCOUNT
FL4 FL4
FL1
FSC
CELLCOUNT
FL4 FL4 FL4
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
80%
100%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
E F G
cIg cIg
cIg cIg cIg
Mock Mock
HCMV HCMV HCMV
RAd + HCMV
HCMV
RAd + HCMV
HCMV
US2
US11
US2
US11
US2
US11
Figure.3. HF’s were co-infected with RAds-US1-US11 (MOI=50) and HCMV deletion mutant ΔUS1-11 (MOI=10) for 2h and analysed by flow
cytometry 48h p.i. A: Live cells were selected and B-C: HCMV infection validated by downregulation of CD155 and CD112. D: Cells co-infected with
RAd-US1-US11 were selected by gating for GFP expression. E-J: Two RAds, corresponding to US2 and US11 showed trend towards recovering
CD112 downregulation function of HCMV, reaching significance threshold of 80% relative staining vs. control HCMV. CD155 expression showed no
similar trend. Isotype matched Mouse IgG (cIg) was used as staining control.
CD155 CD112 MHC Class I
CD155 CD112
CD155 CD112cIg
RELATIVEEXPRESSION(%)
DB C
H I J
A
FSC
SSC
CELLCOUNT
FL4 FL4
FL1
FSC
CELLCOUNT
FL4 FL4 FL4
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11100%
80%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
80%
100%
US1-11
RAdControl
US2
US3
US6
US7
US8
US9
US10
US11
100%
80%
E F G
cIg cIg
cIg cIg cIg
Mock Mock
HCMV HCMV HCMV
RAd + HCMV
HCMV
RAd + HCMV
HCMV
US2
US11
US2
US11
US2
US11
To determinewhich genes within the block modulate of cell surface CD112 expression
we proposed a co-infectiontechnique, whereby fibroblasts infected with HCMV block
deletion mutants would also be infected with a RAd encoding a single deleted gene. This
transgene would then be expressed in target cells; rescuing function and permitting
analysis of CD112 expression.
Optimisationof technique was first necessary to define conditions for best transgene
expression. RAd-GFP, an adenovirus encoding green fluorescent protein was used for
this calibrationdue to ease of protein expression analysis. A time course was performed
to measure transgene expression over time, no difference was observed between 24h
and 48h post infectionusing fluorescence microscopy and western blotting. Titration to
select multiplicityof infectionindicated transgene expression was similar at RAd MOI=5
and MOI=10 when assessed by spectrophotometryand western blotting. Optimalco-
infectionconditions were determinedas 48h incubationat MOI=50 (accountingfor
reduced infectivityof Human Fibroblasts relativeto HFFF-CAR). Analysis of cells exposed
to both HCMV and RAd-GFP showed downregulationof cell surface markers typical of
HCMV infection(CD155, MHC-Class I) with concomitantfluorescence in green channel,
suggesting cells had been co-infected.
Having identifiedthe US1-11 gene loci as putativelyencoding function(s)
involved in modulatingcell surface CD112 expression, the validated co-
infectiontechnique was utilised to screen US1 – US11 individuallyfor
functional modulationof cell surface CD112 expression.
Significantdownregulationof cell surface CD112 expression was defined
as cell surface CD112 staining less than or equal to 80% relative to
control. Cells co-infectedwith RAd’s US2 and US11 showed a trend
suggestive of cell surface CD112 downregulationusing this threshold (73%
and 76% respectively). This indicated US2 and US11 encode functions
involved in cell surface CD112 modulation.No significant modulationof
cell surface CD155 or MHC Class-1 was observed, thus the effect was
likely specific to CD112.
Screening HCMV block deletion mutants highlighted two loci associated with
increased cell surface CD112 expression when removed (US1 – 11 and UL13 -
20). To analyse individualgenes within these blocks, a co-infectiontechnique
was developed and optimised.
Previous research has shown UL141 functionally downregulates cell surface
CD112, although this effect is not seen when expressed alone intracellularly,
suggesting co-operation with other HCMV proteins is required [4]. This
investigationidentifiedUS2, US11 and an unknown gene in UL13-20 (ULX) as
having potentialrole in UL141 complementation.
UL141 downregulates CD155 by targetingthe protein for proteasomal
degredation. It is logicalto assume a similar mechanism exists for CD112 [4].
Indeed, US2 and US11 have also been demonstrated to target MHC Class-1 for
proteasomal degradation, making this function even more likely [5, 6].
Further focussed research should aim to investigatethe function of US2 and
US11. Proteasome inhibition assays should be performed to analyse for
reversal of downregulation in the presence of an inhibitor(e.g. MG132).
Reversal of downregulationwould strongly suggest a functional degredation
of CD112, corroborating previous findings US2 and US11 modulate MHC Class-
1 via this pathway [1, 4, 7, 8, 9].
Additionalconfirmationcould be obtained through conjugation of US2 / US11
with a V5-Strep tag to assess subcellularlocalisation. Sequestration of US2 /
US11 within the endoplasmic reticulum would imply early modificationof
target proteins during post-translationalprocessing [6].
Furthermore,complementationassays using combinations of UL141 and US2 /
US11 / ULX should prove that these proteins act in concert to modulate cell
surface CD112 expression.
References
1. Tomasec, P. Wang, E. Davison, A. Vojtesek, B. Armstrong, M. Griffin, C. McSharry, B. Morris, R. Llewellyn-Lacey, S. Rickards, C. Nomoto, A. Sinzger, C. Wilkinson, G. 2005. “Downregulation of Natural Killer Cell-Activating Ligand CD155 by Human Cytomgealovirus UL141”, Nature Immunology, 6(2), Pg. 181 – 188.
2. Stanton, R. Baluchova, K. Dargan, D. Cinningham, C. Sheehy, O. Seirafian, S. McSharry, B. Neale, M. Davies, J. Tomasec, P. Davison, A. Wilkinson, G. 2010. “Reconstruction of theCompleteHuman CytomeglovirusGenomein a BAC Reveals RL13 to bea Potent Inhibitor of Replication”, Journal of Clinical Investigation, 120(9), Pg. 3191 –3208.
3. Wilkinson, G. Tomasec, P. Stanton, R. Armstrong, M. Prod’homme, V. Aicheler, R. McSharry, B. Rickards, C. Cochrane, D. Llewellyn-Lacey, S. Wang, E. Griffin, C. Davison, A. 2008. “Modulation of Natural Killer Cells by Human Cytomegalovirus”, Journal of Clinical Virology, 41, Pg. 206 –212.
4. Prod’homme, V. Sugrue, D. Stanton, R. Nomoto, A. davies, J. Rickards, C. Cochrane, D. Moore, M. Wilkinson, G. Tomasec, P. 2010. “Human CytomegalovirusUL141 Promotes Efficient Downregulation of theNatural Killer Cell Activating Ligand CD112”, Journal of General Virology, 91, Pg. 2034 –2039.
5. Sugrue, D. Doctor of PhilosophyThesis. 2012. “Modulation of Natural Killer Cell Responseby Human Cytomegalovirus”.
6. Seirafian, S. Doctor of Philosophy Thesis. 2013. “An Analysis of Human CytomegalovirusGeneUsage”.
7. Smith, W. Tomasec, P. Aichleler, R. Loewendorf, A. Nemcovicova, I. Wang, E. Stanton, R. Macauley, M. Norris, P. Willen, L. Ruckova, E. Nomoto, A. Schneider, P. Hahn, G. Zajonc, D. Ware, C. Wilkinson, G. Benedict, C. 2013. “Human CytomegalovirusGlycoprotein UL141Targets theTRAIL Death Receptors to Thwart Host InnateAntiviral Defenses”, Cell Host & Microbe, 13, Pg. 324 –335.
8. Stanton, R. McSharry, B. Armstrong, M. Tomasec, P. Wilkinson, G. 2008. “Re-Engineering AdenovrisuVector Systemsto EnableHigh-Throughput Analysesof GeneFunction”, Biotechniques, 45(6), Pg. 659 –668.
9. Nemcovicova, I. Benedict, C. Zajonc, D. 2013. “Structureof Human Cytomegalovirus UL141Binding to TRAIL-R2 Reveals Novel, Non-Canonical Death Receptor Interactions”, PLOS Pathogens, 9(3), Pg. 1 – 14.

20. Talking about your poster
• Non-specialist audience
• Quick summary
• Don’t worry if experiments or projects
‘failed’
• Convey enthusiasm
• Professional development, plans
• Props?

21. Any Questions?
robertsn1@cardiff.ac.uk
mossmanij@cardiff.ac.uk
@cardiffedu

22. Useful Online Resources
• Poster Templates
– http://www.posterpresentations.com/html/free_poster_templ
ates.html
• Designing academic posters and presenting them at
conferences
– http://colinpurrington.com/tips/academic/posterdesign
• Print Centre Cardiff (www.printcentrecardiff.com)
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