RNA Genetics 
KarlaA. Kirkegaard, Ph.D. — Assistant Investigator 
Dr. Kirkegaard is also Assistant Professor of Molecular, Cellular, and Developmental Biology at the Uni- 
versity 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 genetic and 
biochemical implications of this fact for a virus, 
and in any mechanistic similarities or differences 
in genetic processes between organisms with 
RNA and DNA genomes. In addition, we are ex- 
ploring the mechanisms of RNA packaging, RNA 
replication, and RNA recombination in the ge- 
nome of poliovirus and other viruses. We are also 
interested in the interactions between viruses and 
their host cells, especially in the area of RNA- 
protein biochemistry. 
Many of our genetic studies utilize poliovirus, 
a small icosahedral virus with an RNA genome 
only 7,500 nucleotides in length. We have 
shown, for example, that RNA recombination 
among poliovirus genomes occurs at sufficient 
frequency that 1 out of every 25 is a recombinant. 
In contrast to the breaking and joining of preex- 
isting molecules that leads to DNA recombina- 
tion, RNA recombination occurs during RNA syn- 
thesis. This results from the switching of parental 
template RNAs by RNA polymerase. We are devel- 
oping an in vitro RNA recombination system with 
a view to asking more detailed mechanistic ques- 
tions. For example, can the RNA replication pro- 
teins switch RNA strands themselves or are other 
proteins required? How do the RNA strands in- 
volved in a recombination event find each other 
and align in the proper way? Can RNA polymer- 
ases from RNA viruses other than poliovirus also 
accomplish recombination? 
Further investigation into the incidence of RNA 
recombination among other RNA genomes be- 
sides that of poliovirus will be facilitated by the 
use of physical rather than genetic assays. We are 
using the polynucleotide chain reaction to screen 
the progeny of crosses of various RNA viruses, 
phage, and subviral parasites that are not amena- 
ble to direct genetic analysis. We hope to in- 
crease understanding of the prevalence and mech- 
anism 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, Jim Hogle at 
Scripps Clinic 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 
virus RNA into the final virion structure, nor do 
we know the structural requirements of the RNAs 
and proteins participating in the packaging reac- 
tions. Is the viral RNA threaded into an intact, 
preformed icosahedral 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 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 intact empty particles and smaller subviral par- 
ticles from both mutant and wild-type poliovirus- 
infected cells. We hope to find a correlation be- 
tween the mutant defects in RNA packaging and a 
defect in the binding of one of these particles to 
RNA in vitro. Such a correlation would certainly 
strengthen the idea that the particle is an inter- 
mediate in viral assembly, possibly directly re- 
sponsible for packaging the viral RNA. 
We are also using in vitro RNA-binding assays 
to examine the sequence specificity of the viral 
RNA binding to the subviral protein particles that 
are candidate intermediates in viral assembly, to 
understand any RNA structure or sequence speci- 
ficity of viral packaging. 
To investigate the role of host cells in the prop- 
agation of the genomes of RNA parasites, we are 
extending the study of RNA genetics to yeast. 
Yeast cells, unlike the primate cells in which po- 
liovirus and other RNA viruses of medical interest 
are propagated, are amenable to elegant genetic 
analysis, making it possible to identify quite 
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