ROLE OF PERK PROTEIN KINASE IN THE REPLICATION SUCCESS OF

EBOLAVIRUSby Julie Hestand and Dr. Dye

Overview Background Questions addressed, methods used, and

results Discussion Conclusion Future directions

What is Ebolavirus? A member of the family Filoviridae, or

filament viruses. First discovered in Central Africa in 1976,

though it has recently appeared in West Africa.

Can infect humans and non-human primates.

There are currently five known strains of Ebolavirus. For this research project, the strain Zaire Ebolavirus was used.Image courtesy of www.dailymail.co.uk

What is the UPR? The ER is a multi-functional organelle in the

cell. One such function is the synthesis, storage, and packaging of proteins.

Part of protein synthesis involves folding proteins into their final shapes, which allow them to function properly.

Sometimes proteins do not fold correctly. Mis-folded proteins can build up and stress the ER, triggering the unfolded protein response (UPR).

What is the UPR? The UPR works to fix the problem in several

ways. When the UPR is activated, mRNA translation is

attenuated. This prevents more protein from being generated and possibly mis-folding.

The UPR also increases the amount of chaperones, which aid in proper protein folding.

Some viruses take advantage of this response to create more virions more quickly.

There are three branches of the UPR. One branch is PERK.

Image courtesy of http://www.nature.com/nrd/journal/v12/n9/full/nrd3976.html

PERK PERK, which is short for PKR-like ER kinase,

is phosphorylated to become p-PERK during ER stress.

It phosphorylates the translation factor eIF2α, which inactivates it and attenuates translation in the ER.

This can benefit some viruses because it extends the life of the host cell by preventing rapid accumulation of mis-folded proteins in the ER, which allows the virus to make more virions.

Experimental Question Does Ebolavirus use the UPR to replicate

itself more efficiently? Previous research by Martin Senese, Jess

Boegner, and Kathleen Callahan suggest it induces a mild UPR response

Will inhibiting PERK production reduce the efficacy of Ebolavirus replication?

What is shRNA? A small RNA generated that recognizes

and binds to a specific sequence on an mRNA, preventing that mRNA from being translated.

Can be generated in the lab to inhibit cellular proteins so scientists can better understand how they work.

In this project, shRNA against PERK was introduced into cells using lentivirus particles.

Overview of Methods Used

Generating cell lines

• Tissue culture and counting cells• Polybrene and puromycin concentrations• Lentivirus transduction

Characterizing cells

• RNA and protein isolation• qPCR and Western blotting• p-PERK production

USAMRIID

• Ebolavirus infection• Plaque assay

Tissue culture VeroE6- African green

monkey kidney epithelial cells grown in 10 cm tissue culture-treated dishes.

Susceptible to Ebolavirus.

The cells were split twice a week using trypsin to maintain a healthy stock.

Finding the right concentrations

Before lentiviral transduction could occur, the right concentrations of cells, puromycin, and polybrene had to be determined.

Polybrene- used to make the cells more receptive to transduction.

Puromycin- used to select for successfully transduced cells.

The right concentration of cells would show 50% confluency.

Finding the right concentrations

Hemocytometer- used to count cells.

The correct concentration of cells to use was determined to be 2*10^5 c/well.

100,000

150,000 200,000

Finding the right concentrations

Polybrene: cells seeded in 12-well plates at 2*10^5

c/well. Treated with either 2.5 μg/mL, 5 μg/mL, or 7.5

μg/mL of polybrene. 5 μg/mL was determined to be nontoxic to the

cells and was used.

Finding the right concentrations- puromycin

No puromycin

10µg/ml 15µg/ml 20µg/ml

25µg/ml 30µg/ml 35µg/ml

Viral transduction Seed 2*10^5 c/well in four 12-well plates.

Incubate for one day. Change media to contain 5 μg/mL

polybrene. For each 6-well plate, the media was

removed from each well using vacuum filtration, and 1 mL polybrene media was added to prepare the cells for infection. The cells were incubated at 37°C.

Viral transduction Safety:

Viral transduction There were four conditions for transduction:

control, GFP, mock, and PERK. Control: shRNA that did not inhibit any cellular

proteins. GFP: green fluorescent protein caused cells to

fluoresce green under green fluorescent microscope.

Mock: treated with polybrene but left uninfected. PERK: shRNA inhibited translation of PERK

mRNA.

Observing transduced cells GFP cells (above) and

mock cells (below) were observed using a green fluorescent microscope.

VeroE6 cells have no natural green fluorescence, so any fluorescence observed could be directly correlated to the success of transduction.

GFP cells

Mock cells

Selecting for successfully transduced cells

VeroE6 cells- natural resistance at low levels of puromycin, but not at levels of 20 μg/mL or higher.

Transduced cells- showed higher resistance because that was also conferred by the lentivirus during transduction.

20 μg/mL of puromycin was added to each well of the infected plates to select for successfully transduced cells.

Mock cells were also treated to ensure no spontaneous resistance developed.

20 μg/mL puromycin

GFP cells Mock cells

0 μg/mL puromycin

GFP brightfield GFP green filter

20 μg/mL puromycinGFP brightfield GFP green filter

Is PERK being knocked down?: RNA

All four cell types- RNA isolation cDNA synthesis qPCR

Isolating RNA RNA was isolated from frozen cell pellets

using the RNaqueous kit from Life Technologies. Series of buffer, wash, and spin steps that lyse

cells, bind RNA to column, and elute RNA.

Quantifying RNA To generate cDNA, 1 μg of RNA needed to

be used. A spectrophotometer was used to

quantify the total RNA isolated. For each sample, the absorbance at 260nm was used to determine the concentration of RNA, in μg/μL, present in the sample.

Generating cDNA cDNA Master Mix-

RNA (1 μg) Random primers dNTPs Buffer Water Reverse transcriptase

cDNA reaction- 25°C for 10 minutes 37°C for 2 hours 85°C for 5 minutes

RNA DNAReverse transcriptase

Analysis of cDNA using qPCR

qPCR Master Mix- cDNA (2ul from the cDNA reaction) Taqman primer probe set Master Mix (dNTPs, Buffer, Taq DNA polymerase) Water

2 reactions for each cell type b-actin PERK

qPCR reaction: 2 minutes at 50°C (1 cycle) 10 minutes at 95°C (1 cycle) Alternate 15 seconds at 95°C and one minute at 60°C (40

cycles)

Analysis of cDNA using qPCR

Reactions were completed in triplicate for each cell type.

For the control, water was used in place of cDNA.

StepOne software was used to measure DNA amplification.

Results: Do PERK “knockdown” cells have

decreased PERK expression?

PERK vs. Control

PERK vs. GFP PERK vs. mock

No change

20% 39.5%

Is PERK being knocked down?: protein

PERK shRNA is predicted to have an effect on PERK protein production.

To determine this effect, proteins were analyzed using Western immuno blot analysis.

Isolating protein Soluble protein for all four cell types was

isolated from frozen cell pellets containing 2*10^6 cells via lysis with a mild detergent.

The lysates were cleared by centrifugation, and the isolated soluble protein was used in protein electrophoresis.

Protein electrophoresis Lysates were combined with sample

buffer and a reducing agent, and heated at 70°C for 10 minutes.

Samples were loaded in lanes of a 12% polyacrylamide gel along with a molecular weight ladder.

Analyzing protein After electrophoresis was completed,

proteins on the gels were transferred nitrocellulose membranes using dry electrotransfer and the iBlot system.

Following transfer, the membranes were blocked and then probed for PERK and the endogenous control protein b-actin.

The intensity of each band on the membrane were quantified using ImageJ software. The four cell types were compared after normalizing to b-actin levels.

Results: Do PERK “knockdown” cells have decreased PERK expression?

PERK vs. Control

GFP vs. Control

Mock vs. Control

↓ 50% ↓ 77% ↓ 60%

PERK protein

What if the cells are stressed?

PERK is produced at relatively low levels in un-stresssed cells, so impacts on production may go unnoticed.

PERK is up-regulated during ER stress and phosphorylated to become p-PERK, which is its active form. This may be affected by the PERK shRNA.

Stress can be artificially induced by either tunicamycin or thapsigargin.

What if the cells are stressed?

Tunicamycin- inhibits N-linked glycosylation.

Induces ER stress by causing buildup of un-glycosylated glycoproteins.

Thapsigargin- inhibits the sarco-endoplasmic reticulum Ca2+-ATPase

This induces ER stress by preventing the ER from replenishing its calcium ion stores.

Inducing p-PERK production

To determine if PERK and p-PERK production was reduced in PERK-transduced cells, control and PERK cells were cultured with either Tm or Tg for 6 hours.

After treatment, the cells were harvested and processed for Western blot immuno analysis.

Effect of transduction on PERK and p-PERK production in the presence of ER

StressProtein Treatment Control:

treated vs.

untreated

PERK: treated

vs. untreated

PERK Tunicamycin

↑ 74% ↑ 22 %

Thapsigargin

↑ 77% ↓ 12 %

p-PERK Tunicamycin

↑ 105% ↑ 16 %

Thapsigargin

↑ 103% ↓ 12 %

The meaning behind the results

Un-stressed PERK cells produced 50% less PERK than un-stressed control cells.

Decreases were also observed in GFP and mock cells.

Stressing the cells demonstrated that PERK and p-PERK up-regulation was impaired in PERK cells, suggesting that the PERK shRNA is impacting the production of PERK protein.

Ebolavirus infection Due to the risks associated with

Ebolavirus, infection was carried out at USAMRIID.

For transport, 1 mL of cells was trypsinized and re-suspended in 14 mL VeroE6 media in a T75 non-filter flask. The flask was then filled to the brim with media and capped.

This was done for control, mock, and PERK cells only.

Ebolavirus infection

Seed VeroE6 cells

6-well plates

Infect at MOI = 3

(1 hr)

Remove virus and add fresh

media(incubate 7

days)

Collect media on days 1, 3,

5 and 7

Plaque assay To quantify the amount of virus produced

by each cell type, a plaque assay was performed.

After the plaque assay was completed, the plaques on each plate were manually counted.

It was expected that cells treated with the infected PERK cell media would have less plaques, because the PERK cells would produce less Ebolavirus virions.

Plaque assay

Plaque Assay

Seed VeroE6 cells

6-well plate

(incubate 1-3 days)

Apply supernata

nt(1 hr)

Add agarose-media

overlay to wells

(incubate 7 days)

Add neutral

red overlay to

stain viable cells

Count plaques

Results

Day 1 Day 3 Day 5 Day 750002005000400500060050008005000

100050001200500014005000160050001800500020005000220050002400500026005000

controlmockPERK

Length of infection (days)

Viru

s ti

ter

(pfu

/mL)

What does this mean? PERK production was confirmed to be

knocked down in the cells. However, when infected with Ebolavirus,

this did not seem to have an effect on the virus’s ability to replicate.

This may mean that Ebolavirus does not utilize the UPR to improve replication efficacy, or at least does not utilize PERK.

It could also mean that PERK was not sufficiently reduced.

Future directions The method used to transduce the cells is

questionable because of random integration, which can cause the shRNA to be silenced or have a reduced function while the cell still expresses puromycin resistance.

One way to overcome this is to use a virus with a plasmid that contains integration enzymes and additional sequences. Another method that can be used is nucleoinfection.

Acknowledgements USAMRIID

Sina Bavari Lab Cary Retterer Jackie Benko Sarah Tritsch Kelly Stuthman

John Dye Lab Ana Kuehne

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