Volume 5 Number 10 October 1978
Nucleic Acids Research
Semi-conservative transcription in particles of a double-stranded RNA mycovirus
G.Ratti and ICW.Buck
Department of Biochemistry, Imperial College of Science and Technology, London SW7 2AZ, UK
Received 24 July 1978
ABSTRACT
During transcription ^n vitro catalysed by the virion RNA polymerase
of Aspergillus foetidus virus AfV-S in the presence of tritiated UTP, the
virus double-stranded RNA becomes labelled in one strand, which has the
same sequence as the single-stranded RNA transcripts produced. Most of
the label incorporated into double-stranded RNA could be chased into singlestranded RNA by further reaction with excess unlabelled nucleoside triphosphates. In reactions containing tritiated UTP the single-stranded RNA
transcripts released after the first round of transcription were unlabelled.
It is concluded that transcription in virions of AfV-S occurs by displacement of one of the strands of double-stranded RNA by the RNA strand being
newly synthesised i.e. the reaction is semi-conservative with respect to
double-stranded RNA.
INTRODUCTION
Aspergillus foetidus virus AfV-S is a small isometric virus with a
genome of three dsRNA1 components2, dsRNA 1 (mol.wt. 4.0x10^), dsRNA 2
(mol.wt. 2.6xlO6) and dsRNA 3 (mol.wt. O.26xlO 6 ). ds RNAs 1 and 2 are each
encapsidated separately in S2a and Sla particles respectively, whereas
dsRNA 3 is found only together with either dsRNA 1 or dsRNA 2 in S2b or
Sib particles3. The capsids of all particles are constructed from the same
polypeptides. Recently it has been shown that the virion RNA polymerase of
AfV-S is a transcriptase . Most of the RNA polymerase activity _in vitro
is found in Sla particles, and the major products of transcription, which
are released from the particles, are full length ssRNA1 copies of one of
the strands of dsRNA 2. Re-initiation of transcription occurs and, during
the course of a 48 h reaction, 6 to 8 ssRNA transcripts are produced, on
average, per molecule of dsRNA 2.
In reactions containing 3H-UTP, label is incorporated also into dsRNA 2
within virions, reaching a maximum level after 4 h. Most of this activity
is also associated with Sla particles.
presented that
reaction.
In the present paper evidence is
labelling of dsRNA 2 occurs as part of the transcription
It is shown that transcription in virions of AfV-S occurs by a
O Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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mechanism involving repeated displacement of one of the strands of dsRNA by
the RNA strand being newly synthesised, so that at the end of each round of
transcription, the displaced strand is released from its template and from
the virion, and the dsRNA remaining within the virion contains one conserved
and one newly synthesised strand i.e. the reaction is semi-conservative with
respect to dsRNA.
In this respect therefore AfV-S RNA polymerase activity
proves to be different from the other virion associated, dsRNA dependent,
RNA polymerases until now described.
METHODS
Growth of the fungus, preparation and purification of virus, RNA
polymerase reactions, preparation of virus RNA, separation of ssRNA and
dsRNA, ssRNA/dsRNA hybridisations and analysis of RNA by gel electrophoresis
were carried out as described previously .
Analytical ultracentrifugation.
Equilibrium density gradient centri-
fugation of dsRNA in caesium sulphate solutions was carried out in a Beckman 'Model E ultracentrifuge equipped with a monochromator and a double-beam
ultraviolet absorption optical system, with photoelectric scanner and multiplexer accessory.
Samples were placed in cells with double-sector charcoal
filled Epon centrepieces and -1° wedge top windows in the 4 place, AN-F
rotor, and centrifuged at 34,000 rev/min for 7O h at 25°C. Densities of
caesium sulphate solutions were calculated from refractive indices-' and
buoyant densities of RNA were calculated as described by Szybalski".
RESULTS
Incorporation of ^ H - U M P into one strand of dsRNA 2
It has been shown previously* that during the AfV-S transcription
reaction -Hl-UMP is incorporated into dsRNA 2 within Sla particles the amount
varying with different virus preparations and corresponding on average to
the synthesis of 40 to 80 % of one RNA strand.
Since Sla particles contain
one molecule of dsRNA 2 and no detectable ssRNA this amount of •'H-UMP
incorporation cannot be accounted for either by synthesis of dsRNA on a
ssRNA template, as with nascent subvirions of reovirus?, or by "filling in"
of short ssRNA tails on predominantly dsRNA molecules, as has been found
with the virion RNA polymerase
of bacteriophage <^6. There remain two
other possibilities: (a) transcription could occur by semi-conservative
strand displacement of dsRNA, so that the newly synthesised strand becomes
q
part of the duplex, as is thought to occur in vivo in the replication of
phage 0 6 dsRNA;
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(b) complete replication of dsRNA could occur in some
Nucleic Acids Research
particles, as has been found with the virion RNA polymerase of a PeniciIlium
virus 10 .
In (a) only one strand of the dsRNA duplex should become labelled,
whereas in (b) both dsRNA strands should become labelled.
Hence the two
possibilities can be distinguished by competition hybridisation of denatured
^H-labelled dsRNA 2 with an excess of unlabelled ssRNA 2 transcript.
Theoretically if both dsRNA strands are labelled, 5096 of the label should
be displaced, whereas if only one strand is labelled either 100% or 0%
of the label should be displaced depending on whether the polarity of the
labelled strand is the same or opposite to that of ssRNA 2. Accordingly
total RNA was isolated from an 18 h RNA polymerase reaction with Sla
particles in the presence of ^H-UTP, and labelled dsRNA 2 was separated
from ssRNA 2 by repeated Lid precipitation.
Heat denatured -Hl-labelled
dsRNA 2 was then annealed with increasing amounts of unlabelled ssRNA 2
and the amount of -^H-dsRNA resulting was determined as the amount of RNA
resistant to the action of RNAases 1 A and Tl at high salt concentrations
(buffer A ) 1 , as described previously*' 11 .
The results given in Table 1
show that 97% of the label can be displaced from dsRNA 2 by its ssRNA 2
transcripts.
It is clear that during transcription dsRNA 2 is labelled
in only one strand which has the same sequence as that of ssRNA 2.
Formation of hybrid density labelled dsRNA 2
The tritium labelling experiments show that during AfV-S transcription
dsRNA 2 becomes labelled to the extent, on average, of 40 to 8O % in one
strand, but do not distinguish whether some molecules are completely labelled and others are unlabelled, or whether all molecules become partially
TABLE 1
Competition Hybridisation
Sample
Ratio
ssRNA/dsRNA
(w/w)
RNAase resistant,
TCA' insoluble
label (cts/min)
% RNAase
resistant
^H-dsRNA 2*
-
4000
3
-
3700
92.5
3H-dsRNA 2 +
u n l a b e l l e d ssRNA 2
31
400
10.0
3H-dsRNA 2 +
u n l a b e l l e d ssRNA 2
62
119
3-0
3H-dsRNA 2 +
u n l a b e l l e d ssRNA 2
97***
132
3-3
H-dsRNA 2**
100
* unheated blank value
** denatured and annealed
*•* a different batch of ssRNA 2 was used
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labelled.
Evidence for the complete displacement of one dsRNA 2 strand in
a proportion of the molecules was obtained from density labelling experiments.
An RNA polymerase reaction with AfV-S was carried out with 5-bromo-
UTP in place of UTP.
Transcription occurred with about the same efficiency
as with UTP and after 18 h ssRNA 2 labelled with 5-bromo-UMP was formed in
molar amount equal to about 3 times that of its dsRNA template i.e. reinitiation of transcription also occurred readily with the brominated
substrate.
DsRNA was isolated from the reaction mixture and centrifuged
to equilibrium in a caesium sulphate density gradient in the analytical
ultracentrifuge.
The u.v. scan, shown in Fig. lc, shows that three main
components, all shown to be dsRNA by their resistance to RNAases A and Tl
in buffer A, were resolved.
Scans of unreacted dsRNAs 1 and 2 (Fig. lb)
and dsRNA 2 alone (Fig. la) showed that the two lighter bands correspond
dlRNA2
(b)
(0
diRNAI
d«RNA2
1-602 1-615
DENSITY (f/ml)
Br-d«RNA2
1-6-41
Fig.l.
Equilibrium density gradient centrifugation of dsRNA i n caesium
s u l p h a t e . Scans at 265 no obtained after centrifuging for 70 h at 34,OOO
rev/min. Approximately 2 pg of RNA were used i n each experiment,
(a) unreacted dsRNA 2 from Sla p a r t i c l e s ;
(b) unreacted dsRNA 2 + dsRNA 1
from p u r i f i e d , unfractionated AfV-S; (c) dsRNA obtained from a 24 h AfV-S
polymerase reaction mixture i n which 5-bromo-UTP was used i n place of UTP.
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in buoyant density to dsRNA 2 (47% G+C) and dsRNA 1 (6096 G+C) respectively;
dsRNA 3 which comprised less than 2% of the total RNA was not detected.
It is also clear that after reaction the amount and density of dsRNA 1 was
unchanged, whereas the amount of unlabelled dsRNA 2 had decreased by about
5O% and a corresponding amount of a denser band of bromo-UMP labelled dsRNA
2 has been formed.
The 2.5% increase in density of dsRNA 2 is consistent
with the expected value based on the increased molecular weight of a hybrid
dsRNA containing one brominated and one unbrominated strand, (U:BrU) RNA,
or in comparison with the observed 2.7% increase in density on formation
of hydrid (T:BrU) DNA, after one round of semi-conservative replication of
Escherichia coli DNA in the presence of 5-bromouracil12.
It is concluded
that in this experiment complete strand displacement occurred in about 50%
of dsRNA 2 molecules.
Pulse-chase experiments
The above experiments establish that, during the AfV-S RNA polymerase
reaction, one RNA strand in 40 to 80 % of dsRNA 2 molecules is replaced by
a newly synthesised strand, but do not show that this reaction occurs more
than once.
In order to determine whether the label incorporated into dsRNA
2 was stable or was continually turning over in repeated rounds of transcription, two experiments were carried out.
In the first, an AfV-S prepara-
tion was incubated in an RNA polymerase reaction mixture containing 0.1 mM
ATP, CTP, GTP and 3 H - O T P , and 3 mM Mg 2 * for 1 h and then the reaction
mixture was diluted into 3 volumes of a solution of unlabelled nucleoside
triphosphates such that the final concentrations were 3 mM UTP, 0.1 DM ATP,
CTP and GTP and 7 mM Mg 2 *, and incubation was continued (the chase).
RNA
was prepared by phenol extraction of samples taken at different time intervals during the chase and ^H-RNA resistant and susceptible to RNAase A
digestion in buffer A was measured by using TCA precipitation.
The results
given in Fig. 2, show that 80% of the -hj-UMP incorporated into ribonuclease
resistant RNA during the 1 h labelling period become ribonuclease sensitive
during the chase.
In the second experiment virus was incubated in a standard polymerase
reaction mixture containing all unlabelled nucleoside triphosphates for 4 h
and then -Tl-UTP was added.
After further incubation for 20 mins. (the
pulse), the reaction mixture was diluted as above and incubation was continued (the chase).
RNAase resistant and sensitive RNA were measured in
samples taken at the end of the pulse and after 3.5 h chase as before
(Table 2). It has been shown previously^ that synthesis of ssRNA 2 contin-
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100
u
Z
10
15
TIME(h)
Fig. 2. Turn-over of label in dsRNA. A reaction mixture containing ->H-UTP
(spec. act. 7l4 mCi/nmole) was incubated for 1 h at 30°C and then the specific activity of ^H-UTP was decreased 120 fold by adding UTP (see text).
The high specific activity label incorporated into dsRNA during the first
phase of the reaction was then monitored during the second phase (chase) by
taking samples at various intervals and stopping the reaction with EDTA.
After phenol extraction RNA was dissolved in buffer A and the TCA insoluble
label present after RNAase digestion was determined. This is expressed as
the percentage of the RNAase resistant label (32,000 cts/min) present at
the beginning of the chase (4> C ir the graph).
ues for up to 48 h whereas labelling of dsRNA 2 is maximal after ca. 4 h.
The results in Table 2 show that (a) incorporation of UMP into dsRNA 2 was
still occurring after 4 h of reaction and (b) this incorporation could be
displaced by further reaction.
Further insight was obtained by examination of RNA prepared from
samples at the end of the pulse and after 3»5 h chase by electrophoresis
in polyacrylamide gels containing 8 M urea.
The results showed that at the
end of the pulse -^-labelled R N A w a s found in two bands, one at the top of
the gel and one with the same mobility as dsRNA 2, and in a small proportion
between the two bands (Fig. 3 ) ; no peak of -'H-RNA was found in the position of ssRNA 2, although toluidine blue staining of a similar gel run in
parallel showed that an appreciable quantity of unlabelled ssRNA 2 had been
synthesised, probably mainly during the 4 h of the reaction prior to the
pulse.
If the RNA isolated at the end of the pulse was incubated with
RNAase A in buffer A prior to electrophoresis, the band at the top of the
gel was no longer observed, but the -'H-incorporation into the dsRNA 2 band
had increased from 18,000 counts/min to 34,OOO counts/rain (Fig. 3b). This
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TABI£
2
Pulse-chase experiment
RNAase r e s i s t a n t ,
TCA insoluble label
(cts/min)
RNAase s e n s i t i v e
label
*
(cts/min)
RNA synthesised
between 4 h and
4 h 20 rain (pulse)
20,000
12,800
After 3-5 h chase
5,000
27,800
Sample
(a)
(b)
RNA was prepared by phenol extraction from equivalent amounts of reaction
mixture: (a) at the end of a -'H-UTP pulse (spec. act. 1760 cts min
pinole"1), between 4 h and 4 h 20 min from the start of the polymerase
reaction; (b) after a further 3.5 h of incubation in a reaction mixture
in which the specific activity of ^H-UTP had been reduced 120 times. The
concentration of dsRNA in each sample was adjusted to be the same and 25 pi
(containing 350 ng of dsRNA 2) per assay was used for determination of
ribonuclease resistant and sensitive label. The composition of these RNA
samples was also analysed by gel electrophoresis (Fig. 3 ) .
shows that all the incorporation of 'H-UTP into RNAase resistant RNA was
into structures containing dsRNA 2, presumably with ssRNA tails, of varying
length.
After the chase a peak of RNAase sensitive ^H-RNA with the mobility
of ssRNA 2 appeared (Pig. 3c) and the RNAase resistant 3H-RNA with the
mobility of dsRNA 2 had decreased from 34,200 to 8,800 counts/min (Fig. 3d)
i.e. about 75% of the ^H-incorporation into dsRNA 2 during the pulse was
chased into high molecular weight ssRNA, in good agreement with the results
obtained by direct TCA precipitation.
Displacement of one strand from dsRNA 2
A critical test of the semi-conservative transcription mechanism is to
demonstrate the displacement and release of one strand from parental virion
dsRNA.
If an RNA polymerase reaction, in which transcription occurs semi-
conservatively, is carried out in the presence of ^H-UTP it would be
expected that the ssRNA liberated at the end of the first round of transcription would be unlabelled since it is derived from the unlabelled dsRNA
template;
subsequent rounds would then release
H-labelled ssRNA by dis-
placement of the newly synthesised, labelled strand from the dsRNA duplex.
In contrast the dsRNA template should become labelled in the first round of
transcription.
In order to test these predictions, a preparation of Sla particles,
which contain only dsRNA 2 and no detectable ssRNA, was incubated in a
standard RNA polymerase reaction
containing ^H-UTP and at different time
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sir
Slice
Fig. 3. Pulse-chase experiment: analysis of RNA by electrophoresis in
polyacrylamide gels containing 8 M urea. 0.05 nil of RNA (same samples as
in Table 2) were used for each experiment. After electrophoresis gels were
sliced and counted. The positions of ssRNA 2 and dsRNA 2 were determined
by scanning gels at 260 ran. (a) pulse sample; (b) pulse sample after
RNAase treatment; (c) 3.5 h chase sample; (d) 3-5 h chase sample, after
RNAase treatment.
intervals, samples were taken and ssRNA 2 released into the supernatant was
examined by polyacrylamide gel electrophoresis.
Gels were scanned at 26Onm
and then sliced and counted for 3H-UMP incorporated.
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Incorporation of -'H-
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UMP into dsRNA, after isolation from virions, was examined in a similar vay.
The results (Fig. 4) showed that labelling of dsRNA 2 was greater than that
of ssRKA 2 during the early part of the reaction.
Moreover after 30 min
reaction, gel scans at 260 nm showed that ssRNA 2 was released into the
medium in an amount corresponding to the displacement of one strand from
20% of dsRNA 2 molecules. After this time no label was detected in the
band of ssRNA 2 whereas the band of dsRNA 2 contained more than 30,000 cts/
min.
DISCUSSION
The results show that transcription in virions of AfV-S occurs by a
mechanism involving repeated displacement of one of the strands of dsRNA 2
by the RNA strand being newly synthesised, so that at the end of each round
of transcription the displaced strand is released from its template and
from the virion, and the dsRNA 2 remaining within the virion contains one
conserved and one newly synthesised strand i.e. the reaction is semiconservative with respect to dsRNA.
A diagrammatic representation of the
reaction for two rounds of transcription is shown in Fig. 5. This mechanism
may be contrasted with that of reovirus, in which transcription occurs
conservatively, with no incorporation of labelled nucleoside triphosphate
residues into genome dsRNA^ilA.
The evidence for semi-conservative trans-
cription in AfV-S is summarised below.
(a)
During the polymerase reaction, in the presence of H-UTP, dsRNA
2 becomes labelled in one strand, which has the same sequence as the ssRNA
transcription product (ssRNA 2 ) .
(b)
If 3H-UTP is present from the beginning of the reaction, labelling
of dsRNA 2 reaches a maximum after 4 h reaction . However if the reaction
is allowed to proceed for 4 h with unlabelled OTP and then -Hi-UTP is introduced, dsRNA 2 becomes labelled in the subsequent reaction and the rate of
this labelling is consistent with the rate of ssRNA 2 synthesis after this
period of reaction (Table 2, Fig. 3 ) .
(c) About 80% of the label incorporated into dsRNA 2 can be chased
out by subsequent reaction with unlabelled substrates (Fig. 2 and Table 2 ) ,
resulting in the release of labelled ssRNA 2 (Fig. 3 ) . Failure to chase
out 100% of the label from dsRNA 2 is probably a result of the rapid
deceleration of the initial reaction rate.
It has been observed previously
that the rate of reaction after 6 h is only about 5% of the initial rate.
This may be due partly to failure of some intact particles to re-initiate
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120
TIME(min)
240
Fig. 4. Incorporation of 3 H - U T P into complete ssRNA 2 transcripts and dsRNA
2 template by Sla particles. Sla particles, isolated from purified AfV-S
by two cycles of sucrose gradient centrifugation, were incubated in standard
reaction conditions^ containing ^ H - U T P (spec. act. l80 cts min" 1 pmole" 1 )
at a final virus concentration of 0.O8 mg/ml. At the times indicated 0.1
ml samples were taken and the reaction was stopped with EDTA. The amounts
of label in the ssRNA 2 released into the medium and in the dsRNA 2 remaining within the virus particles were analysed by polyacrylamide gel electrophoresis and plotted against time. The ordinate values correspond to 0.075
ml of original reaction mixture. Open circles, ssRNA 2; closed circles,
dsRNA 2.
the reaction and partly to the disruption of some particles which occurs
during the reaction*.
(d)
The major products of a 20 min labelling period have the
properties of transcriptive intermediates, consisting of dsRNA 2 with long
ssRNA tails, of the type shown in Fig. 5, since in gel electrophoresis they
moved as a slow band near the top of the gel, but after trimming off the
ssRNA tails with RNAase, under conditions where ssRNA is degraded but high
molecular weight dsRNA is stable 11 , moved as a band with the same mobility
as dsRNA 2 (Pig. 3 ) . In contrast intermediates of this type have not been
isolated from a reovirus transcription reaction, which occurs conservatively;
in that case the major products after pulse labelling were incomplete, ssRNA
transcripts which dicKnot remain bound to the dsRNA template after phenol
ext ract i on *'.
(e)
Comparison of the time course of labelling of dsRNA 2 and ssRNA 2
(Fig. 4) with the production of ssRNA 2, measured from u.v. scans of gels
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1
l\
""*"
i
i
\
\
1
1
i
i
i
^_
I
l\
Fig. 5. Diagram of the proposed mechanism of transcription of AfV-S dsRNA,
catalysed, within the virus particle, by the virion associated RNA polymerase. The broken lines represent RNA synthesised during the _in vitro
reaction and do not imply any fragmentation of the product. (For a reaction
carried out in the presence of 3H-UTP, the broken line would represent labelled RNA and the unbroken line, unlabelled RNA). Presumably transcription
starts at the 5' end of the displaced strand. Re-initiation could easily
occur if the ends of the dsRNA template are in close proximity within the
virion e.g. if the RNA is arranged in a circular conformation.
after electrophoresis, shows that labelling of dsRNA 2 was greater than
that of ssRNA 2 during the early part of the reaction and that ssRNA 2
released after 30 min. reaction was unlabelled.
This is consistent with
a semi-conservative mechanism in which unlabelled transcripts would be
released at the end of the first round of transcription and the dsRNA
template would be labelled (Fig. 5 ) .
From the proportion of hybrid (U:BrU) dsRNA 2 molecules produced in
the density labelling experiments (Fig. l) and the amount of -*H-UMP incorporation into dsRNA 2 after 4 h (Fig. 4) it is clear that in these experiments initially 40 to 50 % of Sla particles were involved in semiconservative transcription.
The amount of ssRNA 2 released after 30 min
reaction corresponds to displacement of one strand from dsRNA 2 in 5O% of
the active Sla particles. The reaction was not completely synchronous and
maximum labelling of dsRNA 2 took 4 h. It is probable that either initia3853
Nucleic Acids Research
tion of transcription was asynchronous, or that the rate of transcription
varied in different virions as suggested for reovirus cores
.
The amount of ssRNA 2 released after 30 min reaction was calculated,
on the basis of its content of 25% U and the specific activity of the ^HUTP used in the experiment (see legend to Pig. 4 ) , to be equivalent to
17,000 counts/min if it were fully labelled with UTP as in a conservative
mechanism.
Since no label was detected in the band of ssRNA 2 after this
time and allowing for a sensitivity of detection of 800 counts/min (double
the background in gel slices), it can be deduced that less than 5% of the
transcription, if any, could have occurred by a conservative mechanism.
ACKNOWLEDGEMENT
The work was supported by a grant from the Science Research Council
(to K.W.B.).
REFERENCES
1.
Abbreviations: dsRNA, double-stranded RNA; ssRNA, single-stranded RNA;
RNAase, ribonuclease; TCA, trichloroacetic acid; buffer A, 0.3 M-NaCl
+ O.O3 M-Na citrate, pH 7.0.
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