VIRAL VECTORS FOR GENE TRANSFER

Molecular biologists have been developing the tools needed to build viruses which can carry bits of synthetic DNA into the nuclei of living cells. It may be possible within the very near future to alter the genes of any animal (including the human animal) at will. A principal tool in such "bioengineering" are viral vectors, which are used to carry new genes, or bits of DNA or RNA to a target location. Understanding this process will help explain some of the fundamental mechanisms of cell metabolism and gene expression.

Sports car enthusiasts know that exchanging a stock carburetor for one that gulps more fuel can increase the speed of the car. We are familiar with the idea that simple or complex mechanical changes can permit machines to function in a manner different from their original design. Changing a flat tire on an automobile is an example of replacing a defective component with a functioning component to permit the car to roll, again. Sometimes a change may have an undesirable effect. Removing a good inflated tire from our car and replacing it with a flat tire changes the working automobile into a defective machine, i.e. one that will not go as fast as the one with four functional tires.

The same principle of altering components to change the function of machines can be applied to the functioning of living cells. Most common antibiotics work by interfering with the normal function of bacterial cells. Penicillin, for example, is picked up by most common bacteria and used instead of glucose to manufacture the bacterial cell wall. Unfortunately for the germs, penicillin is an unstable molecule and causes the cell wall to collapse, thus killing the bacterium.

Since most cellular functions are dictated by the DNA of the cell, altering the DNA within a live cell can cause an alteration in cellular function. If a cell or cells of an organism contain a genetic mutation or a defective gene, replacing that defect with a functional gene will repair the cellular function.

Molecular biologists have been developing the tools needed to build viruses which can carry bits of synthetic DNA into the nuclei of living cells. Remember the "machine" model described above. Thinking of cells as molecular machines may change the way we view the practice of medicine as well as alter our definition of species. It may be possible within the very near future to alter the genes of any animal (including the human animal) at will. A principal tool in such "bioengineering" are viral vectors, which are used to carry new genes, or bits of DNA or RNA to a target location. Understanding this process will help explain some of the fundamental mechanisms of cell metabolism and gene expression.

Viruses are usually composed of a protein envelope that surrounds some segments of RNA, its genes. When the virus is picked up by a living cell (sometimes the virus is the one doing the 'picking up'), it works its way into the cell's nucleus.¹ The cell may try to defend itself from the virus but once the viral envelope has opened and the RNA released into the nuclear cytoplasm, the cell can not distinguish viral RNA from its own cellular genetic material. The cellular chemistry treats the viral RNA as if it were supposed to be within the cell. Unfortunately for the cell, the viral RNA usually codes for the production of more virus particles and the cell is destroyed in making more viruses. In other words, the virus uses the cell's enzymes and resources to reproduce, in effect reprogramming the cell.

There is an entire spectrum of virus types and virus actions. We now know that some viruses carry DNA within their envelopes. Some viruses cause the cell to produce substances which are toxic to the cell. Some viruses have elaborate envelopes that alter (or mutate) rapidly, thereby not permitting the cells of an infected organism to muster an immune response. The variety of viral actions parallels the variety of lifeforms and there is evidence that viruses may play an important role in the changing or evolving of DNA and, hence, life on earth. Because of their abilities as natural "bioengineers," viruses seem to be ideal carriers of DNA for the insertion of this DNA into the genome of any living cell.

To construct a virus that will work as a carrier for some specific segment of DNA, we must review some of the basic mechanisms responsible for viral action. The outer structure, the viral sheath or viral envelope, is usually composed of an elaborate protein. Some protein envelopes are almost crystalline. They have very regular structures and are usually, made up of many small identical protein building blocks arranged in some interlocking manner so that they are slightly flexible yet hold their shape. Some protein envelopes are mosaics of several distinct protein subunits. These are patched together and may break apart once within the cell to provide ancillary proteins and enzymes needed for complete viral reproduction. It is important to note that the function of the viral envelope is to carry the viral genetic material into the cell. This may be accomplished in one of several ways.

The viral envelope may be a passive structure, made of some harmless protein that passes through cell walls without disrupting the cell's integrity. Such viruses must be small and can not, therefore, carry much genetic material into a cell.

Another mechanism for viral invasion requires that the viral envelope be a bit active. Consider a virus floating near the cell membrane. The cell is stimulated to ingest this particle. Upon forming a vacuole around the virus, the cell begins to secrete digestive enzymes into the vacuole. If the viral envelope contains proteins that can neutralize the cell's digestive enzymes, the cell will not be able to digest the viral cargo of genetic material. The viral DNA or RNA will pass into the cell much like the working of a timed release medicine capsule.

Some viruses have elaborate attachment mechanisms as complex as grappling hooks and mechanical pinchers for grabbing onto a cell wall or membrane. These hold the virus particle close to the cell while the viral DNA passes through the cell membrane. The advantage of this technique is that the cell is never really invaded by the viral envelope. Thus, the cellular immune response is minimized and the virus gets its genes into the cell. A variation on this theme is an insertion mechanism. Some virus envelopes contain what appears to be a spring-like mechanism that pushes the viral RNA right through the cell membrane.

Once within the cell a virus relies upon the cell's metabolism and enzymes to replicate. The cell processes the viral RNA or DNA (genes) as if they belonged there. The protein of the viral envelope is a different matter. Sheath proteins are not RNA or DNA and are foreign substances to the cell. Depending upon the complexity of the cell, immune responses will begin to try to clear the viral envelop proteins from within the cell. If the cell develops an immune mechanism that can purge the viral envelope proteins from the cell, the virus is in trouble. Even if the viral genes are replicated within the cell, how will the cell allow the formation of new viral envelopes? The proteins needed for these envelopes now evoke an immune response within the cell. If the virus manages to duplicate its genes, complete with a protein sheath before an immune response can be made, then the virus wins. Or, if the cell's immune response is directed to some non-critical portion of the viral envelope proteins, the virus also wins. As an analogy, imagine that you are building a wall of bricks. Each brick has one red side. You begin building your wall (your viral envelope) with the red side of the bricks showing. Snipers (the cell's immune system) can only see your red bricks and each time you pick one up to place it in the wall, a sniper shoots it out of your hand. If you stay with red-faced bricks, you will never complete your wall, but, if you substitute blue and yellow bricks, you will build a solid wall and the snipers will be frustrated. Viruses employ a similar technique to avoid immune destruction of viral sheaths.

Remember that DNA, regardless of its source, introduced into the nucleus of a living cell is treated the same way as the cell's own DNA. The chemistry of the cell can not distinguish foreign DNA from native DNA. This means that all of the processes that would normally be used to replicate the cell's DNA will also be used to replicate the foreign DNA or RNA. All of the subsequent RNAs and proteins developed from the foreign genes will emerge within the cell as if they were always supposed to be there. In fact the foreign DNA may function as if it were a new chromosome within the cell. If the foreign DNA has some segments of base sequences that are similar to the cell's own DNA, the foreign DNA may even be incorporated into the cell's DNA and become indistinguishable from the cell's chromosomes.

Scientists can build a viral vector to deliberately introduce a specific sequence of bases or a replacement gene into a cell. For example:

Cellular segment of DNA: XXXXX-A-A-A-A-(some old gene)-G-G-G-XXXXX
Foreign segment of DNA: A-A-A-A-(some new gene)-G-G-G

These will align naturally as:

XXXXX-A-A-A-A-(some old gene)-G-G-G-XXXXX-A-A-A-A-(some new gene)-G-G-G

and during replication will form some DNA strands that have this configuration: XXXXX-A-A-A-A-(some new gene)-G-G-G-XXXXX.

These processes are not always as straight forward as our example. Some viral vectors need helper viruses to bring ancillary molecules into the cell. These helper molecules act as catalysts for some of the viral reactions. The control of the presence or absence of these helper viruses and their molecules allows biochemists to 'turn on' the viral vectors or deactivate them.

The study of these viral pathways is made possible through the use of markers or tags attached to the viral envelopes or the genes carried into the cell by the viral vector. A genetic marker might be a gene for the production a light emitting protein such as luciferase. Cells that successfully received a new gene would glow under certain circumstances, prove positive that the gene transplant was a success.

In summary, new genes may be introduced into living cells by including this DNA in a viral envelope. The newly manufactured virus then infects the cell and uses the cell's own chemistry to replicate the introduced DNA. This new DNA then directs the cell to produce all the RNA and subsequent proteins encoded within the DNA's sequence of bases. If properly constructed the introduced DNA will also duplicate itself and surround itself with a new viral envelope; prepared to infect the next cell it encounters. This simple process allows us to alter the structure of DNA almost at will.

footnote:
(1) There are some viruses, such as rotavirus, that wreak their havoc within the cytoplasm of a cell, without ever invading the cell's nucleus.