By: Erin HandAdvisor: Dr. Hersperger

CAN THE POXVIRUS PROTEIN E3L RESTORE FULL REPLICATION CAPACITY TO A MUTANT INFLUENZA VIRUS LACKING THE NS1 PROTEIN?

Background• Ectromelia virus (ECTV) also known as “mousepox” • dsDNA in poxvirus family • Wild-type ECTV

Esteban & Buller. Journal of General Virology 2005.

• Influenza virus (Flu) • Contains a genome of

multiple RNA segments (8)

• Wild-type Flu• ΔNS1 Flu

Interferons help to inhibit virus replication

Protein Kinase R (PKR) is induced by interferons

E3L; NS1

normal pathwayfor the

initiation of host cellprotein

translation

Importance• To understand:• the basics of the PKR pathway• PKR is targeted by many viruses• Is the protein that targets PKR from the Flu generally the same as the protein that targets the PKR from the ECTV?

•More generally, is this protein (E3L/NS1) universal to viruses that target the PKR pathway or are specific to the particular virus from which they were derived?

•more about the basics of virus biology & how viruses interact with host cells

Flu NS1 Can Compensate for the Loss of E3L

Guerra, S., Abaitua, F., et al.

Part 1: ECTV E3L Expression Plasmid

E3L043 (E3L)ECTV genome

Mammalian Expression plasmid

E3L E3L

black = no transfectionred = E3L plasmid transfection

E3L

E3L Expression Testing

• Quality control- plasmid created is actually making the protein

E3L expressed in cells is functional and activePlain BSC1/ No Vaccinia

Virus (VACV) Plain BSC1 + w.t. VACV

Plain BSC1 + ΔE3L VACV

BSC1 + E3L/ No VACV

BSC1 + E3L + w.t. VACV

BSC1 + E3L + ΔE3L VACV

E3L expressed in cells is functional and active

BSC1 plain cells: BSC1+E3L cells :

Black: w.t. VACVBlue: ΔE3L VACV

Black: w.t. VACVBlue: ΔE3L VACV

Part 2•Determine if E3L can compensate for the absence of NS1 protein during Flu infection

• Virus types:•Wild-type influenza•Has the NS1 gene•Mutated influenza (ΔNS1)•Does not have the NS1 gene

• RK13 Cells (rabbit cells) •No E3L expression• Stable E3L expression

How can we detect cells that are infected with the influenza virus?

RK13 plain no virus RK13 plain + w.t.Flu RK13 plain + DNS1 Flu

RK13+E3L no virus RK13+E3L + w.t. Flu RK13+E3L + DNS1 Flu

0.20%

42.41%

19.90%

0.37%

34.06%

15.56%

uninfected control DNS1 Flu alone (no transfection)

DNS1 Flu + empty plasmid

DNS1 Flu + NS1 plasmid

DNS1 Flu + VACV E3L plasmid

DNS1 Flu + ECTV E3L plasmid

2.31%

2.62%

2.69%

5.82%

2.03%

0.41%

Summary• NS1 can compensate for the loss of E3L in VACV• E3L cannot compensate for the loss of NS1 in Flu• Appears that the NS1 protein plays a specific role for Flu • The protein has other functions for the Flu virus that E3L does

not have for ECTV, which may be why E3L cannot compensate for the loss of NS1 in Flu replication

• Future• Due to the importance of NS1 for the Flu virus, this suggests

that this protein may be useful for antiviral drugs

Acknowledgments • Carolina Lopez Ph. D.

• School of Veterinary Medicine University of Pennsylvania

• Stefan Rothenburg Ph. D.• Kansas State University

• Adolfo Garcia-Sastre•Mount Sinai Hospital

• Undergraduate Research Committee

at Albright College•Dr. Adam Hersperger • Albright College