Adding Function to Structure
By Jason Socrates Bardi
To
his crown the golden dragon clung,
And down his robe the dragon writhed in gold,
And from the carven-work behind him crept
Two dragons gilded, sloping down to make
Arms for his chair, while all the rest of them
Thro' knots and loops and folds innumerable
Fled ever thro' the woodwork, till they found
The new design wherein they lost themselves...
—from Lancelot and Elaine by Alfred Lord Tennyson,
1858.
When Molecular Biology Professor John Johnson started working
with cowpea mosaic virus (CPMV) in 1978, he was aggressively
pursuing what was then one of the cutting-edge problems in
structural biology—solving the complete structure of
an intact virus. In 1986, when Johnson published the first
complete structure of CPMV, it was one of the first such structures
solved.
Johnson was concerned then with the relationship of structure
to function. How is the viral genome packaged inside the viral
capsid (shell), and how does that shed light on how the virus
works?
Now, decades later, Johnson knows the structure of CPMV
very well, and he is asking how the virus can be made to work
for us.
In recent years, he has collaborated with Dr. Tianwei Lin,
Assistant Professor of Molecular Biology, and Dr. George Lomonossoff
of the John Innes Institute in England to change the genetic
makeup of the virus to modify the capsid proteins and change
a few amino acids on the outside of the virus. More recently,
Johnson has collaborated with two other TSRI researchers,
M.G. Finn of the Department of Chemistry and The Skaggs Institute
for Chemical Biology and Marianne Manchester of the Department
of Cell Biology.
These researchers have been able to attach a wide range
of molecules to the CPMV surface, essentially enhancing the
virus with the properties of those molecules. This has led
to a program, which Johnson, Lin and Finn are pursuing, in
molecular electronics—aiming to create logic elements
out of viral particles. And, with Manchester, they have been
experimenting with adding proteins and peptides to the virus
surface to create viral warheads that can attack infectious
agents, like measles.
"We never in our wildest dreams imagined that [the virus]
would have these kinds of applications when we started working
on it," says Johnson.
Anatomy of a Cowpea Virus
Cowpea mosaic virus withers and stunts the leaves and pods
of the Vigna unguiculata plant—an important crop
and source of protein in many parts of the world. Like most
plant viruses, CPMV is delivered by insects into plant cells,
and like most plant viruses, CPMV has little need for its
viral envelope to facilitate entry into cells. All these envelopes
are, basically, are a rigid, stable container—shells.
The shell of a CPMV particle is some 30 nanometers in diameter
and is formed by 60 identical copies of a viral protein surrounding
a single strand of viral RNA. These 60 copies constitute 60
equivalent sites for attaching molecules through molecular
genetics.
With molecular genetics Johnson and Lin have developed a
general technique for inserting particular amino acids of
interest onto the surface of the virus by making relatively
conservative mutations in a loop of viral protein on the outside
of the virus. The loop can tolerate different amino acid sequences
without altering the basic structure of the virus.
In fact, by replacing a few amino acids like threonine and
serine with cysteines, the researchers have been able to make
minimal variations to the capsid architecture while putting
these highly reactive groups on the surface of the virus.
These cysteine-containing groups can then be used to attach
other molecules. All of Johnson's years working on the structure
of CPMV help him direct the mutations to specific sites on
the viral surface.
"We know what we are changing," says Johnson.
In what he calls a wonderful "Scrippsian story," Finn describes
the beginning of his collaboration with Johnson as the day
they sat down a few years ago to look at Johnson's crystal
structures. "I was agog," says Finn. "And as soon as I got
it into my head that the viruses were obtainable in gram quantities
and the crystal structures were known, I immediately began
to think of them as molecules."
Molecules to a chemist are also molecular subunits—scaffolds
upon which higher order molecules can be built—and treating
the virus particles as molecular subunits meant that these
viruses could be used to build higher order structures. Finn
immediately proposed that he and Johnson collaborate.
"My laboratory is fortunate enough to have some funds from
the Skaggs Institute for Chemical Biology, so we had some
resources available that we could put to this immediately,"
says Finn. "That was crucial."
In a recent study by the two laboratories, Qian Wang, Tianwei
Lin, Liang Tang, Johnson, and Finn reported the first results
showing that CPMV particles can be used as chemical scaffolds.
Through chemical manipulations, the team attached fluorescent
dyes and clusters of gold molecules to the cysteine residues
because the dyes and the gold clusters could be easily imaged.
The study was a proof-of-principle—an aperitif for
the more hearty applications that they are working on at the
moment. A particularly tantalizing one is to build circuits
of conducting molecules on the surfaces of the viruses to
form a component of a molecular-scale computer—a new
type of "nanowire."
Molecular Electronics
"The ultimate goal in this part of the program," says Finn,
"is to create virus particles that have a function that is
useful in electronic or computational applications."
The primary advantage of a viral wire would be one of scale,
potentially reducing the size of logic elements by orders
of magnitude. Another potential advantage would be cost. Because
the materials are biological, they could possibly be constructed
through self-assembly.
The home run, Finn says, would be to engineer a virus particle
to be a logic element in a circuit—in other words, to
lay down conducting material on the surface of the virus in
a pattern that allows one to probe at one end of the virus
and get an answer at the other end. But, he adds, they are
nowhere near there yet.
Johnson and Finn are currently working on the preliminary
problem of mastering control over the conductive properties
of the virion. Viruses are natural insulators, and the researchers
are attempting to turn them into not just conductors, but
conductors that can be asymmetrically patterned and connected.
Crystallizing the particles could potentially give larger
circuitry.
The crucial first step will be to see if the researchers
can make contact points on the surfaces of the CPMV particles
with elemental gold and then connect these gold contact points
with conducting organic molecules in order to make molecular
circuits.
Another possible application the researchers are pursuing
is blocking viral infection.
Attacking a Virus with a Virus
Manchester, like Finn and Johnson, comes to the collaboration
from a diverse past and sees in CPMV a potential fountainhead
of applications that address her interests, which range from
understanding how viruses attach to and enter cells to developing
new antiviral agents and vaccines. Manchester is particularly
interested in the measles virus.
Measles is a highly infectious virus that causes a maculopapular
rash, fevers, diarrhea, and, in one to two cases out of a
thousand, death. Measles is also highly contagious, and until
the advent of mandatory vaccination programs in the United
States, there were an estimated three to four million cases
annually. Some 90 percent of the U.S. population had had measles
by the age of 15.
"It's basically the most contagious infectious agent there
is," says Manchester.
There has been a commercially available vaccine for measles
in use since 1963, and, though effective, this vaccine is
expensive and must be kept refrigerated for the duration of
its one-year shelf life. This is problematic in tropical climates
like southern Asia and sub-Saharan Africa, which continue
to support endemic measles infection and millions of cases
a year.
The viral receptors that facilitate the entry of measles
into cells are known, and one of these receptors, called CD46,
is of particular interest to Manchester. "We have done a lot
of studies to characterize the binding of the virus to the
outer part of the receptor," says Manchester.
CD46 is expressed on virtually all cells in the body, and
the measles virus has a hemagglutinin glycoprotein that binds
to a single, broad surface on one side of CD46. The area of
CD46 that measles binds to is quite large, and this has allowed
Manchester to make a series of peptides that correspond to
the different regions to which measles binds and test the
peptides for efficacy against measles infection.
"We asked whether they could prevent the virus from infecting
by competing for binding," says Manchester. "And they did."
Since the peptide bound to the virus, preventing the virus
from binding to CD46, Manchester and Johnson wondered what
would happen if they could introduce these peptides onto the
surface of the cow pea mosaic virus using the same general
technique. Could the peptide expressed on the surface of the
virus attack the measles just as the free peptide had?
It could.
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