This document summarizes research on two proteins involved in late gene expression of Epstein-Barr virus (EBV): BGLF3 and BGLF4. BGLF3 regulates late gene expression but its function was unknown. It was expressed in E. coli and purified using nickel column chromatography. BGLF4 is the only kinase encoded by EBV and phosphorylates proteins to trigger late gene expression. A mutant, inactive version of BGLF4 (BGLF4(K102I)) was created and shown to inhibit expression of a late protein (FR3) but not an early protein (EA-D). This mutant can be used to study BGLF4 protein-protein interactions and

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