Viral Workhorses

DeepOcean

Viral Workhorses

From:    Scientific American   


September 02, 2002

Viral Workhorses

Emptied of their infectious nucleic acids, viruses make surprisingly
adaptable tools for nanoengineers

       By Anne M. Rosenthal

A virus, essentially nucleic acid clothed in a protein coat, or capsid, is
well designed for its lifestyle as a cellular parasite. Targeting, packaging
and delivery have all been optimized over billions of years of evolution.
To search out target cells, the viral coat incorporates recognition and
docking sites for specific cell types. To stabilize its negatively charged
genetic package, a virus may carry a remarkably high positive charge on
the capsid interior. And once it arrives at its destination, a virus delivers
its genes into the interior of the targeted cell, where it usurps cellular
machinery for viral purposes. Now researchers are taking advantage of
these viral systems to develop clever nanotechnology applications in
medical imaging and drug delivery, as well as new approaches to building
electronic devices

Mark Young and Trevor Douglas, both at Montana State
University, Bozeman, in conjunction with Jack Johnson’s group
at the Scripps Research Institute in La Jolla, Calif., spent
a number of years sleuthing the structure and assembly of
viruses. They focused on the well-studied Cowpea chlorotic
mottle virus (CCMV). The viral coat of CCMV, like that of many
viruses, is composed of identical protein subunits that
self-assemble into a quasispherical shape known as an
icosahedron. This geometry forms the largest volume of a given
size that can be constituted from identical subunits, notes
Young. The subunits are organized into five-sided and six-sided
capsomeres, which are arranged to form a pattern similar to that
on a soccer ball. CCMV has gated pores that open and close
according to the chemistry of its surrounding environment.
Armed with an arsenal of CCMV knowledge, the researchers began
to explore whether they could redesign the capsid to both
incorporate an imaging agent and zoom in on new targets. In
addition, they wondered, what could be packaged inside the
viral capsid in place of nucleic acid? And how could the gates
be triggered to make deliveries?

It turns out that the capsid, assembled without its nucleic acid
and thus no longer infectious, can serve as a highly modifiable
and versatile addition to the nanoengineer’s toolbox.
Conveniently, the empty capsid even self-assembles in the test
tube or in yeast cells genetically engineered to produce
subunits.
Visualizing the Threat
To conquer a metastasized cancer, physicians must identify the
sites of new tumors and then selectively kill the wayward cells.
CCMV capsids can potentially be engineered to achieve both
goals.

For example, CCMV capsids could improve detection of tiny new
tumors by magnetic resonance imaging (MRI). MRI identifies the
differing responses of hydrogen atoms of water to the presence
of a powerful magnetic field. Prior to being scanned, the
patient may receive an injection of an imaging agent, most
commonly gadolinium. The agent as currently given makes areas
of interest more distinct but usually cannot resolve extremely
small metastases.
Over the past two years, Young, Douglas and their colleagues
significantly raised contrast levels in MRI images by
incorporating the gadolinium atoms into CCMV protein shells.
This promotes gadolinium抯 interaction with water molecules.
That's because the gadolinium molecules--180 of which are woven
into the 28-nanometer-diameter capsid--tend to be in higher
concentration at any given location. Also, unlike
today’sgadolinium agent, which tends to clump, the gadolinium
bound to the capsid surface keeps the atoms evenly distributed
and available to interact with water.
To attach gadolinium to the capsids, the scientists exchanged
the agent for the usual calcium--normally, during capsid
assembly, calcium binds to the protein shell at sites between
the subunits. To further knit gadolinium to the capsid, the
researchers genetically engineered changes on the viral genome
that optimized the binding sites for gadolinium.

Now that they had an improved imaging agent, the scientists
wanted to specifically light up metastases in the MRI images.
To do this, the investigators placed protein-based docking
molecules on the capsids. These docking sites would bind with
proteins expressed on the surface of cancer cells, so the
gadolinium-bound capsids would collect at tumor sites.
Again, the investigators turned to genetic engineering, making
changes in the viral genome. In fact, they found that different
types of docking sites could be placed on one capsid,
potentially making it possible to search for several cancer
types simultaneously.
In one experiment to test the technique, the researchers
attached lamanin peptide 11, a docking site for lamanin-binding
protein. This protein is expressed in large quantities on the
surface of many types of breast cancer cells. Tested in a cell
culture system, viral capsids were able to locate the cancer
cells and bind to them; cancer cell location was detected by
using a laboratory technique, nuclear magnetic resonance
(NMR),
which works on the same principles as clinically used MRI.
By combining docking sites and gadolinium onto each capsid, the
investigators could cluster the capsids around tiny clumps of
cancer cells and image them in experimental systems. But what
about eradicating the metastasized cancer?
Killing Cancer Cells
Bereft of its nucleic acid, the viral capsid could be a handy
suitcase for transporting potent anticancer compounds to tumor
sites. Over the past four years, the researchers have shown that
a variety of compounds can be placed inside the capsule. They
showed that some therapeutic agents used to treat cancer can
be encapsulated through the viral gates or, in a few cases, can
actually be manufactured in situ using the capsid as a tiny
reaction vessel.
That left a final puzzle: Once docked at the tumor with the
drugs, how would the capsid deliver its toxic package? The viral
gate with which nature has endowed CCMV is controlled by pH,
which isn抰 a useful trigger for delivering medication to
specific sites.
Again, the scientists reengineered the evolutionary solution
and designed gates controlled by redox potential (the oxidation
state of a local environment, which influences the tendency of
a molecule to lose or gain an electron). For initial work, the
scientists have used CCMV, which, as a plant virus, does not
enter human cells; however, the final delivery vehicle could
be a reconfigured human virus that does slip into human cells.
Since cellular interiors have a higher redox potential than the
bloodstream, viral capsids could be shut tight in transit but
will open their redox-controlled gates once they enter targeted
cancer cells. The scientists are also developing another type
of gate that is triggered by a type of radiation commonly used
in cancer therapy.
The team is currently exploring how the modified virus capsules
work in a mouse model system and is encouraged by promising
initial results. Taken together, the four capabilities of the
newly engineered capsids--high-sensitivity imaging, target
finding, drug transport and controlled delivery--add up to a
potentially powerful, yet minimally toxic, way to fight
metastasized cancer.

Anne M. Rosenthal is based in the San Francisco area.



More to Explore:

Johnson/Schneeman Laboratory of Structural and Molecular Virology, The Scripps Research Institute
Garry Lab, Tulane University page, The Big Picture Book of Viruses
Mark Young's Web site
Ask the Experts: Is it possible to engineer viruses so that they would be helpful to a human host?
Articles from Scientific American's single-topic issue on Nanotechnology, September 2001, are
available for purchase from the Scientific American Archive
Linda M. Stannard, Virus Ultra Structure