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
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