A virus reproduces itself by co-opting its host cell's own replicating mechanisms. Essentially, the virus enters the cell nucleus and splices its own DNA into that of the host. The modified host DNA now contains instructions for producing, not only the proteins needed by the cell, but the proteins required to assemble a new virus particle as well. In the case of HIV-1, one of these viral proteins, the Rev protein, must fit (bind) like a key into the RBE "lock" before viral replication can proceed. If this binding can be blocked, perhaps by a different molecular key of the right shape, the infected cell will no longer be able to produce new virus particles, thus slowing the spread of the infection.

The RBE is a highly structured region in the virus's messenger RNA, which is a mobile copy of the genetic information stored in DNA. Previous studies have shown that the RBE takes the form of a double helix that includes two "noncanonical" base pairs of nucleotides: guanine-adenine (G-A) and guanine-guanine (G-G). Normally, guanine will pair only with cytosine (G-C). The two noncanonical base pairs are separated by a single (unpaired) uracil (U). This arrangement distorts the double helix and presents a unique, high-affinity binding site for the Rev protein.

To study the structure of the RBE crystallographically, the researchers used synthetic RBE molecules. They heated solutions containing RNA strands that were chemically synthesized to have the requisite nucleotides in the proper order. The solutions were then purified, concentrated, and crystallized. A brominated derivative (where 5-bromouridine replaced one of the uracils) was also produced and used for crystallographic phasing. Analysis of the data, obtained using multiwavelength anomalous diffraction (MAD) techniques at Beamline 5.0.2, revealed four unique RBE structures that could be grouped into two types (I and II) distinguishable by structural deviations of up to several angstroms. The largest deviations (and thus the greatest flexibility) occurred at the "internal loop" where the noncanonical base pairs create an asymmetrical bulge in the double helix.

Another Key to Blocking HIV?

The human immunodeficiency virus type 1 (HIV-1) is the primary form of HIV found worldwide. In recent years, great progress has been made in finding drug mixtures ("cocktails") that have proven effective in slowing the advance of the illness. Each drug in a cocktail interferes in a different way with a different stage of the virus's life cycle, a strategy necessitated by HIV's high mutation rate. While a random mutation may render one of the drugs ineffective, it is unlikely to do so to all of them at once. The more drugs we have in our arsenal, the better the odds.

The Rev binding element (RBE) of HIV-1 is an attractive target for such a drug. It is a highly structured area of the HIV-1 gene that serves as a binding site for a key viral protein (Rev). Just as a key must be inserted and turned to open a lock, the Rev protein must bind to the RBE before the virus can begin reproducing itself. Researchers hope that understanding the structure and mechanism of the RBE "lock" will help them find a drug that can deliver a molecule that will bind to the RBE in place of the Rev protein, thus effectively locking it out.

Left: Superposition of type I (black) and type II (red) RBE structures. Right: Superposition of type I RBE structure (black) and NMR model of unbound RBE (orange). The unpaired uracil (U25) is located between two noncanonical base pairs in an asymmetrical bulge in the double-helix structure.

The four structural variants were found to differ in several respects from models previously derived using nuclear magnetic resonance (NMR) techniques. For example, in the type II crystal structures, the bonding between the noncanonical G-A base pair was unexpectedly bridged by water molecules. Furthermore, in both the type I and type II structures, the noncanonical G-G pair formed a novel asymmetrical bond, in contrast with the symmetrical bond indicated by NMR-based models of the RBE in complex with (i.e., bound to) the Rev protein. This means that the G-G bonds would have to be broken and re-formed to accommodate Rev protein binding. The observed flexibility of the bulged internal loop region may facilitate this kind of extensive structural change. These results suggest that the NMR model provides only a partial picture of how Rev binding occurs. More detailed studies will be needed before we can begin identifying or designing potential drugs to exploit the RBE-Rev interaction.

Asymmetrical bonding between noncanonical G-G base pair.

Research conducted by L.W. Hung, E.L. Holbrook, and S.R. Holbrook (Berkeley Lab).

Research funding: National Institutes of Health; Office of Biological and Environmental Research, U.S. Department of Energy (DOE). Operation of the ALS is supported by the Office of Basic Energy Sciences of the DOE.

Publication about this research: L.W. Hung, E.L. Holbrook, S.R. Holbrook, "The crystal structure of the Rev binding element of HIV-1 reveals novel base pairing and conformational variability," Proc. Natl. Acad. Sci. USA 97(10), 5107 (2000).