S L I D E 1

Expression of Late Regulators in the Epstein-Barr Virus

Newlyn Joseph

El-Guindy Lab

August 21, 2014

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.

S L I D E 3

Latent State Lytic cycleReactivation

Very Early StageZEBRA & Rta (transcription activators)

Early StageViral Replication Proteins

DNA ReplicationZEBRA (Origin binding protein)

Late StageViral capsid proteins

Packaging& Egress

EBV-infected cell

Dependent on replication

BGLF3BGLF4“BGLF4 and BGLF3 are both

indispensable for late gene expression.”

EBV Life Cycle

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.

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.

S L I D E 6

The General Procedure

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.

S L I D E 8

G3 Elution Results

S L I D E 9

SDS-PAGE Nickel Column Purification Products

Increasing amount of Imidazole

150100

75

50

37

25

20

1510

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.

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.

S L I D E 12

Creating BGLF4(K102I)

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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)

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.

S L I D E 15

Western Blot Results (I-125)

EA-D

FLAG

ZEBRA

FR3

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.

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