any viral proteins can independently accomplish 
this inhibition. In vitro protein secretion assays may 
reveal the identity of the host components whose 
altered function during poliovirus infection inhibits 
protein secretion. This project was also supported 
by a grant from the National Institutes of Health. 
Recombination and Replication 
of Polioviral RNA 
One mechanism by which such a wide variety of 
RNA viruses is generated is thought to be genetic 
recombination. In poliovirus, recombination be- 
tween RNA genomes occurs via the switching of the 
viral RNA replication complex between parental 
templates. The Kirkegaard laboratory has developed 
a polymerase chain reaction (PGR) assay for RNA 
recombination to detect and quantify the formation 
of recombinant molecules at frequencies as low as 1 
in 10^. A collection of temperature-sensitive poly- 
merase mutants generated by charged-cluster-to- 
alanine scanning mutagenesis is currently being 
screened with this quantitative assay for any changes 
in recombination frequency. With the PGR assay it 
was found that, in contrast to previous reports, RNA 
recombination occurs throughout the infectious cy- 
cle of poliovirus, increasing in frequency late in in- 
fection. Thus the drastic reorganization of the cyto- 
plasm during infection does not restrict the access 
of replicating RNAs to each other, and RNA recombi- 
nation can occur whenever RNA polymerization oc- 
curs in the infected cell. This point is underscored 
by the increasing number of RNA viruses for which 
genetic recombination has been reported; the quan- 
titative PGR assay developed in the Kirkegaard labo- 
ratory is suited for use in studying recombination 
frequencies between defined markers in any RNA 
genome. 
Double-stranded RNA Viruses 
of S. cerevisiae 
Three S. cerevisiae genes whose products are re- 
quired for L-A maintenance have been identified in 
the laboratory of Dr. Reed Wickner (National Insti- 
tutes of Health) . One of these genes encodes an 7V- 
acetyltransferase that may stabilize the icosahedral 
capsid; the functions of the other two are unknown. 
A genetic screen in the Kirkegaard laboratory to 
identify more L-A maintenance mutants has yielded 
more than 100 candidate strains; complementation 
analysis is in progress to determine the number of 
new loci identified. 
To engineer mutations into the genome of an RNA 
virus and to study the effects of those mutations on 
the normal replicative cycle it is necessary to initi- 
ate the replicative cycle either with cloned DNA or 
with RNA molecules transcribed in vitro from 
cloned DNA. One goal of the Kirkegaard laboratory 
is to study the effects of defined mutations in the 
RNA genome of L-A on its replication in its S. cerevi- 
siae host and, in turn, to be able to study the effects 
of defined host mutations on mutant and wild-type 
L-A replication. First, with RNA molecules encoding 
the firefly luciferase protein, methods to introduce 
RNA directly into yeast cells were developed. Then 
an L-A cDNA clone provided by Dr. Wickner was 
modified such that transcribed RNA nearly indistin- 
guishable from single-stranded L-A RNA present in 
yeast cells could be synthesized. By using condi- 
tions under which yeast cells grow very poorly in 
the absence of the L-A virus, colonies of cells in 
which the L-A replicative cycle had been initiated by 
the direct introduction of RNA could be identified 
and characterized. Thus "infectious" L-A RNA can 
be synthesized from wild-type and, presumably, 
mutated cDNA clones, and the yeast RNA virus will 
now be accessible to direct genetic analysis. 
Dr. Kirkegaard is also Assistant Professor of Mo- 
lecular, Cellular, and Developmental Biology at 
the University of Colorado at Boulder and Ad- 
junct Assistant Professor of Cellular and Struc- 
tural Biology at the University of Colorado Health 
Sciences Center, Denver. 
Articles 
Jarvis, T.G., and Kirkegaard, K. 1992. Poliovirus 
RNA recombination: mechanistic studies in the 
absence of selection. EMBO / 11:3135-3145. 
Kirkegaard, K. 1992. Genetic analysis of picorna- 
viruses. Curr Opin Genet Dev 2:64-70. 
Maynell, L.A., Kirkegaard, K., and Klymkowsky, 
M.W. 1992. Inhibition of poliovirus RNA synthe- 
sis by brefeldin A. / Virol 66:1985-1994. 
Russell, P.J., Hambidge, S.J., and Kirkegaard, K. 
1991. Direct introduction and transient expres- 
sion of capped and non-capped RNA in Saccharo- 
myces cerevisiae. Nucleic Acids Res 19:4949- 
4953. 
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