July 25, 2005 SAN FRANCISCO -- It's one of the most common viruses in America, and one that causes the most guilt and shame. It can get inside almost any kind of human cell, reproduce in vast numbers, and linger for years in the body, causing everything from recurrent genital blisters to sores around the mouth. Its complications can kill, and it may increase susceptibility to many nerve and brain disorders.
But until now, scientists haven't fully understood how the herpes simplex virus (HSV) manages to do all of this. And that has stood in the way of developing more targeted, effective treatments against it to help those infected.
New research from the University of Michigan Medical School may help change that.
An estimated 45 million Americans have genital herpes and millions more have the more visible oral variety. Once someone is infected, they're infected for a lifetime. New medicines for herpes infection are badly needed; currently, antiviral drugs can quell symptoms of an outbreak, but not eliminate the virus. And, there's increasing evidence that HSV may damage the nerve cells in which it hides between outbreaks, possibly contributing to neurological disease.
In a presentation Sunday at the International Congress of Virology and in two new papers in the Journal of Virology, U-M researchers are reporting the discovery of a receptor that appears to function as one "lock" that HSV opens to allow it to enter human cells. They've also found the gene that controls the production of that receptor, deciphered some aspects of the receptor's structure, and developed a pig-cell system that could be used to test new anti-herpes drugs.
The findings may help explain why the oral and genital herpes virus has such a successful track record: The receptor, dubbed B5, is made by most cells for another purpose not yet understood. HSV appears to have evolved a way to latch onto it, and fool the cell into letting the virus in. And since most cells express the gene for the B5 receptor, this may be a reason HSV can get into most kinds of cells.
"This may be one central part of the Achilles' heel in interactions of herpes virus with a cell to start infection. We can use the receptor molecule to try to understand the process and perhaps combat infection at this vulnerable site," says A. Oveta Fuller, Ph.D. the leader of the U-M team, senior author on the two papers and an associate professor in the U-M Medical School's Microbiology and Immunology Department. "While we're still a few years away from being able to use this new knowledge to find effective drug candidates, this is a very exciting confluence of discoveries."
The U-M holds a patent on the system and methods that the team used to make the discoveries.
Coincidentally, the U-M team's findings about the B5 receptor are being published at about the same time as an Italian team's reports about a possible 'key' on the herpes simplex virus surface that may match the 'lock' found by the U-M team. The Italian team has identified a region of a viral surface protein that matches the U-M team's predictions of what the virus likely would use to bind and engage the B5 receptor.
"It appears that B5 is a new class of viral receptor. Unlike other viruses so far, HSV seems to have evolved to take advantage of a broadly present cellular protein that has properties like that of known cellular fusion machinery," says Fuller. "No other virus has been shown to use a cellular fusion protein for entry into cells."
She explains that the search for the mechanisms by which HSV enters cells has been hindered by the fact that the virus is very good at entering so many kinds of cells. The many possibilities for virus binding to cells make deciphering the entry process a difficult problem to solve.
The gene that encodes B5 had in fact been sequenced, but not characterized, as part of the Human Genome Project. Discovering its role and studying the HSV entry mechanism was tricky and near impossible until Fuller's team discovered a type of pig kidney cell that isn't vulnerable to infection by human herpes virus. They searched the genome library to find genes essential to HSV infection, isolated the B5-coding sequence, and figured out how to get pig cells to express the human B5 protein to allow the pig cells to be infected with human herpes virus.
For these studies, Fuller credits the persistence of research team members in working with the genomic library and culture of human and pig cells, especially U-M doctorate graduate Aleida Perez and postdoctoral fellows Qingxue Li and Pilar Perez-Romero. Perez-Romero is first author of one of the two new papers, and a co-author on the other.
The two new papers show that the B5 receptor has important features that could explain why it is important to HSV's ability to fuse with the fluid membrane that encloses every human cell. The researchers were able to show that by placing only the DNA sequence that encodes B5 into HSV-resistant pig cells, they could make the pig cells susceptible to HSV. They were also able to block viral infection of both human cells and susceptible pig cells by adding to cell cultures a synthetic peptide made to mimic the structure of a smaller region of the B5 receptor. This peptide looks like a functional region of B5 and apparently interferes with virus engaging of the cell receptor.
The papers detail how the team isolated and characterized the gene that encodes B5, called hfl-B5, and used the DNA sequence to find out more about the protein structure of the B5 receptor. In the presentation at the International Congress for Virology, Fuller will describe recent findings that further confirm B5's importance in HSV infection.
The virology team reports that the B5 molecule appears to form a shape called a coiled coil. This intricately wound structure, they believe, may be similar to the structure of some fusion proteins of viruses and also to cellular proteins called SNAREs. Typically, SNARE proteins help cells to manage the fusion of membranes of vesicles inside the cell with other specific vesicles. Vesicles are tiny membrane-encased packets that encapsulate neurotransmitters, enzymes or other important substances and allow them to be transported within and between cells.
The researchers were able to show that B5 sits in the cell membrane with one end of the protein exposed outside of the cell ready to link up with viruses -- or to serve the receptor's "real" function, which still remains to be discovered. They also showed that HSV does not enter into pig cells that have an altered human B5 protein that is changed by mutations that affect a functional region important to forming a coiled coil.
"If B5 is a SNARE-like cell fusion receptor", Fuller says, "it may turn out to be useful for more than HSV drug treatment. It could act as a way to link vesicles containing drugs with cells, and deliver them inside". She is currently collaborating with U-M nanotechnology researchers on this concept.
The findings suggest that B5 or its viral ligand could be a target for antiviral treatment, much like cell receptors for the entry of human immunodeficiency virus (HIV) into cells have become targets for new AIDS drugs.
The research team included Gregory DeLassus and Santiago Lopez, graduate students recruited by the Program in Biomedical Sciences and the NIH-supported Genetics Training Program. Both programs are part of the U-M's graduate training for biomedical researchers. Funding for the studies was provided by the National Institutes of Health, the Herpesvirus Foundation and pilot grants from the U-M.
Reference: Journal of Virology, June 2005, p. 7419-7430 and p. 7431-7437, Vol. 79, No. 12.
Session 80V, Symposium: Virus Receptors, Congress of Virology, International Union of Microbiological Societies, San Francisco CA
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