Evidence for 30~40S RNA as Precursor of the
60~70S RNA of Rous Sarcoma Virus
P. DUESBERG, P H . D . , E. CANAANI, M.S.,
Department
AND K. von der HELM, P H . D .
of Molecular Biology and Virus Laboratory,
Berkeley, California 94702
University
of
California,
ABSTRACT
Duesberg, P., Canaani, E., and von der Helm, K.: Evidence for 30-40S RNA
precursor of the 60-70S RNA of Rous sarcoma virus. Am. J. Clin. Pathol.
60: 57-64, 1973. Rous sarcoma virus (RSV) harvested from cells at intervals of 3 minutes has the same density, sedimentation coefficient, and DNA
polymerase as virus harvested at hourly intervals. The RNA of such RSV
consists of a minor class of 60-70S RNA, a major class 30-40S RNA and a
4-12S class of RNA present in variable concentration. Upon incubation of
virus harvested at 3-minute intervals at 40 C. in cell growth medium or
Tris-saline, most of the 30-40S RNA is converted to 60-70S RNA. The electrophoretic mobility of the 30-40S RNA of RSV harvested at 3-minute
intervals is lower than that of the 30-40S subunits of completely dissociated
60-70S RNA; after heating their mobilities are identical. Heating also releases some small RNA's from 30-40S RNA of RSV harvested at 3-minute
intervals, but five times more 4S RNA is released if the 30-40S RNA is
allowed to convert to 60-70S in the virus. It is suggested that association of
30-40S RNA's with some RNA's of the 4-12S class may take place simultaneously with their conversion to 60-70S RNA.
40S RNA in the virion.12- " • " Yet, free 3040S RNA has not been observed in avian
tumor viruses propagated under similar
conditions. However, it was found recently
that Rous sarcoma virus (RSV), an avian
tumor virus, if harvested from cells at intervals of 5 minutes, contains heterogeneous RNA, smaller than the 60-70S RNA of
virus harvested at hourly intervals.4 The
RNA of such viruses was described to consist of a principal 55-60S component and
(RNA) of all
known groups of RNA tumor viruses consist of a major 60-70S class and a minor
4-12S class of polynucleotides. 6 ' 16 The 6070S RNA has a subunit structure. 6 It can
be dissociated into several major 30-40S
RNA'sB> 10 ' 15 and several small heterogeneous and distinct RNA's. 3 -" There is also
evidence that certain RNA tumor viruses
contain, in addition to 60-70S RNA, free 30THE
RIBONUCLEIC
ACIDS
lueppo7tedDbyTont«« No!*' 71-2173 within the
several smaller
components. Conversion of
Special Virus-Cancer Program of the National
Cancer Institute, National Institutes of Health,
Public Health Service; Public Health Service
Grants CA 11426 from the National Cancer Institute; and Grant AI 01267 from the National Institute
of Allergy
andentire
Infectious
Diseases.
Reprints
of this
Research
Symposium are
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57
58
DUESBERG ET
AJ.C.P.— Vol. 60
AL.
o
IO
FRACTION NUMBER
FIG. 1. Velocity and equilibrium density gradient sedimentation of Rous sarcoma virus
(RSV) harvested at intervals of 3 minutes. A (left), [3H] uridine-RSV harvested at 3-minute
intervals (see text) was precipitated from 100 ml. of medium with an equal volume of saturated
ammonium sulfate and rcdissolved in 10 ml. of standard buffer.3 After centrifugation for 3
minutes at 3,000 X g. the supernatant was reprecipitated and centrifuged at 30,000 X g. for
5 minutes. T h e pellet was redissolved in 1 to 1.5 ml. of standard buffer. It was then mixed
with an appropriate amount of [32P] RSV, harvested at intervals of 3 hours and with a TMV
marker, and was layered on a preformed sucrose gradient (10 to 25%, w/v) in standard buffer
but containing 0.2 M NaCl. Centrifugation was for 70 minutes at 25,000 r.p.m. in Spinco SW
25.3 rotor at 2 C. Radioactivity was determined in a Tricarb spectrophotometer by placing
a 100 til. aliquot of each fraction in 5 ml. toluene-based scintillation fluid containing 20% NCS
(Nuclear-Chicago). T h e TMV marker was located by its absorbance at 260 nm. B (middle), f H ]
uridine RSV harvested at 3-minute intervals was purified from 170 ml. of medium and sedimented as described for A. A 100 p.1. aliquot of each fraction was incubated with 100 /j\. of a solution containing 12 mM MgCl2, 10 mM dithiothreitol, 0.1 M tris, p H 8.0, 0.2% Triton X-100, 2 ng.
poly rA, 1 ng. oligo dTis_18, and 5 M M[ 3 H]dTTP for 2 hours at 40 C. ["H] poly T was determined as described previously 7 ' 8 and was plotted after subtraction of the fH] of the virus.
C (right), identical aliquots of fractions 8 to 15 (Fig. IB) were pooled and diluted with an
equal volume of standard buffer and layered on a preformed sucrose density gradient, prepared from 4 ml. of 55% sucrose and 4 ml. of 20% sucrose in standard buffer. Centrifugation
was for 12 hours at 25,000 r.p.m. in a Spinco SW 25.3 rotor at 2 C.
three to six times more 30-40S RNA than
60-70S RNA and that most of the 30-40S
RNA may be converted to 60-70S RNA in
the virion. We have also asked whether
the viral 4-12S RNA's and the small RNA's
associated with 60-70S RNA 3>1J result only
from partial degradation of 60-70S RNA in
the virion as proposed by some 1 or whether
at least a fraction of these small RNA's
are present in virus freshly budded from
infected cells. Recent studies in this laboratory indicated that in several strains of
avian tumor viruses harvested at hourly
intervals both free and 60-70S RNA-associated small RNA's were present (unpub-
lished). The relative concentrations of
these small RNA's were only little decreased with decreasing age of the virus,
making it unlikely that all of these small
RNA's result from degradation of the 3040S subunits. We report here that even
virus harvested from cells at intervals of
3 minutes contains free 4-12S RNA's and
that the 30-40S RNA of this virus is associated with some small RNA's.
Results
Properties of Rous Sarcoma Virus Harvested at Short Intervals. [3H] RSV in
medium harvested at intervals of 3 rain-
July 1973
59
PRECURSOR OF 60-70S ROUS RNA
utes from infected cultures was concentrated and sedimented in a sucrose gradient together with a [32P] RSV harvested at
intervals of 5 hours (Fig. 1). It can be
seen in Figure 1A that both viruses had
the same sedimentation-coefficient of about
600S, based on a 200S tobacco mosaic virus
(TMV) marker. The distribution of viral
DNA polymerase activity after sedimentation was determined on another aliquot in
a parallel rotor tube but without other
viral markers. It is shown in Figure IB
that peaks of poly(rA) oligo d T dependent
DNA polymerase activity and [ 3 H]uridine
label of virus harvested at 3-minute intervals coincide at about 600S. The buoyant
density of [3H] RSV sedimenting at 600S
(Fig. IB, fractions 8 to 15) in a preformed
sucrose density gradient was about 1.17
to 1.18 Gm. per ml. (Fig. 1C). These experiments indicate that virus released from
cells at intervals of 3 minutes has the same
sedimentation coefficient and buoyant density as virus harvested at intervals of several hours 1 6 and that it contains viral
DNA polymerase.2-18
The sedimentation distribution of the
RNA of viruses harvested at intervals of
3 minutes is seen in Figure 2A. A major
class of RNA sedimented at 30-40S ahead
of a 3IS TMV RNA marker. Only 25%
of the RNA sedimented with the 60-70S
marker of [32P]RSV harvested at intervals
of 3 hours. In addition, a 4-12S peak of
1
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FRACTION NUMBER
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Fie. 2. Sedimentation analysis of the RNA of RSV harvested at 3-minute intervals before and
after incubation of the viruses in cell growth medium at 40 C. A (left), 400 ml. of medium
containing [aH] uridine-RSV harvested at 3-minute intervals was divided in halves. One half
was purified and its RNA extracted in the presence of carrier TMV as described previously. 6
Subsequently an appropriate amount of 60-70S [**P] RNA of RSV harvested at intervals of
several hours was added. Centrifugation was in a 15 to 30% glycerol gradient containing
standard buffer and 0.1% sodium dodecyl sulfate (SDS) for 90 minutes at 40,000 r.p.m. in a
Spinco SW41 rotor at 20 C. Radioactivity was determined as described for Figure 1. B (right).
T h e other half of the [°H] RSV harvested at 3-minute intervals was incubated for 45 minutes
at 40 C. Subsequently the RNA was analyzed as for A.
<
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A.J.C.P.—Vol.
DUESBERG ET AL.
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Fic. 3. Kinetics o£ the conversion at 40 C. of the 30-40S RNA to 60-70S RNA in purified
RSV harvested at intervals of 3 minutes. Purified ["H] uridine-RSV harvested at intervals of
3 minutes was concentrated in the presence of about 0.5 mg. of carrier RSV harvested at
intervals of 5 hours by sedimentation through a 2 cm. column of 30% to a cushion of 80%
glycerol in standard buffer containing 1 mM mercaptoethanol. The pH] virus was then
diluted five-fold in standard buffer and identical aliquots were incubated for 0(A), 5(B),
15(C), and 30(D) minutes at 40 C. prior to extraction of the RNA (cf. Fig. 2). Centrifugation
of the RNA was for 55 minutes, at 50,000 r.p.m. in a Spinco SW 50.1 rotor and otherwise as
described for Figure 2.
slowly sedimenting RNA was present at
approximately the same relative concentration as in viruses collected in periods of
several hours. Storage of purified or unpurified viruses at 0 to 4 C. for 48 hours
did not affect the sedimentation pattern
of the viral RNA.
Conversion in the Virion of 30-40S to
60-7OS RNA. T o test whether the 30-40S
RNA in freshly budded virus may be a
precursor of 60-70S RNA or perhaps the
RNA of abortive virus particles, the following experiments were done. Medium
containing viruses released at intervals of
3 minutes was divided into two aliquots.
One aliquot was incubated for 45 minutes
at 40 C ; another aliquot was kept at 0 C.
Viruses were then purified from both aliquots and the RNA's were extracted and
analyzed. It is shown in Figure 2A and B
that in viruses that were incubated at
40 C , most of the 30-40S RNA had disappeared and that an approximately equiv-
alent amount of radioactivity had appeared in the 60-70S region of the gradient.
In addition, it was observed in this and
other experiments that RNA sedimenting
faster than 60-70S was present in viruses
incubated at 40C. This result suggests that
most 30-40S RNA is converted to 60-70S
RNA and some to even faster sedimenting
forms in the virion.
T o determine whether components of
the tissue culture medium are required to
convert 30-40S RNA of viruses harvested
at intervals of 3 minutes to 60-70S RNA,
purified viruses were incubated for various times in standard buffer 3 containing
10 to 20% glycerol. The results (Fig. 3)
showed that after 5 to 15 minutes at 40 C.
most of the 30-40S RNA, and after 30 minutes practically all of the 30-40S RNA, had
been converted to 60-70S and faster sedimenting RNA. The relative amount of
4-12S RNA was not significantly affected
by incubating the viruses at 40 C.
July 1973
61
PRECURSOR OF 60-70S ROUS RNA
In some viral preparations the rate of
conversion was lower than that shown in
Figure 3 (experiment not shown). This
may have been due to the presence of
virions in such preparations in which the
capacity to convert the RNA was lost or
impaired during purification of the viruses. Attempts to convert 30-40S RNA
after disruption of the viruses by detergents or phenol have not been successful
so far, suggesting that the structural components of the virus must be intact for
this conversion.
Electrophoretic Analyses of 30-40S RNA
of Virus Harvested at Intervals of 3 Minutes. It appeared from sedimentation
analyses such as those shown in Figures
2 and 3 that the 30-40S RNA of viruses
harvested in 3-minute periods sedimented
50
slightly faster than the 30-40S RNA obtained by complete melting of 60-70S RSV
RNA. As shown in Figure 4, the 30-40S
[ 3 H]RNA of viruses harvested at intervals
of 3 minutes (c/. fractions 7 to 10, Fig. 3A)
also had a lower electrophoretic mobility
than a 30-40S [32P]RNA marker, obtained
by complete melting of 60-70S [32P]RSV
RNA. However, after heating to 100 C , the
electrophoretic mobilities of the two RNA's
were identical. We conclude, therefore,
that the primary structures of the 30-40S
RNA's of viruses harvested at 3-minute intervals and of heat-dissociated 60-70S RNA
are very similar or identical.
As shown in Figure 5, the increase of
the electrophoretic mobility of native 3040S RNA after heating to 100 C. (Fig. 4)
is accompanied by the appearance of low
60 0
DISTANCE MOVED
10
30
50
60
(mm)
FIG. 4. Electrophoresis of the 30-40S RNA of [°H] RSV, harvested at intervals of 3 minutes,
before (A) and after (B) heating to 100 C. A (left), the RNA of [=H] RSV harvested at intervals of 3 minutes was analyzed as described for Figure 3 and fractions (cf. 7 to 10, Fig. 3A)
corresponding to the 30-40S peak were pooled. T h e RNA was precipitated with ethanol, redissolved in 50 /xl. electrophoresis sample buffer 0 and divided into two aliquots. A 25 /J.
aliquot was mixed with 5 / J . of an appropriate amount of f""P] 60-70S RSV RNA that had
been heated at 100 C. for 45 seconds in the same buffer. Electrophoresis was done after the
addition of a methylene blue marker for 6 hours at 7 v. per cm. in a 2.1% bisacrylamide
crosslinked polyacrylamide gel.9 Radioactivity of 1-mm. gel sections was determined in a
Tricarb spectrophotometer after incubation in 3 ml. of toluene based scintillation fluid containing 20% NCS and 2 % H s O at 60 C. for 2 to 12 hours. B (right), another 25 M l. aliquot of
the 30-40S [8H] RNA was mixed with the above described [MP] 60-70S RSV RNA. T h e mixture
was then heated to 100 C. for 45 seconds and analyzed as for A.
62
DUESBERG ET
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A.J.C.P.—Vol.
AL.
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DISTANCE MOVED
20
40
60
60
(mm)
FIG. 5. Electrophoresis of the small RNA's associated with the large RNA's of RSV harvested at 3-minute intervals before (A) and after (B) incubation of the viruses for 2 hours
at 40 C. A (left), medium containing [3H] uridine-RSV harvested at intervals of 3 minutes
was divided in halves. T h e 30-40S RNA of one half was prepared as described for Figure 2
and heated at 100 C. as described for Figure 4. It was then mixed with an appropriate amount
of chick [32P]tRNA and with methylene blue and analyzed by electrophoresis for 2 hours at
15 v. per cm. in a 6-cm. gel consisting of an upper 2 cm. section of 2.5% and a lower 4 cm.
section of 6% polyacrylamide. Radioactivity was determined as described for Figure 4. B (right),
the other half of the [3H] RSV collected at 3-minute intervals was incubated in medium for
2 hours at 40 C. T h e 60-70S RNA was then prepared from purified viruses and analyzed as
described for A.
amounts of small RNA species detectable
by electrophoresis in a 6% polyacrylamide
gel. Thus, the difference in electrophoretic
mobility between native 30-40S RNA and
heated 30-40S RNA may be due either to
the association of native 30-40S RNA with
small RNA's or to differences in the conformations of the two forms of 30-40S
RNA's, or both. After heating, the ratio of
4S RNA relative to 30-40S RNA from viruses harvested at 3-minute intervals was
about five times lower than that obtained
previously after analysis of heated 60-70S
viral RNA. 3 This result suggests that the
30-40S RNA's must associate with additional
4S RNA's as they are converted to 6070S RNA in the virus at 40 C. T o test this
directly, we incubated an aliquot of the
virus harvested at 3-minute intervals,
used above, for 2 hours at 40 C. in cellgrowth medium to allow conversion of its
RNA to the 60-70S form, and determined
the ratio of 30-40S to small RNA's after
heating 60-70S RNA to 100 C. It was found
(Fig. 5B) that the ratio of 4S RNA to 3040S had increased to about the same level
as that found previously in 60-70S RNA. 3
Since it was shown above that 30-40S RNA
was not detectably degraded by the conversion to 60-70S RNA, it appears likely
that these additional 4S RNA's were
picked up from the viral pool of slowly
sedimenting 4-12S RNA.
July 1973
63
PRECURSOR OF G0-70S ROUS RNA
Discussion
The experiments described in this paper
suggest that the 60-70S RNA of avian
tumor viruses is probably the result of an
aggregation of 30-40S subunits and of some
small RNA's, most or all of which may
occur in the virus shortly after it is released from the cell. These findings are
in aggreement with the suggestions of
others that certain RNA tumor viruses contain, in addition to 60-70S RNA, free 3040S RNA, 1 2 ' 1 4 , " possibly also as precursors
of 60-70S RNA. Our data also confirm the
finding of Cheung and associates4 that free
30-40S RNA is detectable in viruses harvested at short intervals. However, we find
a principal 30-40S RNA component which
is readily converted to 60-70S in such viruses, whereas Cheung and associates4 observed a principal 55-60S RNA component
which is not converted to 60-70S RNA in
partially purified viruses. Further work
will be necessary to explain this divergence.
The formation of 60-70S RNA from 3040S and smaller subunits in the virion
appears to rule out the argument that the
subunit structure of 60-70S tumor virus
RNA is the result of posttranscriptional
cleavage of a putative 60-70S polynucleotide. This is compatible with failures to
find a single 60-70S polynucleotide in
RNA tumor viruses 6 and in virus-infected
cells.13'10
Our data are consistent with the view
that the conversion of 30-40S to 60-70S
RNA is a physical rather than a chemical
event. For example, the primary structure
of 30-40S RNA is not detectably changed
after conversion to 60-70S RNA. In addition, the finding that there are fewer 4S
RNA's associated with 30-40S RNA before
than after conversion to 60-70S RNA is
likely to be the result of an association
reaction. Association of pre-existing 30-40S
and 4-12S RNA's may take place in the
virion simultaneously with the conversion
to 60-70S RNA. This is suggested because
cleavage of 30-40S RNA's as a possible alternate source of small RNA's was not detectable and cleavage of 4-12S RNA's was
not determined. Nevertheless, the possibility exists that a minor cleavage of 30-40S
RNA or of some of the 4-12S RNA's in
the virion may accompany the conversion
of 30-40S to 60-70S RNA. The role of
small RNA's in the formation of 60-70S
RNA cannot be deduced from our experiments. It is conceivable that some small
RNA's function as linkers of 30-40S subunits in the 60-70S complex.
The results, reported here, also raise the
question whether the association of 30-40S
subunits and small RNA's to form a 60-70S
complex is essential for viral infectivity.
Acknowledgments.
Marie Stanley and Jeff Weber
assisted in the preparation of this paper.
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