It's almost like that famous trick where a magician drops pieces of rope into a hat, then pulls out the whole length intact: Scientists can now smuggle up to 17 snippets of DNA into a cell and--presto!--out comes an infectious influenza virus. This sleight-of-hand, reported in the 3 August issue of the Proceedings of the National Academy of Sciences, may give researchers a tool for developing new flu vaccines.
One goal of vaccine developers is to create strains of pathogens that are less virulent, or attenuated. To accomplish this feat, scientists tinker with the virus's genetic material, a step that, for RNA viruses, is most easily done by making a DNA copy of the viral genome. This DNA version can be put back into cells, which then manufacture a tailor-made virus. But some viral oddballs such as measles, rabies, Ebola, and influenza are much trickier to manipulate. That's because they are made of so-called negative-strand RNA, which, in order to yield protein products and hence viral particles, must first be copied into its mirror image, or messenger RNA version--a task that can only be accomplished by a viral enzyme. So any DNA version of the viral genome would only be copied into negative-strand RNA by the cell's gene-reading machinery but would never churn out any virus protein. Complicating matters for influenza, the virus consists of eight RNA segments, all of which have to end up in the same cell to make new viral particles.
Virologist Yoshihiro Kawaoka and his colleagues at the University of Wisconsin, Madison, figured that they could make influenza virus from scratch by inserting DNA versions of the eight viral segments--in negative-strand orientation--into circles of DNA, called plasmids, and shooting them into a cell along with four other elements: the genes--in positive-strand orientation--for the three subunits of the enzyme that copies the viral RNA, plus the gene for a protein that kick starts the enzyme.
In a tour de force, the team slipped a cocktail of the 12 plasmids into cultured cells. After 24 hours, about one in 1000 cells spat out infectious virus particles. What's more, by adding the genes for all the other viral structural proteins, the team could boost virus production more than tenfold. They also managed to recover viruses that harbored engineered mutations in some of their proteins, the first step in generating attenuated strains. Previous techniques only allowed the manipulation of a single influenza virus segment at the time.
"This opens up the door for us to analyze all the functions of the influenza virus in a simple, straightforward way" by mutating every single gene, or combinations thereof, says virologist Andrew Pekosz of Northwestern University in Evanston, Illinois. Besides contributing to a better understanding of the virus's life cycle, Pekosz hopes the findings will speed up vaccine development. If a new virulent influenza strain emerges in the population, he says, "we can isolate the [underlying] genes, substitute them in current vaccine strains, and almost immediately we can produce the new vaccine."