RNA Viral Genetics 
KarlaA. Kirkegaard, Ph.D. — Assistant Investigator 
Dr. Kirkegaard is also Assistant Professor of Molecular, Cellular, and Developmental Biology at the 
University of Colorado at Boulder and Adjunct Assistant Professor of Cellular and Structural Biology at the 
University of Colorado Health Sciences Center, Denver. After receiving a B.S. degree in genetics from the 
University of California, Berkeley, she developed her doctoral thesis in the Department of Biochemistry and 
Molecular Biology at Harvard University with James Wang. Her postdoctoral work in virology was in 
association with David Baltimore at the Massachusetts Institute of Technology and the Whitehead Institute. 
FOR numerous viruses and other subcellular 
parasites, RNA rather than DNA is the mole- 
cule used for storage and transmission of genetic 
information. We are interested in the conse- 
quences for a virus of having an RNA genome and 
in any mechanistic similarities or differences in 
genetic processes between RNA and DNA organ- 
isms. In addition, we are exploring the mecha- 
nisms of RNA packaging, replication, and recom- 
bination in the genome of poliovirus and other 
RNA viruses. We are also interested in the interac- 
tions between viruses and their host cells, espe- 
cially in the realm of RNA-protein biochemistry. 
Many of our genetic studies utilize poliovirus, 
a small icosahedral virus with an RNA genome of 
only 7,500 nucleotides. We have shown, for ex- 
ample, that RNA recombination occurs among 
poliovirus genomes with sufficient frequency 
that 1 out of every 25 is a recombinant. In con- 
trast to the breaking and joining of molecules 
that leads to DNA recombination, recombination 
of RNA occurs during its synthesis. Genetic re- 
arrangement results from the switching of paren- 
tal templates by the viral RNA polymerase. 
Thale Jarvis, a postdoctoral fellow in our labo- 
ratory, has developed a sensitive quantitative as- 
say for RNA recombination using the polymerase 
chain reaction (PGR). Recombinants can be de- 
tected at a frequency as low as 1 in 1 0*^ parental 
genomes. Using this assay, we have been able to 
learn a great deal about the process of RNA recom- 
bination in poliovirus-infected cells. For exam- 
ple, we discovered that recombination frequency 
increases exponentially throughout the course of 
a single infectious cycle. It was surprising that 
the extensive cytopathic changes during the 
course of poliovirus infection do not inhibit the 
access of the parental RNA templates to each 
other. This work was also supported by a grant 
from the National Institutes of Health. 
Taken together with other data we have ob- 
tained, our results suggest that the process by 
which recombinant RNA genomes are generated 
may be quite simple, and possibly universal 
among RNA viruses. We are using the PGR assay to 
test this idea by looking for RNA recombination 
among other RNA viruses that are less amenable 
to genetic analysis than poliovirus. We hope to 
gain a better understanding of the prevalence and 
mechanism of genetic recombination among RNA 
genomes, a process that is certainly responsible 
for much of the variability and rapid evolution of 
RNA viruses. 
Using x-ray crystallography, James Hogle at 
Scripps Glinic has determined the three-dimen- 
sional structure of the poliovirion. However, an 
appreciation of functional interactions between 
the viral RNA and the virion proteins calls for the 
application of genetics as well as structural bio- 
chemistry. We do not know, for example, exactly 
which subviral protein particles package the po- 
liovirus RNA into the final virion structure, nor 
do we know the structural requirements of the 
participants in the packaging reactions. Is the 
viral RNA threaded into an intact, preformed ico- 
sahedral capsid, or do smaller parts of the capsid 
condense around the viral RNA to form the final 
icosahedral structure? 
We have constructed several mutants in the po- 
liovirus RNA genome and have characterized 
them in great detail. Two of these mutants have 
pointed out a region of the viral capsid, quite 
internal to the virion, that is involved both in RNA 
packaging and RNA uncoating. We are examining 
the RNA-binding properties of subviral particles 
from both mutant and wild-type poliovirus- 
infected cells in hopes of identifying which sub- 
viral particles bind to RNA during intracellular 
assembly. 
To investigate the RNA uncoating defects dur- 
ing cell entry of these mutant viruses, we have 
devised an assay to detect interactions between 
poliovirions and their cellular receptor directly. 
The laboratory of Vincent Racaniello (Golumbia 
University) has identified the poliovirus receptor 
as a cellular adhesion protein of the immunoglob- 
ulin superfamily. Using antibodies prepared 
against peptides from the receptor's amino and 
carboxyl termini, we can analyze proteolytic di- 
gestion patterns of the receptor in the presence 
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