Viruses entrust their most fundamental function - reproduction - to the host cells they infect. But it turns out this highly economical approach also creates vulnerability.
Researchers at Rockefeller University, with input from a team at the Medical Research Council's National Institute for Research (NIMR; now part of the Francis Crick Institute), have found an unexpected way the immune system exploits the flu virus's dependence on its host's machinery to create new viruses capable of spreading infection. This discovery suggests a new approach to combating flu.
"Influenza A, the virus we studied, relies on a host's protein-cutting machinery to put the final touches on new viral particles. Our research has shown that the host immune system fights back by turning off this machinery," said Professor Charles Rice of Rockefeller University. "This concept, that a host would inhibit its own protein-cutting enzymes in order to fight off a virus, is entirely new."
The research reveals a new function for the well-studied protein known as PAI-1 (plasminogen activator inhibitor 1) as the key to this defensive strategy. PAI-1 shuts down proteases, which are enzymes that break the chemical bonds within protein molecules. PAI-1 is best known for inhibiting proteases involved in the break down of blood clots. After seeing evidence of a new role for PAI-1, the researchers found that human and mouse cells unable to properly produce it appeared more vulnerable to infection by influenza A. In experiments, they used the subtype H1N1, a derivative of the 1918 pandemic flu and a member of a large family of flu viruses that include seasonal flu.
A cell infected by a virus releases chemical signals known as interferons, which turn up the volume on a legion of defensive genes. "The hundreds of host proteins produced by these interferon-stimulated genes are like an army. We know that, together, they can effectively defend against a viral infection, but we don't know how the individual soldiers fight back, particularly those that interfere with later stages of viral replication, when the virus exits the cell and spreads the infection," said Dr Meike Dittmann, also of Rockefeller University.
They started out by testing a large suite of genes activated by interferon and introduced these individual genes into cells, then infected the cells with the flu. They then watched to see which genes blocked the ability of influenza to spread. As expected, numerous genes inhibited late stages of infection, but one stood out: SERPINE1, the gene that codes for PAI-1.
Given what was already known about PAI-1, Dr Dittmann suspected how it might help cells fight flu.
"A virus attacks a cell using fusion proteins, and if these don't work properly, new virus particles get out of an infected cell just fine, but they cannot spread the infection to other cells. Proteases activate fusion proteins by clipping them, but on its own influenza A doesn't have the gene for the protease it needs. As a result, the virus relies on the host proteases to do the job," Dr Dittmann said.
Subsequent experiments confirmed PAI-1 did indeed prevent the cutting of the fusion protein, known as hemagglutinin, and that high levels of PAI-1 prevented the virus from producing particles capable of spreading the infection. Furthermore, mice that lacked the gene for PAI-1 generally fared worse than their peers when infected with the influenza A virus. Experiments led by Andreas Wack of NIMR used tissues cultured from mice's tracheae to confirm PAI-1's role in fighting off the infection.
Human cells were the final step. Cells derived from patients with mutations in SERPINE1 were infected and accumulated higher loads of the virus than cells derived from people without mutations in the gene.
Dr Andreas Wack of NIMR said: "This was a great collaboration as we were able to build a bridge between Dittmann's findings on cell lines in the lab and what was seen in PAI-1 deficient animals, thereby contributing to the discovery of a novel way to fight virus infection. PAI-1 has been studied for a long time but was never linked to influenza. Since PAI-1 is a member of a larger family of protease inhibitors and many viruses rely on host proteases during their life cycle, this will become an interesting field for future antiviral strategies."
The paper, A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation, is published inCell.