The gene we are going to engineer is one of the proteins that the rabies virus needs to set up a productive infection. It is a glycoprotein, which is carried in gene form by the virus that infects Neuron A. If the virus can manufacture this glycoprotein after it enters Neuron A, it can make progeny and spread the infection to an adjacent nerve cell. If the virus cannot make the glycoprotein, it will not be able to jump to an adjacent nerve cell, even if Neuron A is otherwise infected. The virus will simply stay inside its host.
Today, researchers can be quite clever about forcing the hand of the rabies virus. For example, they can gut the gene that encodes the glycoprotein from the virus, rendering it impotent to spread infection. The virus can get into Neuron A but it cannot get out.
While we are working on the rabies virus, we are also going to genetically modify Neuron A. We will engineer the nerve cell to make the glycoprotein by extracting its gene from the virus and inserting it into the nucleus of Neuron A. This process is called transfection, and there are many ways to perform the procedure. Soon, Neuron A will be flooded with the glycoprotein.
Why do we go through these steps? This double manipulation allows the virus to set up a productive infection in just 1 cell. If the hobbled rabies virus is allowed to infect the modified Neuron A, the cell will allow the virus to make new progeny, which can then escape Neuron A and infect all its neighbors. But that is where everything stops. The neighbors will be able to create a new virus just like Neuron A; however, these progeny are still reproductively incompetent (remember, the viral genetic information is gutted from the glycoprotein). Neuron A will serve as the birth mother, and the virus will spread to neighboring nerve cells. If the neighbors do not have the viral glycoprotein already working in the background, the virus will not be able to spread outward from them. The buck, as it were, stops there.
This finding could be of great value to a neurobiologist, especially if Neuron A is embedded in a thicket of normal neurons that are not genetically engineered. It is the neurobiologist’s job to find out how they are connected. This concept will become very important as I further discuss this technology.
ASLV-A
The second virus in our discussion goes by the tongue-twisting name avian sarcoma and leukosis virus subgroup A (ASLV-A). The virus attacks via a surface grappling hook—a protein called EnvA. If EnvA finds a receptor protein called TVA, ASLV-A will set up an infection. This will occur even if the TVA protein is found on a cell other than an avian cell.
Why am I bringing this up? It is possible to mix and match viral proteins and to alter their host ranges. For example, a researcher could remove an EnvA gene from ASLV-A and stick it to the genetic background of a rabies virus. The rabies virus now has a grappling hook for avian cells. This means that the rabies virus is now fully capable of binding to an avian cell or any cell that carries the TVA receptor protein. The virus could be directed to go anywhere the researcher wanted it to, as long as the EnvA-TVA matching conditions were met. This, too, turns out to be a critical part of this story.
Two fluorescent proteins
The fluorescent proteins are probably the least complex piece of molecular biology in our discussion. There are a number of proteins that glow in the dark if given the proper substrates at different wavelengths when inserted into cells. There are genes whose proteins glow red and genes whose proteins glow green (green fluorescent protein, GFP). These genes can be stitched into the genetic machinery of viruses, which are then allowed to infect cells. Researchers can trace not only the success of the infection but also the routes of infection just by looking for the presence of the fluorescent color in the cell. For example, if the GFP gene is inserted into the rabies genetic information, every cell in which the rabies virus sets up an infection will turn green. As you might suspect, this phenomenon provided researchers with everything they needed to trace neural interactions.
With this information in mind, we are prepped to discuss the data, which will occur in Part 2. We will see that the clever use of these viruses, genes, and manipulated cellular backgrounds has allowed researchers to create their own Lilliputian Sistine masterpiece.
They did it all by answering that simple question, “Are the neighbors green yet?”
