RNA binding site differs significantly from other viruses
By Graciela Flores | December 6, 2006
Researchers have unraveled the structure of the influenza A virus nucleoprotein, a development that might lead to the functional design of protein inhibitors used in antiviral therapeutics, according to a report in Nature.
The crystal structure of the nucleoprotein is a trimer, with each monomer molecule shaped like a crescent. "Each molecule has a head and a body, two domains that are linked together by a single polypeptide chain that winds back and forth between them," senior author Yizhi Jane Tao of Rice University in Houston, Texas, told The Scientist. There is also a small tail at the back of the molecule that plays an important role in the oligomerization of the protein.
The viral nucleoprotein has two well known functions: It provides the scaffold for the ribonucleoprotein double helix, which is the complex in which viral RNA is packaged, and it also regulates viral RNA replication through interaction with the viral RNA polymerase. But the topology of the nucleoprotein has eluded structural biologists for years.
"Mine is one of many, many labs who tried previously to get a high-resolution structure of this protein, and we failed," Paul Digard of the University of Cambridge, United Kingdom, who was not involved in the new research, told The Scientist. "Jane's lab found a virus strain that worked better than others. It's fantastic what she managed to do."
Tao and colleagues cloned and expressed the nucleoprotein gene in E coli, obtaining large amounts of highly purified protein. Then, they grew the protein crystals and determined their atomic structure using multiple isomorphous replacement and anomalous scattering (MIRAS.) "In this way, we were able to get the coordinates for each of the nucleoprotein's few thousand atoms," Tao said. "We were very lucky."
"It's a nice piece of work," Adolfo García-Sastre, from Mount Sinai School of Medicine in New York, told The Scientist. "We had the crystal structure for the viral glycoproteins, but in terms of the replication machinery of the virus, there was nothing available," added García-Sastre, who did not participate in the new study.
The visualization of the protein at the atomic level allowed Tao to test specific hypotheses about specific functions, such as that of an internal salt bridge allegedly involved in protein-protein interaction. "The tail loop, which is inserted inside a neighboring molecule, contains about 30 amino acids where the internal salt bridge forms," Tao explained. "With a single amino acid mutation of the residues making the bridge we were able to block out completely the oligomerization of the nucleoprotein molecules."
The team was also able to rule out one of two potential nuclear localization signals previously identified in the nucleoprotein. "In the influenza virus, the nucleus is the site for virus replication," Tao said, "but of the two nuclear localization signals identified in the nucleoprotein polypeptide chain, only one makes sense with our structure."
Finally, they visualized a highly positively charged canyon, an RNA-binding groove, which is found between the head and body domains. One implication of this structure is that the RNA binds to the outside in the ribonucleoprotein complex.
Digard remarked that the topology of the nucleoprotein is completely different from that of other viruses. "It's got the tail, involved in binding other nucleoprotein molecules, and it's got a groove, involved in binding RNA, but the way that groove is made up is completely different from, for example, the rabies virus nucleoprotein," Digard said.
"Now we want to understand how the nucleoprotein regulates viral RNA replication. Studies suggest that it probably interacts directly with the viral polymerase, and we want to confirm that by mutagenesis and by enzymatic activity assays," said Tao.
Links within this article:
"Devious and deadly: influenza through the ages," The Scientist, Jan.19, 2004.
Q. Ye, "The mechanism by which influenza A virus nucleoprotein forms oligomers and binds RNA," Nature, online publication, Dec. 6, 2006.
C. Holding, "Polymerase may be key to flu's virulence," The Scientist, Dec. 6, 2005.
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