Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

The overall goal of the following experiment is to model the lifecycle of the highly pathogenic Ebola virus under biosafety level two conditions. This is achieved by producing virus-like particles, which have the same protein composition as Ebola virus particles, but carry a mini genome instead of the viral genome. A mini genome is a miniature version of the viral genome in which viral genes have been removed and replaced by a reporter gene such as luciferase.

Importantly, the mini genome used for this experiment also still carries a number of viral genes, which encode the viral proteins. VP 40 GP one, two, and V VP 24 virus like particles are produced by expressing a mini genome in 2 9 3 T cells called producer cells. Together with the viral proteins, np, VP 35 V, VP 30, and L, which together are also called RNP proteins.

The mini genome is then replicated and transcribed into mRNAs by the viral proteins. The mRNAs encode the Luciferase reporter and the additional viral proteins VP 40 GP one two, and VP 24. Together these three proteins lead to the production of virus-like particles, which are called transcription and replication.

Competent virus-like particles or TR vlp. Since the nucleo capsids in them are able to undergo genome replication and transcription. Since TLPs carry the Ebola virus glycoprotein G one two, they are able to infect target cells.

These target cells have been pret transfected with expression plasmid for np, VP 35 V, VP 30, and L together. These RNP proteins recognize the mini genome that was brought into the target cells inside the T RVPs. The mini genome is then replicated and transcribed leading to reporter activity, as well as the production of new TRV LPs, which can again be used to infect new target cells.

Importantly, the mini genome does not carry the genetic information which is required for its replication. Therefore, T RVPs cannot productively infect cells which do not express the four RNP proteins. This makes the system safe to be used under biosafety level two conditions.

The major advantage of this method is that it allows us to model the complete virus lifecycle, including genome replication, expression of viral gene products, production of virus particles, and the infection of target cells over multiple inject factors cycles under biosafety level two conditions. This means that this work can be done without a maximum containment laboratory, which is otherwise needed for work with live Ebola viruses. This method can be combined with other techniques such as RA mediated knockdown of cellular factors or overexpression of those factors to study the role of these factors.

Then in virus biology also we can introduce mutations in the virus genes to study the role of these virus genes and the gene products for virus biology. Split cells by removing the medium from 80 to 90%confluent 2 9 3 T cells, washing them twice with PBS and adding two mils of trypsin to the cells. Wait until the cells are rounded off.

Then dislodge them by tapping the flask and adding eight mils of medium with 10%FBS to the cells. Count the cells using an automated cell counter, then dilute the cells to two times 10 to the fifth cells per mil and seed two mils into each well of a six. Well plate return the cells to a humidified 37 degree incubator with 5%CO2 after 24 hours.

Transfect the cells with the plasmids required for production of T RVPs in A BSC pipette the DNA into a tube using filter tips as a negative control in one well the plasmid encoding the viral polymerase L should be omitted. Instead, a plasmid encoding GFP can be transfected to visualize the transfection efficacy. Then add 100 microliters optimum per well to the DNA vortex the tube and spin it down.

Next, add 7.5 microliters of transit LT one transfection reagent, the diluted DNA gently vortex the tube and incubate it at room temperature for 15 minutes. After 15 minutes, gently mix the transfection mixture by pipetting it up and down. Then add 100 microliters to each well of a six well plate in a dropwise fashion.

Once you have added the transfection complexes to the cells, rock the plate forward and backwards and from side to side. Do not swirl the plate as this will result in uneven transfection. After this, return the cells to the incubator 24 hours after transfection, remove the supernatant from the cells, then add four mils of medium with 5%FDS to the cells and return them to the incubator.

24 hours after transfection of the producer cells split 2 9 3 T cells as previously described 24 hours after splitting. Transfect them with the plasmids, encoding the Ebola virus, RNP proteins, as well as Tim one, which serves as an adhesion factor for Ebola viruses. 72 hours after transfection of the producer cells and 24 hours after transfection of the target cells, harvest the supernatant from the producer cells and pipette it into a 15 mil tube.

After you have harvested the supernatant, remove any remaining liquid and give 250 microliters of glow lysis, buffer onto the cells and incubate them for at least 10 minutes at room temperature. After this, determine the luciferase activity as described in step three of this video. In the meantime, spin down the supernatant from the producer cells for five minutes at 800 G and room temperature.

Then remove the medium from the target cells and add three mils of the clarified supernatant from the P zero cells to the target cells. Do this one well at a time. When you are done, return the target cells to the incubator 24 hours after infection.

Remove the supernatant from the target cells. Then add four mils of medium with 5%FBS to the cells and return them to the incubator to determine reporter activity in producer or target cells. These cells are harvested 72 hours after transfection in the case of producer cells and 72 hours after infection in the case of target cells.

To do so, remove the supernatant from the cells and add 250 microliters of glow lysis, buffer to the cells and incubate them for at least 10 minutes at room temperature if desired. The supernatant can be used to infect a fresh set of target cells. In this case, harvest the snat and infect target cells as described in step two of this video.

In the meantime, thaw 40 microliters per well of luciferase assay buffer and add one 100th volume of luciferase substrate to the buffer vortex the finished luciferase reagent, and make sure that it has reached room temperature before you proceed. Then pipette 40 microliters of reagent into a white opaque 96 well plate. In order to avoid problems with crosstalk, it is advisable to leave one well free between sample wells after the 10 minute incubation is over.

Wash the cells into the lysis buffer by pipetting and transfer the cell lysate into a tube. Spin down the lysates for three minutes at 10, 000 G and room temperature. After this pipette 40 microliters of the lysates into the 96 well plate with the luciferase reagent.

Incubate the samples for 10 minutes at room temperature. After the 10 minutes, measure luciferase activity using a luminometer. The integration time for the luminescence measurement should be one second with a modulus microplate luminometer.

Typical luminescence signals for the positive control should be around one times 10 to the sixth to one times 10 to the seventh, whereas the signal for the negative control should be at least two to three logs lower in subsequent passages. Reporter activity in the positive controls should remain stable, whereas reporter activity in the negative controls should drop to background levels. After watching this video, you should be able to produce Ebola virus replication and transcription competent virus-like particles used them to infect target cells and measure report activity in producer and target cells.

Importantly, this method can be combined with other techniques such as ated, knockdown of cellular factors to study the role of these factors in virus biology. Also, one can introduce mutations in the viral components employed in this method in order to study the role of these components for the Ebola virus lifecycle. Further experimental details and specific reagents can be found in the manuscript accompanying this video.