1. S L I D E 1
Expression of Late Regulators in
the Epstein-Barr Virus
Newlyn Joseph
El-Guindy Lab
August 21, 2014

2. S L I D E 2
Epstein-Barr Virus (EBV/HHV-4)
• A very common virus in humans, responsible for infectious
mononucleosis.
• It has also been associated with various forms of cancer including
several lymphomas.
• Early infections are asymptomatic.
• Genome consists of about 85 genes.

3. S L I D E 3
Latent State Lytic cycle
Reactivation
Very Early Stage
ZEBRA & Rta (transcription activators)
Early Stage
Viral Replication Proteins
DNA Replication
ZEBRA (Origin binding protein)
Late Stage
Viral capsid proteins
Packaging
& Egress
EBV-infected cell
Dependent on replication
BGLF3
BGLF4“BGLF4 and BGLF3 are both
indispensable for late gene
expression.”
EBV Life Cycle

4. S L I D E 4
Why Study EBV?
• EBV’s only mode of propagation is via lytic replication.
– Studying the lytic cycle gives us insight into the general behavior of
EBV.
• While much is known about ZEBRA and Rta (which play a role in
lytic activation), the mechanisms that trigger late gene expression
need to be further understood.
• There is a near universal presence of EBV in certain tumors.
– Oncolytic therapy is said to assist in the treatment of cancers.

5. S L I D E 5
The BGLF3 Gene
• BGLF3 regulates late gene
expression (viral structural
proteins).
• BGLF3 encodes for a protein of
an unknown function.
• The pEX system was used to
express BGLF3 in E. Coli.

6. S L I D E 6
The General Procedure

7. S L I D E 7
After Expression
• Cells were lysed and the protein extract was suspended in binding
buffer.
• The histidine tag present on the expressed protein allowed for
nickel column affinity chromatography.
• SDS-PAGE was used to view the eluted protein purity.

8. S L I D E 8
G3 Elution Results

9. S L I D E 9
SDS-PAGE Nickel Column Purification Products
Increasing amount of Imidazole
150
100
75
50
37
25
20
15
10

10. S L I D E 10
Final Steps
• The purified BGLF3 protein will then be cleaved of the Histidine
and GST tags.
• An antibody will be generated from the protein.

11. S L I D E 11
The BGLF4 Gene
• BGLF4, the only kinase encoded by EBV, phosphorylates various
proteins, which in turn trigger late gene expression.
• Changing the 102nd amino acid (within the catalytic domain) from
Lysine to Isoleucine yields an inactive kinase.
• This mutant is used to analyze the substrates and phosphorylating
activity of BGLF4.

12. S L I D E 12
Creating BGLF4(K102I)

13. S L I D E 13
Expression of Mutant BGLF4
• After the mutation was confirmed via sequencing, analysis of the
mutant kinase via Western Blot was used to verify inactivity.
• EBV Positive cells (without endogenous BGLF4) were exposed to
the following:
– CMV (empty vector)
– ZEBRA
– ZEBRA + FLAG-BGLF4
– ZEBRA + FLAG-BGLF4(K102I)

14. S L I D E 14
Hypothesis
• EA-D (an early protein) should not be hyper-phosphorylated.
– EA-D is a critical component of EBV DNA Polymerase.
• FR3 (a late protein) expression should be inhibited.

15. S L I D E 15
Western Blot Results (I-125)
EA-D
FLAG
ZEBRA
FR3

16. S L I D E 16
Conclusion
• BGLF4(K102I) is an inactive kinase.
• EA-D is hyper-phosphorylated by BGLF4.
• BGLF4 regulates the expression of the late protein FR3.
• FLAG-BGLF4(K102I) can be used in pull-down experiments to
further study protein-protein interactions.

17. S L I D E 17
Acknowledgements
• El-Guindy Lab
– Dr. Ayman El-Guindy
– Jessica McKenzie
– Kid’s Meeting
• George Miller
• Discovery To Cure
– Dr. Gil Mor
– JoAnn Bilyard