Scientists at MIT and Northwestern University Medical School have uncovered the structural basis of an elusive replication technique that allows viruses, especially retroviruses, to commandeer cells to manufacture the proteins they need for their own survival, according to an article published in the March issue of Nature Structural Biology.
For many years, scientists have studied the virus's ability to create an RNA structure called a pseudoknot, which allows it to control its genetic material for its own purposes via a process called ribosomal frameshifting. Until now, the detailed three-dimensional structure of the pseudoknot (so called because the RNA is not truly knotted, but tightly bound together) has not been known.
The RNA pseudoknot formed by the beet western yellow virus has been crystallized, and the three-dimensional structure reveals many unusual features, the authors of the study report. Ribosomal frameshifting also is used in the AIDS virus.
"This research will help us uncover some of the methods that viruses use to regulate the production of components that are essential to viral replication. Knowledge of this mechanism may allow us to develop ways to modify that process and thus lead to the development of new antiviral agents," said Alexander Rich, the William Thompson Sedgwick Professor of Biophysics at MIT and one of the study's authors.
The work provides information that will allow researchers to understand which features of the pseudoknot formation facilitate ribosomal frame-shifting by introducing mutations or changes in the pseudoknot and measuring their effect on ribosomal frameshifting.
Viruses have developed ingenious systems for invading cells and making more copies of themselves. One of the systems used in many viruses, including most retroviruses (the most well-known is the one responsible for AIDS) involves inducing changes in the way the virus's genetic material is translated to produce the next generation.
The virus needs to synthesize two different proteins. Typically, the first protein is involved in building the virus and the second is an enzyme, usually a polymerase, used in replicating the virus's nucleic acid, or genetic building blocks.
The problem is that the virus needs many copies of the structural protein and a smaller number of the polymerase proteins. The virus has developed a novel system for regulating the production of these two proteins, involving the use of ribosomal frameshifting.
In all cells, the ribosome is used to translate messenger RNA, adding one amino acid for every three nucleotides. Thus, its reading frame involves clusters of three nucleotides along the viral messenger RNA chain.
In most retroviruses, however, the gene encoding the second polymerase protein slightly overlaps the end of the gene encoding the structural protein, and it is "out of frame" with the structural protein. To translate that protein, the ribosome must move back one nucleotide to continue to translate the polymerase protein fused to the structural protein. Otherwise, it will simply reach the "stop codon" at the end of the structural protein and complete its synthesis.
The virus has adopted a system for frameshifting that involves a so-called "slippery sequence" in which the ribosome can be pushed back, but only when it encounters a folding or secondary structure obstacle in the RNA itself. The most frequent secondary structure obstacle that the ribosome encounters for frameshifting is called an RNA pseudoknot.
A pseudoknot is formed by folding back the RNA upon itself to make a so-called stem loop structure, where the stem is held together by Watson-Crick base pairs. However, an adjacent set of nucleotides also forms base pairs to nucleotides in the loop. This produces a structure which then has two stems and two loops.
The three-dimensional structure of the RNA pseudoknot formed by the beet western yellow virus makes it possible to determine the organization of all the nucleotides in the pseudoknot as well as the organization of the surrounding water molecule and ions. The pseudoknot is held together by an unusual configuration of hydrogen bonds.
The decision to frameshift or not to frameshift depends on whether the pseudoknot unravels when it collides with the ribosome, Professor Rich said. If it does not unravel, the ribosome can slide back one nucleotide and then make a fusion protein, involving both the structural protein and the polymerase. If the pseudoknot does unravel, then only the structural protein is made, but not the polymerase.
This work is supported by the National Science Foundation, the National Institutes of Health, the National Foundation for Cancer Research and NASA.
A version of this article appeared in the March 17, 1999 issue of MIT Tech Talk (Volume 43, Number 23).