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Roland Rueckert
on 04/12/2000 at 04:27 PM
Rhinoviruses
Rhinoviruses

The following comments are from Roland Rueckert who until recently was a Professor
in the Institute of Molecular Virology, University of Wisconsin, Madison, Wisconsin.


Well, my first reaction to your history request was "That's a long time ago;
I'm a different person now", meaning I'm not a virologist anymore. For three
years I've been thinking and breathing forestry and ecology.


But he relented and continued:


OK what's recorded below began as a short description, but as I typed it all
came back pretty vividly.

One part of Michael's history is all wrong. We were not foggy about the
protomer concept in 1985 - Michael was, but I wasn't. We published a paper
in 1969 (The Structure of Mouse-Elberfeld Virus: A Model, R.R. Rueckert, A.K.
Dunker and C.M. Stoltzfus, Proc. Natl. Acad. Sci. 62: 912-919, 1969) and a
second paper in 1971 (Fragments Generated by pH Dissociation of ME-virus and
their Relation to the Structure of the Virion, A.K. Dunker and R.R. Rueckert,
J. Mol. Biol. 58: 217-235, 1971) proposing the protomer concept for picornaviruses.
The only uncertainty was whether VP4 was still situated close by but,
even there, the simplest possibility was that it was part of the protomer.
The crystallographic structure settled it.

As for the relationship with Jim Hogle, you remember he was a student here
with Sundaralingam. I had crystallized poliovirus by accident while
post-docing with Wendall Stanley at Berkeley in 1964. Stanley made it clear
that computing wasn't up to the task of determining a virus structure at
that time. In 1978 when I learned Steve Harrison had solved the protein
subunit structure of Tomato Bushy Stunt Virus I realized the time was ripe.
I asked Sundaralingam, our only biological crystallographer at Wisconsin and
himself crippled by polio. He was tempted but ultimately unwilling to make the
necessary commitment. But Jim, then working on the structure of lysozyme,
leapt at the opportunity. I encouraged him to do post-doctoral work in
Steve's lab to learn virus crystallography; we'd send him poliovirus and if
things looked promising maybe I could convince the Biochem Department to
hire him with a joint appointment in our Institute (then called the
Biophysics Laboratory). Because we planned to mail the virus, I suggested
we'd be less liable to run into trouble with the post office if we used
type 1 vaccine virus rather than Mahoney (Mahoney had been crystallized by
Finch and Klug in 1960). Jim got crystals but they were very fragile in
the X-ray beam. We tried lots of things to improve crystal stability,
fruitlessly. I didn't want to risk sending mg quantities of polio through
the mail. Finally, after considerable trepidation, I suggested to Jim he
talk to David Baltimore about supplying him with Mahoney. That resulted in
his collaboration with Marie Chow.

Within months of the time Jim got high quality crystals he received a very
attractive offer from the Scripps Laboratory in La Jolla.

I tried to convince the Biochem Department to hire Jim. Neither Biochemistry
nor the Biophysics Laboratory at Wisconsin were able to marshal an adequate
counteroffer and, to my keen disappointment, it became clear Hogle could
hardly refuse the Scripps offer. I did arrange for Joseph Icenogle, a graduate
student from my laboratory, to join Hogle at La Jolla and this enabled Jim
to get off to a fast start at Scripps. (Icenogle later joined the virology
section at the Centers for Disease Control in Atlanta.)

Meanwhile at the Strasbourg Virology Congress in 1981 Michael Rossmann,
having learned of Hogle's success, told me that he planned to proceed
vigorously with poliovirus crystallography. In Michael's hotel room that
evening I argued against this plan on the grounds that it might jeopardize
Hogle's research career and that it made better sense to study a
picornavirus belonging to a different family and thereby benefit from the
comparisons that would emerge as the structures were complete. I suggested
human rhinovirus 14. I knew it could be produced and purified in sufficient
bulk, was stable enough for crystallization and posed no health hazard to
lab workers. The latter point ultimately proved crucial in a race between
Hogle and Rossmann to solve the first crystallographic structure of an
animal virus. I agreed to provide virus for preliminary studies and to
train key personnel in Rossmann's laboratory with skills necessary for
growing and purifying virus. To promote communication and maintain
momentum we initiated regular annual meetings at Wisconsin.

I was interested in correlating picornavirus structure with its functions.
How did they attach to cells and what kind of structural rearrangements are
involved in the infection step, i.e. release of its RNA genome from the
shell into the cytoplasm of the host cell? I hoped that neutralizing
monoclonal antibodies might provide a key tool. That antibodies might be
useful for this purpose was first suggested by Dulbecco and coworkers in
their 1956 paper on neutralization of animal viruses. They interpreted
kinetic evidence, much like that used in radiation target theory, to mean
that a single antibody molecule could neutralize a poliovirus particle.
Moreover they argued this was not at an attachment step. But because of the
complexity of antibodies in antiserum, purification of the antibodies in
question was technically infeasible. The arrival of monoclonal antibody
technology changed all that. Inspired by Phil Minor's preliminary work on
neutralization resistant mutants of poliovirus, we launched a program aimed
at defining neutralization sites on HRV14.

Ann Mosser began with poliovirus and then taught Barbara Sherry how to
make neutralizing monoclonal antibodies against HRV14. Barbara eventually
isolated 35 B-cell clones producing neutralizing antibodies. These she
used to isolate neutralization-resistant ("escape") mutants. These mutants
were then used to classify the panel of antibodies into four groups using a
microtiter neutralization assay. Sequencing the RNA showed that most of the
escape mutants differed by substitution of one amino acid for a patch at
the surface of the virus. Moreover each patch was correlated with one of
the four antigenic groups previously defined in a novel way by screening
for cross-resistance.

Within a month of the time Barbara located the four escape mutation
patterns, Rossmann and his coworkers had solved the rhinovirus structure to
a resolution good enough to see that at least two of the mutable residues
resided on the surface of the protein shell. He invited us to bring our
collection of mutations to Purdue and help them trace their location in
the electron density map. The map consisted of over a hundred planes
stacked in a pile some 15 inches high. We color coded each alpha carbon in
the chain, blue for VP1, green for VP2 and VP4, and red for VP3. It was a
tedious process - and a tense one! One by one, over the course of a long
day, we identified one after another of Barbara's mutants. At the end our
hearts jumped for joy. Every single one of the escape mutations was
located at the surface of the shell. Moreover the mutations were organized
in four patches with each mutable side chain projecting into the patch to
which it had been assigned by the cross-resistance mapping. No exceptions.
It was an incredible high - total intoxicating elation. It gave us an
unanticipated conviction of certainty in our results that one almost never
experiences in experimental science.

I realized at this moment the privilege of working with Michael Rossmann
- and the luck of it. Michael accepted the disadvantages of working on the
rhinovirus project, thus protecting Hogle's career. Poliovirus was known to
form crystals of high quality, its sequence was known and antibody
resistant mutants were already accessible. None of these advantages were
available for HRV14. Yet Michael conquered these disadvantages,
including a late start by his daring, determination and skill. Oh,
and the element of luck? Rossmann was able to use the synchrotron to
collect diffraction data while Hogle was not. That is because HRV14,
unlike the Mahoney strain of poliovirus, was not considered a safety
hazard. That, in the end, may have been the clinching factor in that
exciting race for the first animal virus structure.

There is more about Roland in the Viruses From Structure to Biology site at:
http://medicine.wustl.edu/~virology/rueckert.htm


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