The aim of this experiment is to demonstrate how a primary neuron cell culture system can be utilized to study herpes simplex virus latency and reactivation. This is accomplished by first isolating superior cervical ganglia neurons from RA embryos and culturing neurons isolated from dissociated ganglia after elimination of non neuronal cells. A latent HSV infection is established using an EGFP virus to infect neurons in the presence of a lytic replication inhibitor.
The next step is to induce reactivation of the latent infected cultures. Finally, the efficiency of reactivation is assessed by monitoring EGFP expression over time. Ultimately, results can be obtained that show how different neuronal signaling pathways regulate herpes simplex virus latency and reactivation through the use of chemical inhibitors, RNA interference and gene delivery.
The main advantage of this technique over existing methods is that it permits detailed studies of the interactions regulating herpes simplex forest latency in the host neuron at the molecular level. This method can help answer key questions in the herpes simplex forest latency field, such as what are the signals that act to repress the latent genome and how is reactivation triggered at the molecular level? In neurons, though this method can provide insight into HSV one latency.
It may also be applied to other neurotrophic alpha herpes viruses Demonstrating the procedure will be marigo, Kochi and Julian Kim, both graduate students in our laboratories Superior cervical ganglia or SCG neurons for this protocol are isolated from E 21 rat pups that were removed from the uterus of a pregnant female rat. Working at a dissection hood, sacrifice each pup by aiming the scissors at the base of the neck just above the shoulders and shearing the head from the torso. To expose the ganglia, use 23 gauge needles to pin down the head, neck side up in three locations.
First pin down the spinal cord. Second, pull the anterior skin over the nose and pin and third pin the esophagus trachea away from the back of the neck. Look for the SCG by locating the carotid artery bifurcation.
The carotid artery is a paired structure. There are two bifurcations on either side of the neck, thus two s CGS per pop. The SCG is located just underneath the branch.
Separate the SCG from the arteries by pulling the bifurcation apart using the two ends. The SCG is translucent and colorless compared to the opaque yellowish adipose tissue that surrounds the bifurcation. Place the ganglia into 12 milliliters of L 15.
Media supplemented with glucose in a 15 milliliter conical tube on ice. After all s SCGs are harvested from the embryos and pulled gently centrif the ganglia for one minute. At 600 RPM, remove the excess media Reese.
Suspend the ganglia in one milliliter of L 15. Media containing trypsin and collagenase. Incubate the 24 minutes at 37 degrees Celsius.
Agitate every eight minutes. Next, add 10 milliliters of sea media to inactivate the trypsin centrifuge for one minute. At 600 RPM, remove as much of the tryin collagenase media as possible and wash the ganglia with 10 milliliters of C media centrifuge for one minute at 600 RPM.
Repeat this wash once. Remove the wash media and resuspend the ganglia in one milliliter of C media to dissociate t tritrate using a 21 gauge needle attached to a five milliliter syringe for a maximum of 15 up and down cycles. Finished tri rating using a 23 gauge needle until the majority of clumps have dissociated.
Remove any remaining clumps of cells by filtering the dissociated neurons through a 70 micron nylon filter into a 50 milliliter conical tube. Withdraw a 10 microliter aliquot of the filtered cell suspension, a mix of triam blue to determine the number of live cells. Count the number of cells that actively exclude the dye using a hemo cytometer.
Next, dispense 5, 000 to 6, 000. Total live cells in 100 microliters of C media into each well of a 96 Well tissue culture plate pre-coated with collagen and laminin include 50 nanograms per milliliter nerve growth factor in the C media. Incubate overnight, a 37 degrees Celsius on the following day, day one in vitro or DIV one, check for proper attachment of neurons and growth of some axonal projections.
Replace the C media in the wells with 50 microliters of neuro basal medium or NBM containing NGF Acho and five fluoro two doxy uridine. Replace media one row at a time to ensure the cells do not dry out. Incubate the cultures at 37 degrees Celsius for five days.
Monitor the culture daily to confirm the death of non neuronal cells and growth of the neurons. To begin this part of the procedure at day in vitro six, check that the majority of non neuronal support cells have been eliminated. It is exceedingly important to minimize unnecessary physical manipulation of the neuron at all times.
Removing and replacing media must be done very gently and slowly, otherwise resulting mechanical stress can affect neuron viability. Add aciclovir or a CV to the existing media in each well to a final concentration of 100 micromolar. Add a CV slowly and gently to ensure that mechanical force does not negatively affect the cells on the following day.
At DIV seven, infect the SCG cultures with a recombinant HSV one that expresses an EGF reporter fused to the viral lytic protein US 11 at a multiplicity of infection between one to two. Add the diluted virus directly to the existing media in the well. Include a mock infected control and a lytic infection.
Positive control in media lacking a CV.Allow the infection to proceed for two to three hours at 37 degrees Celsius. Next, replace the infection media with fresh NBM containing NGF and a CV.It is critical to be extremely gentle when changing the infection media. Tilt the plate towards you and slowly remove the media from the bottom of the well.
Then aim the tip of the pipette at the wall of the well rather than at the bottom of the well and allow the media to gently slide down onto the cells. Replace one row of media at a time to avoid the cells from drying out. Return the cultures to the incubator, handling them as gently as possible to avoid compromising their ability to support HSV one latency.
Maintain the cultures for six days at 37 degrees Celsius during which time the virus will establish latency. During this period. Use a fluorescent microscope to monitor us 11 EGFP expression as an indicator of lytic replication.
As expected, there should be no GFP expression in the mock infected control. S 11 EGFP should be detected only in the infection. Positive control wells lacking a CV treatment indicating successful primary infection and productive lytic replication.
To assess reactivation at DIV 14, carefully replace the a CV containing media with fresh media lacking a CV as before. Aim the tip of the pipette at the wall of the well rather than at the bottom of the well and allow the media to gently slide down onto the cells if appropriate. Include pharmacological or biological variables such as LY 2 9 0 0 4 at 20 micromolar.
No GFP expression should be detected in the A CV treated latently infected cultures. Return the cells to 37 degrees Celsius over a five to six day period. Monitor the live cultures using a fluorescent microscope to detect neurons undergoing reactivation.
The reactivation frequency is calculated by counting the number of wells containing EGFP positive neurons and expressing this as a proportion of the total number of sample wells. This figure illustrates an example where trigger statin, a a known inducer of reactivation is applied to the late the infected SCG cultures. The reactivation was monitored and scored using fluorescence microscopy each day after drug treatment for a period of five days.
With TSA reactivation reaches 50%of the maximum within two days by five days, reactivation reaches 60%baseline or spontaneous reactivation represented by the DMSO. Treated control cultures is approximately 10%in this experiment and typically ranges from five to 20%using this in vitro system. Late in the infected SCG cultures were also treated with LY 2 9 0 0 4.
A known inducer of reactivation cultures with reactivation induced by LY were compared to those treated with the viral DNA synthesis inhibitor, phospho, no acetic acid, and the two compounds together as illustrated here, US 11 EGFP expression is impaired by PAA in cultures induced to reactivate with LY 2 9 4 0 0 2 indicating that US 11 EGFP expression is dependent upon viral DNA replication. While attempting this procedure, it's important to pay close attention to sterile technique and to handle the CG culture as gently as possible to avoid increased spontaneous reactivation induced by stress or mechanical movements. Following this procedure to establish HSV one latent infections and cultured neurons, other methods like S-H-R-N-A, knockdown and ectopic expression of wild type and mutant gene loss can be performed in order to characterize neuronal functions that regulate HSV one latency and reactivation.
Don't forget that HSV one is a human pathogen that requires PSL two safety precautions. You should be familiar with working on their PSL two conditions before performing this procedure. After watching this video, you should have a good understanding of how to establish SCG cultures to set up latent HSV one infections, and to effectively induce reactivation in the neurons.
