translation of the picornavirus RNA or genome replication for a review, see Carrasco, 1994. In
contrast, only a very small number of compounds have been described that inhibits the poliovirus
assembly or morphogenesis. Among them are 2- amino-4,6-dichloropyrimidine Py 11 and 2-
amino-5-2-sulfamoylphenyl-1,3,4-thiadiazole G 413. Py 11 reversibly prevents the assembly of the
P1 proteins the precursor of the structural proteins into 14S particles La Colla et al., 1976,
1977. G 413 probably prevents the assembly by interacting directly with the structural proteins
Bonina et al., 1982.
Recently, it has been shown that 5-3,4- dichlorophenyl methylhydantoin referred to here
as hydantoin possess anti-picornavirus activity, specifically against the three serotypes of po-
liovirus, several coxsackieviruses and a less pro- nounced activity against human rhinoviruses
Gerzon et al., 1974; Vance et al., 1997. The mode of action of hydantoin against poliovirus
the best-studied model virus of the picor- naviruses has been studied in detail. It was
shown, that hydantoin is an inhibitor of the mor- phogenesis of the virus Vance et al., 1997.
Poliovirus morphogenesis can be described as a stepwise assembly process Putnak and Phillips,
1981; Hellen and Wimmer, 1992. In this cascade of events, hydantoin affects at least one of these
assembly steps. In hydantoin-treated cells a new putative assembly intermediate 110S could be
detected. As this intermediate has properties con- sistent with that of a packaging intermediate, it
has been suggested that hydantoin inhibits the encapsidation of viral RNA Vance et al., 1997.
Molla et al. 1991, 1993 has developed a cell- free system for de novo synthesis of infectious
poliovirus. This cell-free system is an ideal tool to study the assembly of poliovirus. The cell-free
system consists of an extract of uninfected HeLa cells and is programmed with viral RNA. Conse-
quently, there is direct access to the replication machinery, unhampered by intact cellular mem-
branes. As no extraction procedure is required for the detection of the assembly intermediates,
the isolation and characterisation of such particles is relatively easy. Moreover, assembly intermedi-
ates can be studied without membrane passage. Making use of these properties, we have studied
the mechanism of action of hydantoin in this cell-free system.
2. Materials and methods
2
.
1
. Cells and 6irus HeLa S-3 cells in suspension were grown in
SMEM medium, supplemented with 0.03 glu- tamine and 5 calf serum.
Purified type 1 Mahoney poliovirus was used throughout the experiments. All virus purification
procedures were as previously described Everaert et al., 1989.
2
.
2
. Drug solutions 5-3,4-dichlorophenyl
methylhydantoin was
kindly obtained from Lilly Research Laborato- ries. Stock solutions 10 mgml of the product
were made in dimethyl sulfoxide.
2
.
3
. Preparation of 6RNA The vRNA was extracted from purified po-
liovirus Mahoney strain using an RNA isolation kit PUREscript, BIOzym. In a sterile tube, 1250
m l cell lysis solution PUREscript, BIOzym was
added to 500 ml purified virus 100 mg100 ml, see Section 2 and mixed by vortexing for 5 min.
Afterwards, 500 ml protein-DNA precipitation so- lution PUREscript, BIOzym was added and
mixed by gently inverting the tube 10 times. The mixture was cooled on ice for 5 min and cen-
trifuged for 15 min at 13 000 × g
max
and 4°C in a centrikon A8.24 rotor. The RNA containing su-
pernatant was collected and centrifuged for 15 min at 13 000 × g
max
and 4°C in a centrikon A8.24 rotor. To the supernatant, 1 ml of a 20
mgml glycogen solution and 1500 ml of iso- propanol were added. The tube was gently in-
verted 50 times and centrifuged for 15 min at 13 000 × g
max
and 4°C in a centrikon A8.24 rotor. The supernatant was poured off and the vRNA
pellet was washed with 1500 ml 70 ethanol. Finally, the vRNA was collected by centrifuga-
tion; air dried and resolved in 50 ml diethyl py- rocarbonate treated water RNase free water.
The concentration and the A
260
A
280
ratio of the vRNA preparations were spectrophotometrically
measured and the ratio was found to be 1.9, as expected for pure vRNA. The vRNA was
stored at − 80°C. Before use, the integrity of the vRNA was determined by agarose gel elec-
trophoresis 0.8.
2
.
4
. Preparation of cytoplasmic HeLa extract Cytoplasmic HeLa extract HeLa S-10 was
prepared as previously described Molla et al., 1991, 1993; Cuconati et al., 1998. Briefly, HeLa
cells were harvested en washed with HBSS buffer. Afterwards, the cells were resuspended in
a hypotonic buffer 10 mM K-HEPES, pH 7.4, 10 mM potassium acetate, 1.5 mM magnesium
acetate and 2.5 mM dithiothreitol, cooled on ice for 10 min and lysed with a Dounce Ho-
mogenizer. The degree of lysis was determined visually by phase-contrast microscopy. Cell de-
bris and nuclei were removed by centrifugation and the supernatant was dialysed against 2 l of
dialysis buffer 10 mM K-HEPES, pH 7.4, 90 mM potassium acetate, 1.5 mM magnesium ace-
tate and 2.5 mM DTT. The dialysed lysate was centrifuged and 50 glycerol was added to a
final concentration of 10. After treatment with a micrococcal nuclease to remove the endoge-
nous mRNA’s, the cytoplasmic HeLa lysate was aliquoted and stored by − 80°C.
2
.
5
. Cell-free synthesis of polio6irus The preparations of the cell-free systems were
carried out as described previously Molla et al., 1991, 1993; Cuconati et al., 1998 with slight
modifications. Rabbit Reticulocyte Lysate 10 Promega
and pirodavir 10 mgml, a stabiliser of the viral capsid synthesised by the Janssen Research
Foundation Rombaut and Boeye´, 1991; Rom- baut et al., 1994, was added to each cell-free
system Verlinden et al., in preparation. These additives enhanced the efficiency of the cell-free
system. The final volume of each cell-free system was 25 ml, including 17.5 ml mastermix contain-
ing 55 cytoplasmic HeLa lysate, 1.5 mM ATP, 296.5 mM GTP, 284 mM CTP and UTP, 14.8
mM creatine
phosphate, 37
m gml
creatine phophokinase, 28 mM K-HEPES pH 7.4, 37 mg
ml calf liver tRNA, 1.85 amino acid mix mi- nus methionine, 370 mM spermidine, 545 mM
magnesium acetate, 1.31 mM magnesium chlo- ride, 159 mM potassium acetate, 88 mCi tran
35
S- label™, 1 ml 250 mgml pirodavir solution, 2.5
m l Rabbit Reticulocyte lysate and 4 ml DEPC
treated water, vRNA and other reagents. Incu- bation at 34°C for 15 h was followed by an
RNase treatment.
2
.
6
. Analysis of labelled proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis
SDS-PAGE The SDS-PAGE was essentially as described
by Laemmli 1970 with the following modifica- tions. After denaturation, the proteins were
analysed by
SDS polyacrylamide
gelelec- trophoresis in 12.5 slabgels 0.1 SDS. Gels
were fixed in an acetic acid – methanol – water 104545 solution, fluorographed with Amplify
Amersham, dried and exposed to BioMax MR-1 film Kodak at − 80°C.
2
.
7
. Analysis of assembly intermediates by sucrose gradient ultracentrifugation
In each sample, free
35
S-methionine was re- moved by dialysis. For the analysis of the pro-
capsids 74S and the virus 160S, the dialysed samples were layered onto a 15 – 30 sucrose
gradient in PBS buffer 137 mM NaCl; 2.7 mM KCl;
8.1 mM
Na
2
HPO
4
· 12H
2
O; 1.4
mM NaH
2
PO
4
· H
2
O pH 7.4 and centrifuged for 2 h 15 min at 180 000 × g
av
and 4°C in a centrikon TST 41.14 rotor. For the analysis of the 5S and
14S particles, dialysed samples were layered onto a 5 – 20 sucrose gradient in PBS buffer
and centrifuged for 17 h at 55 000 × g
av
and 4°C in a MSE SW 30.3 rotor. After centrifuga-
tion, 400 ml fractions were collected for analysis of the radioactivity.
2
.
8
. Plaque assay Human Embryonal Kidney HEK cells in
monolayer were grown in 55 mm diameter petri dishes. Serial dilutions of each sample were pre-
pared in SMEM medium. From each dilution, 250 ml was added to a petri dish. After 30 min at
room temperature, 5 ml of a first overlay MEM medium, supplemented with 1.1 agar and 10
calfserum was added. After the petri dishes were incubated for 2 days at 37°C, the HEK cells were
fixed with 5 ml of 10 formaldehyde for 2 h at room temperature. The first overlay was removed
and the cells were stained with a crystal violet solution 0.2 crystal violet, 2 ethanol, and 10
formaldehyde in water for 1 h. The cells were washed with water and the plaques were counted.
3. Results
