Biologists at Purdue University have found how the dengue virus matures and becomes infectious. "This is possibly the most detailed understanding of how any virus matures," said study co-author Michael Rossmann, Ph.D., the Hanley Distinguished Professor of Biological Sciences at Purdue University, and NIAID grantee.
The study, published in two back-to-back research papers in the journal of Science, pertains to all viruses in the family of flaviviruses, which includes insect-borne diseases such as dengue, West Nile, yellow fever and Japanese encephalitis. Dengue, the most prevalent mosquito-borne viral disease affecting humans, results in about 50-100 million cases of dengue fever and 250,000 to 500,000 cases of the more severe dengue hemorrhagic fever/dengue shock syndrome each year, with about 20,000 deaths. "Ultimately, researchers might want to find ways to treat or prevent viral infections, such as dengue, but in order to do that we first have to learn how viruses work, how they mature and initiate infection," said Dr. Rossmann.
The research team detailed critical changes that take place as the virus is assembled and moves from the inner to the outer portions of its host cell before being secreted so that it can infect other cells. Virus particles are exposed to progressively less acidic conditions as they traverse this "secretory pathway," and this changing acidity plays a vital role in the maturation of the virus.
The dengue virus moves through compartments inside the cell called the endoplasmic reticulum and the trans-Golgi network. While immature, virus particles are incapable of fusing with cell membranes, preventing them from infecting their own host cells and ensuring their maturation. Once mature, however, the virus particle is able to fuse to cell membranes, a trait that enables it to infect new host cells. As a virus particle matures along the pathway through the host cell, it changes the protein structure, or “conformation,” in its outer shell. The team mimicked the trans-Golgi network environment in test tubes, enabling them to study the virus’s changing structure with increasing acidity.
They noted that the surface of each virus particle contains 180 copies of a component made of two linked proteins called the 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.
Using a technique called cryo-electron microscopy to gain a more detailed view of virus structure; the researchers learned that the precursor membrane protein remains in place until the virus is ready to exit the original host cell. Therefore, the precursor membrane protein is retained on the virus surface even after the enzyme detaches the two proteins. 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. They 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. To produce the complex of the two proteins, they 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. They also had to mutate the protein to remove sites where furin normally attaches, preventing the proteins from being snipped apart. A better understanding of this structure has enabled scientists to learn why the immature form does not fuse with membranes.
These studies to define the various metamorphic structures that flaviviruses exhibit during the viral replication cycle could well lead to the design of drugs for treatment of diseases caused by this family of viruses.
Li et al, Science, 319:830-1834 (2008); and Yu et al, Science, 319:1834-1837 (2008)
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Last Updated November 10, 2009