A Common Cold for the Cure?

By Jason Socrates Bardi

"A cold in the head causes less suffering than an idea."

—Jules Renard (1864–1910)

Several years ago, Associate Professor Glen Nemerow of the Department of Immunology at The Scripps Research Institute went looking for a new virus.

He had been interested in the interactions of viruses and cells and the pathways by which DNA viruses enter cells. And trying to elucidate these mechanisms, he had been working with a common type of herpesvirus called the Epstein-Barr Virus (EBV).

EBV was proving to be extremely difficult to work with. It was difficult to culture and produce in large quantities, and it was unstable and would fall apart in the test tube.

So, to simplify his life, Nemerow decided to switch viruses, and in the early 1990s, he began working with a pathogen that causes the common cold—the adenovirus. These adenoviruses, he found, can be grown in large amounts and they are highly stable and can last for years if stored properly.

But working with them has done anything but simplify his life. Now, because adenoviruses are of interest to structural biology, immunology, and molecular medicine alike, Nemerow is at the center of a half dozen collaborations with investigators at Scripps Research and at other institutions.

"Over the years the virus has proven to be a useful tool for understanding a whole host of problems," says Nemerow.

A Large and Complex Virus

Adenoviruses are a collection of very old viruses that infect a number of vertebrate species, including humans. There are at least 51 distinct types, which are found all over the world and have the ability to infect cells all over the body.

Typically, adenoviruses cause acute infections in the upper respiratory tract, and these infections manifest as common colds. They can also cause gastroenteritis by infecting cells in the gastrointestinal tract, and conjunctivitis (pink eye) by infecting cells in the eye. Normally, the body gets rid of these infections readily.

The viruses have a simple structure of a protein shell assembled from 240 hexon proteins and 12 penton proteins in a compact icosahedron—a structure consisting of 30 edges, 12 vertices, and 20 triangular faces.

This icosahedral capsid is about 90 nanometers in overall diameter and contains a tightly packed single piece of double-stranded DNA about 35,000 nucleotides long surrounded by associated proteins.

"It's a very large and complex virus," says Nemerow.

Each of the 12 vertexes of the virus is anchored by a penton protein, and each penton protein is decorated with a fiber. Nemerow has shown that the long fiber protein on the viral shell is an important element in the virus because it allows the virus to enter a cell.

How the adenovirus enters cells is a major focus of Nemerow's laboratory at the moment. "Although the virus is very good at [entering cells]," he says, "we still don't have a good molecular explanation of how it's happening."

Nemerow is also interested in what happens to adenoviruses once they are inside cells. The whole viral particle does not seem to enter the nucleus. Rather, it disassembles and the viral DNA and perhaps a few associated proteins enter the nucleus.

Once inside the nucleus, the virus expresses "early" genes that it uses to subvert the cell's machinery for its own uses. These early genes turn on the cell's replication machinery, which then begins transcribing the virus into mRNA, which is transported outside the nucleus and translated into viral proteins. These large proteins are then imported back into the nucleus where new viral particles are assembled.

Nemerow collaborates with Scripps Research Professor Larry Gerace to look at how viral assembly is linked to the transport of viral proteins into the nucleus. More broadly, they are interested in how nuclear transport occurs in general.

Size Does Matter

Nemerow has worked out the broad picture of adenovirus entry in the last couple of years. The effort was significant because it provided the first example of a virus that could gain entry into a host cell through multiple receptors—something that has since been observed in other viruses, including HIV.

This fact has major implications for medicine, and it has allowed adenoviruses to emerge as an important tool for gene therapy—the field of medicine that seeks to provide vehicles for inserting new genes into the cells of patients who need them.

Because adenoviruses are so efficient at getting their DNA into cells, they make effective tools for basic research and medicine.

One of the main areas of application of the virus is gene delivery, and the virus can reliably deliver a large gene into a target cell. That is, after all, what they are designed to do. Scientists like Nemerow have simply to redesign the virus so that it cannot replicate and so that it delivers a therapeutic gene instead of a viral one.

There are other viruses that can do the same thing, but adenoviruses are popular because they can deliver a large piece of DNA. More importantly, because adenoviruses can target various cell types, they can be designed to efficiently deliver DNA into particular cells by modifying the viral fiber proteins to interact with particular cellular receptors.

"Various forms of the virus are being harnessed for gene delivery applications," says Nemerow.

Certain types of adenoviruses, for instance, are associated with severe eye infections, and several years ago, Nemerow and his colleagues began asking why these particular adenoviruses were so adept at infecting the eye.

Much of this work was done in Nemerow's laboratory by Eugene Wu, who is set to graduate from The Kellogg School of Science and Technology next week. Wu did his thesis work on understanding the molecular basis of why these particular adenoviruses are associated with eye infections.

"What he found was that the coat protein of these types of viruses was different from the coat protein of other types of adenoviruses that affect the respiratory track," says Nemerow. "It turns out that one of these adenoviruses has the ability to deliver a gene to photoreceptor cells in the retina."

The adenoviruses that infect the eye have a shorter and more rigid fiber protein connected to each of their 12 vertices. The fiber is about a third the length of the normal fiber protein, and this subtle morphological change restricts the virus's ability to enter human cells by binding to the same receptors other adenoviruses use.

Wu showed that the normal fiber protein is flexible and that this is essential for the binding of the virus to its normal receptor—a protein on the surface of human cells called CAR.

Wu went on to identify an alternative receptor that adenovirus uses to bind to cells in the eye. Called CD46, this human receptor is broadly expressed on the surface of many human cells and is also the receptor used by a number of other viruses to gain entry. Measles and one type of herpes, for instance, use CD46. A number of bacterial pathogens also use the CD46 receptor to enter cells, such as Streptococcus pyogenes and Neisseria gonorrhoeae.

Nemerow is now looking to develop compounds that will block the interaction of the adenovirus fiber protein with the CD46 receptor or will interfere with some other downstream event and block entry into cells.

An Eye for Collaborations

Nemerow is also part of a $9.6 million National Eye Institute (NEI) grant titled Fragments of TrpRS to Treat Neovascular Eye Disease that was awarded to a group of Scripps Research investigators a few years ago to deliver therapeutic genes to the retina.

The grant is led by Martin Friedlander, associate professor in the Department of Cell Biology and chief of the Retina Service in the Division of Ophthalmology, Department of Surgery at Scripps Clinic. It also includes Scripps Research investigators Paul Schimmel, who is Ernest and Jean Hahn Professor of Molecular Biology and Chemistry and a member of The Skaggs Institute for Chemical Biology; Dale L. Boger, Richard and Alice Cramer Professor of Chemistry; David Cheresh, professor in the Scripps Research Department of Immunology; and Gary Siuzdak, adjunct associate professor of the Department of Molecular Biology.

"The collaborative nature of this project is extremely important," says Nemerow. "Having a relatively large number of collaborators with expertise in different areas allows us to explore a wide range of options and bring our combined knowledge to bear on a complex problem."

Nemerow's role in the grant is to explore the potential of using his special eye-targeting adenovirus to deliver a gene to treat various diseases of the eye such as macular degeneration. "The idea would be to replace [certain] proteins and prolong the life of those photoreceptors," he says.

The trick is to get the right receptors on the adenovirus to match the receptors on the surface of the human eye cells.

"By changing virus coat proteins, we have in preliminary studies been able to get more efficient gene delivery to photoreceptors in vivo," says Nemerow.

In preliminary studies, Nemerow and his colleagues have also had success delivering a reporter gene called green fluorescent protein to retinal cells using his modified adenovirus vectors that target photoreceptors on these cells. And they are planning to look at the efficacy of the vectors to deliver a normal gene (peripherin) to correct macular degeneration in murine models of ocular disease.

Collaborations are one thing that Nemerow does well. A number of other investigators at Scripps Research are interested in the virus because of its utility for getting genes into cells or because of what the virus can teach about how viruses get into cells and traffic to the nucleus.

"One of the great advantages of being here at Scripps is that you do not have to solve every problem yourself," he says. "It's much more fun to establish collaborations to help answer these questions.


Send comments to: jasonb@scripps.edu



"One of the great advantages of being here at Scripps is that you do not have to solve every problem yourself," says Associate Professor Glen Nemerow. Photo by Jason S. Bardi.













A CRYO-EM image reconstruction of a whole human adenovirus (top) and details of a single vertex (bottom). In the image, Hexon proteins are blue, penton base proteins are yellow, and the the fiber proteins are green. Because the fiber proteins are flexible, only their base tips can be resolved by the instrument. Image by Phoebe Stewart, Vanderbilt University.