Findings reveal how dengue virus matures, becomes infectious

"There are many membranes in this trans-Golgi network, so the immature virus is always surrounded by membranes," Chen said. "In fact, the environment of the secretory pathway is very similar to what the virus encounters while it enters and infects a new host cell. So the question is, why doesn't the virus fuse to membranes on the way out""

The researchers have examined the crucial role played by the changing acidity as the immature virus travels through the compartments.

"This change in acidity was already known, but its impact on the maturation process was not known until these new findings," Rossmann said.

As a virus particle matures along the pathway through the host cell, it changes the protein structure, or "conformation," in its outer shell.

Yu mimicked the trans-Golgi network environment in test tubes, enabling the researchers to study the virus's changing structure with increasing acidity.

The surface of each virus particle contains 180 copies of a component made of two linked proteins called precursor membrane protein and envelope protein.

The precursor membrane protein prevents the immature virus from fusing with membranes by covering an attachment site in the envelope protein. During maturation, an enzyme called furin snips the connection between the two proteins, eventually exposing the envelope protein site and enabling the virus to fuse with membranes.

Yu learned, however, that the precursor membrane protein remains in place until the virus is ready to exit the original host cell. The researchers used a technique called cryoelectron microscopy to gain a more detailed view of the virus.

"So, the precursor membrane protein is retained on the virus surface even after the enzyme detaches the two proteins," Chen said. "This is a critical step because the virus is ready to mature but still is incapable of fusing with membranes until after it exits its own cell."

The researchers also determined that the environment must be acidic before the enzyme will snip the two proteins, and they examined the structure to learn specifically why the increased acidity is needed.

Li used fruit fly cells to produce large quantities of the linked proteins so that researchers could study them with a method called X-ray crystallography. Using crystallography, the researchers were able to visualize and study the combined structure of the precursor membrane and envelope proteins.

"Having a better understanding of this structure will enable us to learn why the immature form does not fuse with membranes," Rossmann said. "Ultimately, researchers might want to find ways to treat or prevent viral infections, but in order to do that we first have to learn how viruses work, how they mature and initiate infection."

To produce the complex of the two proteins, Li first had to replace the insoluble "transmembrane region" of the protein with a soluble segment, a step essential for using the fruit fly cells to manufacture the proteins. He also had to mutate the protein to remove sites where furin normally attaches, preventing the proteins from being snipped apart.