SCIENTISTS DISCOVER HOW NIPAH VIRUS ENTERS CELLS

From: National Institutes of Health

Working independently, two research teams funded by the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), have identified how Nipah and Hendra viruses, closely related viruses first identified in the mid-1990s, gain entry into human and animal cells.

Nipah and Hendra are emerging viruses that cause serious respiratory and neurological disease. People can get these deadly viruses from animals. Beginning in 1994, public health officials have recognized disease outbreaks in Malaysia, Singapore, Bangladesh and Australia. Both viruses use a protein essential to embryonic development to enter cells and begin their often-fatal attack, report researchers at the University of California, Los Angeles (UCLA) and the Uniformed Services University of the Health Sciences (USUHS) in Bethesda, Maryland.

The UCLA team, headed by Benhur Lee, M.D., describes its findings in a "Nature" paper posted online on July 6. The report by the USUHS researchers, led by Christopher Broder, Ph.D., is appearing online the week of July 4 in the "Proceedings of the National Academy of Sciences".

Using different methods, both teams identified a specific cell surface receptor, Ephrin-B2, as the doorway used by Nipah and Hendra viruses to get inside cells. This receptor is found on cells in the central nervous system and those lining blood vessels. It is crucial for the normal development of the nervous system and the growth of blood vessels in human and other animal embryos. Ephrin-B2 is found in humans, horses, pigs, bats and other mammals, which explains the unusually broad range of species susceptible to Nipah and Hendra virus infection.

The team narrowed the search for the Nipah/Hendra receptor by first sifting through the genetic sequences of 55,000 possible receptors using microarray technology as a molecular "sieve."

The scientists compared microarray signals from the 55,000 genetic sequences in one set of Nipah virus-resistant human cells with microarray signals from three sets of human cells that the virus can infect. This enabled the research team to narrow the possible number of receptor proteins to 120 by identifying those present in the virus-susceptible cells but absent in the virus-resistant cells. They winnowed the possibilities further -- to just 21 -- by selecting only those candidate receptors within the molecular weight range they expected. They selected 10 expressed at high levels in the susceptible cell lines and inserted them, one by one, into the cells that resisted Nipah virus to identify the receptor. When they inserted the gene for Ephrin-B2, the previously Nipah-resistant cells admitted the virus.

The UCLA team, with collaborators at the University of Pennsylvania, took a different approach, using tools of advanced molecular biology as well as old-fashioned detective work to identify the Ephrin-B2 receptor. They knew the receptor would be abundant among the type of cells Nipah virus attacks, that is, nerve cells and cells lining blood vessels.

To identify the human cell receptor, they created a bait: the Nipah protein that docks to that unknown receptor was attached to part of a human antibody, like a worm on a fishing hook. When they placed this bait onto cells susceptible to Nipah virus infection, it attached to a protein on the cell surface. When placed on Nipah-resistant cells, however, the antibody did not attach to the cells. The scientists used an instrument that sorts molecules by weight to identify that Ephrin-B2 was the cell receptor protein that bound to the antibody/Nipah protein "fishing pole" they had made.

They wanted to confirm their findings, but since they did not have access to a high-level biosafety laboratory as Dr. Broder's team did, the UCLA researchers engineered a harmless virus with Nipah virus proteins embedded in its coat. The UCLA team found that this artificial construct could infect cells vulnerable to Nipah virus but was unable to infect Nipah virus-resistant cells. They also showed that this engineered virus could infect nerve cells and cells lining blood vessels using Ephrin-B2 as the receptor, in the same way as actual Nipah virus would infect these cells.