If it seems like you catch a cold every time someone sneezes on the subway, you'll probably be surprised to learn that most virus particles fail in their attempts to successfully infect their hosts. Only one out of every 100 influenza particles, for example, manages to infect respiratory passage cells. The other 99 virus particles may bind to the cell, or even make it inside the cell, but fail to replicate.
Inefficient viruses may be good news for subway riders, but for a researcher like Dr. Ila Singh, assistant professor of pathology, who studies how viruses enter cells, viral inefficiency only makes it harder to understand how these microscopic entities do their job. "There's no way to find out how a successful virus is better' than the others," says Dr. Singh. "Using biochemical techniques, you'd likely look at those 99 out of 100 particles that are unable to infect. We need to use genetics to find out why successful viruses succeed, but conventional genetic techniques are time-consuming. They often consist of making, isolating, sequencing and studying each mutant individually, which can be both time and labor intensive. So for most viruses what happens immediately after their entry into cells is not well known."
To circumvent this problem, Dr. Singh has developed a new technique, genetic footprinting, which allows researchers to efficiently identify essential regions of a gene. Instead of making and analyzing one mutation at a time, the researchers produce a pool of thousands of mutations by inserting a small, defined DNA sequence at random positions in a viral gene. These viral mutants are then tested as a pool for their ability to enter the cell and replicate.
DNA extracted from these cells contains DNA from all viruses that successfully infect cells and is analyzed by PCR. One PCR primer corresponds to the mutational insert and the other to a specific site in the gene. For each mutant, a PCR product of unique length is generated, the length depending on the location of the insert. For the whole pool, this results in products of different lengths, giving rise to a ladder of bands on a gel. Mutants, in which the insert disrupts a structure required for viral replication, result in missing bands in the ladder called "footprints." The footprints mark essential regions of the gene (see diagram).
Recently, Dr. Singh applied genetic footprinting to Moloney murine leukemia virus, a virus popular in gene therapy research. The results, which show that even familiar viruses can offer surprising information, were published in the Sept. 30 issue of Proceedings of the National Academy of Sciences.
The new technique, applied to the virus's gag gene, provided some remarkable results. It identified novel regions essential for viral replication. Some of these regions are as short as 5 to 7 nucleotides and lie in unexpected regions and remained undiscovered despite many years of conventional mutational analysis. Another surprising finding of this study was that for some gag genes, three quarters of the positions in the genes were able to tolerate 36-nucleotide insertions. This was unexpected because viral genes are presumed to have evolved to be small, efficient and intolerant of further changes. Another gag protein, though, did not tolerate any insertions at all, showing that adjacent regions in a gene can have very different requirements for function.
"Genetic footprinting might be most useful when applied to new genes, where a good functional map of the gene might be a very useful place to start any analysis," says Dr. Singh. "In order to apply this technique to a gene, the gene needs to be cloned and there needs to be a method to select for its function. We expect that applying this technique will supply us with a lot of information about relatively unknown viruses like hepatitis C and West Nile viruses. We hope genetic footprinting can tell us things about viral entry that may be useful for designing drugs and vaccines."