volume 9 Number 201981
Nucleic Acids Research
Separation of the plus and minus strands of cytoplasmic polyhedrosis virus and human reovirus
double-stranded genome RNAs by gel electrophoresis
Robert E. Smith, Maureen A.Morgan and Yasuhiro Furuichi
Roche Institute of Molecular Biology, Nutley, NJ 07110, USA
Received 28 July 1981
ABSTRACT
The complementary strands of most of the genome double-stranded RNA
segments of insect cytoplasm!o polyhedrosis virus (CPV) and human reovirus are
separated for the first time by agarose gel electrophoreais in the presence of
7 M urea. CPV (+) strands and most reovirus (-) strands migrate faster than the
corresponding strands of opposite polarity. Glyoxal treatment, which modifies
guanine residues and prevents G-C basepairing, results in a loss of strand
resolution and concomitantly a significant decrease in electrophoretic mobilities. Reovirus mRNAs synthesized in vitro with ITP substituted for GTP show
similar decreased electrophoretic mobilities as the glyoxalated mRNAs. These
results clearly indicate that the basis for (+) and (-) strand resolution is
the presence of secondary structure formed mainly by G-C(U) base-pairs that are
maintained during gel electrophoresis in the presence of 7 M urea. When the
plus and minus strands of CPV genomes were separated and compared for protein
synthesizing activity, it was found that only the plus strands were able to
form stable 80S ribosome-RNA initiation complexes in wheat germ cell-free
extracts.
INTRODUCTION
Cytoplasmic polyhedrosis virus (CPV) of the silkworm Bombyx mori is the
prototype of many insect CPVs (1). The silkworm virus causes a disease which
results in the formation of virus polyhedral inclusion bodies, accompanied by
diarrheal symptoms and eventual death.
Like human reovirus, CPV is a member of
the Reoviridae and contains ten double-stranded genome RNA segments (2,3).
Parts of the 5'- and 3'-terminal sequences of these viral genome RNAs common to
all the segments have been determined and contain end-to-end base-paired
structure as shown:
Human reovirus type 3 (4-7)
CPV (8-10)
(+)
5' m7GpppGm-C-U
A-U-C
(-)
3'
U-A-Gpp5'
(+)
5' m7GpppAm-G-U
(-)
C-G-A
U-C-A
G-C-C
3'
3'
C-G-Gp(p)5'
In addition to a segmented genome both reovirus and CPV contain RNA
polymerases which copy the minus strand of each of the duplex segments to form
© IRL Press Limited. 1 Falconberg Court. London W1V 5FG. U.K.
5269
Nucleic Acids Research
the viral messenger RNAs (plus strands) (1,11,12).
contain
nucleotide
Virus particles also
phosphohydrolase, guanylyltransferase
and methyltrans-
ferases which are necessary to produce a 5'-terminal cap structure similar to
those found at the 5'-termini
of most eukaryotic viral and cellular mRNAs
(13,14).
In order to eventually decipher the coding properties of each CPV genome
RNA segment, we have recently optimized the conditions of in vitro transcription yielding capped, full-size transcripts of each of the segments, which were
separated into discrete bands by agarose gel electrophoresis (15).
In this
paper, we report that most of the complementary strands of both CPV and
reovirus genome dsRNAs, which were labeled at their 3'-termini with
P-pCp by
RNA-ligase, can be separated by gel electrophoresis. The physicochemical basis
for separation of the plus and minus strands of virus genome dsRNA and the
biological significance of these observations are discussed.
In addition, these basic findings have now made it possible to isolate, on
a preparative scale, each of the complementary RNA strands.
The method will
facilitate the sequencing of genome RNA, cloning of dsRNA by recombinant DNA
technology and other studies of dsRNA which include investigation of the roles
of the plus and minus strands in translation as well as genome replication. As
an application of the findings, we separated
P-pCp labeled plus and minus
strands of CPV genome VIII and X, and compared their ability to form translation
initiation
complexes
(80S
ribosome-mRNA)
in
wheat
germ
cell-free
extracts.
MATERIALS AND METHODS
Separation of mRNAs and double-stranded RNAs by agarose gel electrophoresis.
Preparation of 1.75$ agarose gels for electrophoresis was essentially
same as described by Rosen et^ al.. (16). In brief, 1.75 g agarose (Seakem) was
mixed with 100 ml of 0.025 M Na citrate buffer (pH 3.5) containing 7 M urea
(Schwarz-Mann, Ultrapure grade) and boiled to melt the agarose.
After the
solution was cooled to 40°C, the gel was cast as a 28 cm slab (Hoefer) and
allowed to solidify at 5°C for at Iea3t 4 hr.
Samples of mRNA or double-
stranded RNA were denatured by boiling for 1 min in 1.5 M urea or by incubation
in 90$ dimethyl sulfoxide (DMSO) at 37°C for 30 min.
The cooled samples were
applied to the agarose gel, and the RNAs were separated by electrophoresis for
30 hr at 25 mA unless otherwise noted.
Bands containing
P-labeled RNA were
detected by autoradiography of the wet gel with pre-flashed Kodak XR-5 film at
4°C according to the method of Laskey & Mills (17).
5270
Nucleic Acids Research
Labeling of the 3'-termini of CPV and reovlrus double-stranded RNAs by 5'32
P-pCp using RNA ligase.
The
reaction
mixtures
(50 pi)
contained
2.3
uM
5'-
P-pCp
(specific
activity 3,000 Ci/mmol, New England Nuclear), 1.2 uM dsRNA, 6.0 uM ATP, 50 mM
N-2-hydroxy-ethyl-piperazine-N'-2-ethanesulfonic
acid (Hepes-buffer, pH 7 . 5 ) ,
20 mM magnesium chloride, 3-3 mM dithiothreitol, 0.5 Ug bovine serum albumin,
1056 (v/v) DMSO, and 6.25 units RNA ligase (18). After 4 hr incubation at 5°C,
the RNAs were recovered by phenol extraction and filtered through a Sephadex G100 column (0.7 x 25 cm) to remove the unreacted
P-pCp.
3'-end-labeled RNAs,
eluted in the void volume fractions, were recovered by ethanol precipitation in
the presence of 0.2 M sodium acetate (pH 5 ) .
32
5'-Terminal
P-labeling of the minus (-) strands of CPV and reovirus double-
stranded RNAs.
Double-stranded
RNAs of CPV and reovirus were first treated with phos-
phatase in order to remove 5'-terminal phosphates.
The reaction mixtures (50
Ul)
(pH
contained
100 ug
pyroearbonate, 0.1
dsRNA,
50 mM
Tris-acetate
M sodium chloride and
phosphatase (Boehringer Mannheim).
8 ) , 0.0075$ diethyl-
1 unit of calf intestine alkaline
The reaction mixtures were incubated for 30
min at 37°C and RNAs were recovered by phenol extraction followed by ethanol
32
precipitation.
The phosphatase treated dsRNAs were
P-labeled at the 5'termini
of the minus
strands with y-
P-ATP by T1) polynucleotide kinase as
described by Miura et^ al. (19) •
Treatment of mRHAs and double-stranded genome RNAs of CPV and reoviru3 by
glyoxal.
The reaction mixtures (20 pi) contained about 0.1 ug of
or
double-stranded
RNA,
0.025 M
sodium
citrate
buffer
(nitrogen flushed) and 1 M glyoxal (deionlzed) (20).
incubated
(pH
P-labeled mRNA
3 . 5 ) , 50$
DMSO
Control reactions were
in parallel but with H_0 in place of glyoxal.
The mixtures were
incubated for 1 hr at 50°C and the RNAs were then separated by electrophoresis
in 1.75$ agarose gels.
Synthesis of ITP-mRNAs of reovirus in vitro.
32
P-labeled
CPV and
reovirus mRNAs were synthesized
described previously (15).
32
with a-
P-UTP as
Reovirus mRNAs that contained inosine in place of
guanosine were produced similarly except that GTP was replaced by ITP (21).
Ribo3ome binding assay.
Ability of the separated complementary strands of CPV genome dsRNA to form
protein
synthesis initiation complexes was examined by incubating RNAs with
wheat germ extracts under the conditions described before ( 2 3 ) . In brief, the
5271
Nucleic Acids Research
3'- P-labeled HNAs (containing approximately 20,000 opm) were mixed with
wheat germ translation extract (50 yl) containing 20 mM Hepes buffer (pH 7.4),
3 mM magnesium acetate, 70 mM KC1, 10 uM concentration each of 20 amino acids, '2
mM dithiothreitol, 0.2 mM GTP, 1 mM ATP, 5 mM ereatine phosphate, 40 ug/ml
creatine phosphokinase, 200 uM sparsomycin and 0.72 O.D. 2 g Q n m units of wheat
germ extracts prepared as described previously (24). After a 10-min incubation
at 25°C, mixtures were diluted with 4 volumes of cold 20 mM Tris-HCl buffer (pH
7.5) containing 70 mM KC1 and 3 mM magnesium acetate. mRNA-ribosome complexes
(80 S) were separated from unbound radioactive mRNA by centrifugation (SW50.1
rotor, 48,000 rpm, 4°C, 90 min) in a glyoerol gradient (10-30$, v/v), in 20 mM
Tris-HCl buffer (pH 7.5) containing 70 mM KC1 and 3 mM magnesium acetate.
Fractions (0.18 ml) were collected and counted in 10 ml of Aquasol (New England
Nuclear).
RESULTS
Separation of the complementary strands of genome dsRNA.
Each CPV genome dsRNA segment separated by polyacrylamide gel electrophoresis contains the 3'-terminal nucleotide sequences C-C-OH, in (+) strands
and C-U-OH common to all (-) strands in the ten duplex segments (8). In a
mixture of CPV dsRNA segments labeled at the 3'-termini with 5'- P-pCp by T^induced RNA ligase (18), both 3'-termini in each dsRNA segment are labeled
•
32
equally as measured by nearest neighbor transfer of P phosphate from pCp to
the penultimate C and U residues (• denotes radioactive phosphate). When the
P end-labeled CPV dsRNA mixture is heat-denatured in 4.5 M urea and subsequently subjected to agarose gel electrophoresis in citrate buffer (pH 3.5)
containing 7 M urea, it is resolved into as many as 18 discrete band3 (Fig. 1,
lane B). At least 7 of the bands comigrate with CPV mRNAs synthesized in vitro
(Fig. 1, lane A ) . The (-) strands of CPV genome d3RNA were labeled at the 5'32
32
termini with
P phosphate by incubation with y- P-ATP and polynucleotide
kinase, and their mobilities were compared by gel electrophoresis. The 5'termini of all the (-) 3trands in CPV dsRNA consist of phosphorylated (p)pGpG
and are selectively labeled after removal of the phosphate(s) by phosphatase
digestion; the 5'-termini of the (+) strands are not labeled because they
contain a phosphatase resistant cap structure, m GpppA pG (10). As shown in
Fig. 1, the 5'-labeled (-) strands comigrated with the 3' 32P-pCp-labeled minus
RNAs and did not comigrate with the plus-stranded mRNAs (lane C ) . These
results clearly demonstrate that both the (+) and (-) strands of CPV dsRNAs are
separated by gel electrophoresis. For example, the RNA of the fastest mobility
5272
Nucleic Acids Research
ABC
•
mRNA
SPECIES
DE
•—ORIGIN
GENOME RNA
SPECIES
2. 3— ^T
4—B .
5 -
*
— (-)IV
~(-!v'
\+)V
«
—(-)VI
6—
*
— (-)VII
7-^J,
—(-) VIII
s-i
—(+1VIII
— ( - ) ix
"-(+) IX
9—•
1
• —(-)x
10—
Figure 1: Separation of the complementary strands of CPV genome dsRNAs. CPV
genome dsRNAs
P-pCp-end-labeled were denatured either by heating or incubation in 90$ dimethyl sulfoxide, and separated by electrophoresis on a 1.75$
agarose gel in the presence of 0.025 M citrate (pH 3.5) ajid 7 M urea. Lanes
A,D: CPV mRNAs synthesized in vitro in the presence.of o- P-UTP. Lanes B,E:
CPV genome dsRNAs labeled at their 3'-termini by i pCp using RNA ligase and
separated into (+)_and (-) strands. Lane C: CPV genome dsRNAs with (-) strands
P-labeled by y- P-ATP using W polynucleotide kinase after treatment with
alkaline phosphatase. RNAs in lanes A, B and C were denatured by heating in 1.5
M urea for 1 min at 100° prior to sample application. RNAs in lanes D and E
were denatured by incubating in 90$ dimethyl sulfoxide for 30 min at 37°.
(in lane B) that comigrates with mRNA-10 (in lane A) is the (+) strand of genome
X [(+)X] (genome dsRNAs are numbered by I-X in decreasing order of molecular
weight) and the second band from the bottom of lane B is the (-) strand of
genome X [(-)X]
because it comigrates with 5'-labeled (-) strand of genome X
(lane C, the fastest migrating band).
Several faint bands marked by asterisks
5273
Nucleic Acids Research
apparently result from incomplete strand separation of duplex RNA (lane B) or
reannealing of mRNAs with contaminating non-radioactive dsRNAs (lane A) that
occurs during heat-denaturatlon and subsequent chilling on ice. Consistently,
these bands disappear or fade under more rigorous denaturation conditions such
as preincubation of RNAs at 37°C for 30 mln in 90$ DMSO (lane D: mRNAs; lane E:
3 P-pCp-labeled genome RNAs).
In addition to the separation of the (+) and (-) 3trands of each dsRNA
segment, it should be noted that the (+) strands of CPV genome always migrate
faster than the corresponding (-) strands.
Significant differences in the
rates of migration of the two genome RNA strands occurs in the segments of
smaller molecular weight.
The (+) strand of genome X, for example, migrates
about 20$ further than the (-) strand, while the differences in migration of
strands in genome V, with a molecular weight 4.5-fold larger than that of
genome X (1), remains less than 4.5$ of the total distance migrated.
In an attempt to determine if these findings are general for viruses which
contain dsRNAs, we tested genome RNAs from purified human reovirus (type 3),
the prototype of Reoviridae.
Human reovirus, like CPV, contains ten dsRNA
genome segments which are classified by molecular weight into three categories,
L (1-3), M (1-3), and S (1-4). As seen in Fig. 2, reovirus mRNAs synthesized in
vitro are clearly separated by agarose gel electrophoresis into 9 bands which
are classified to correspond to the three large (.1 1-3), three medium (m 1-3)
32
P-
and four small (s 1-4) species (lane A ) . Reovirus dsRNAs 3'-labeled with
pCp and heat-denatured prior to electrophoresis are resolved into 13 bands
(lane B ) . Reovirus (-) strands
P-labeled at the 5'-termini are resolved into
8 discrete band3 including a band that contains two comigrating medium-size
strands [(-) M-2 and (-) M-3l (lane C ) .
The separation of reovirus genome RNAs which were denatured into singlestranded RNA was incomplete due to comigration of several RNAs derived from
different genome segments.
For example, the third and eighth bands from the
bottom of gel in lane B result from comigration of (+) S-3 and (-) S-2, and (-)
M-2 and (-) M-3, respectively.
the other bands.
Note that they have stronger intensities than
Nevertheless, it is clear that each of the dsRNA segments is
separated into its component two strands. Most of the (-) strands of reovirus
genomes, contrary to CPV genomes, migrate faster than the corresponding (+)
strands with the exception of segment M-3 and perhaps L-3 whose (+) strands,
like CPV, migrate faster.
These results demonstrate that the complementary
strands of the genome dsRNAs of both CPV and reovirus are separable by gel
electrophoresis.
5274
The greater migration of CPV (+) strands that occurred for
Nucleic Acids Research
A B C
D
E
I—ORIGIN
( )
(-1M3.M2
( + )S4
(-)S4
Figure 2: Separation of,^.he complementary strands of reovirus genome dsRNAs.
Reovirus genome dsRNAs
P-pCp-end-labeled were separated by electrophoresis
on a 1.75$ agarose gel similar to CPV dsRNAs described in the legend to Fig. 1.
Lanes A,D: Reovirus mRNAs labeled by a-A P-UTP. Lanes B,E: Reovirus genome
P-pCp using RNA ligase and resolved
dsRNAs labeled at their 3'-termini by
into the (+J_ and (-) strands. Lane C: Reovirus genome dsRNAs with the (-)
strands 5'- P-labeled by y- P-ATP and Ti polynucleotide kinase after treatment with alkaline phosphatase. RNAs in lanes A, B and C were heat denatured in
1.5 M urea for 1 min at 100° before applying to the gel. RNAs in lanes D and E
were treated with 90$ dimethyl sulfoxide for 30 min at 37° before applying to
the gel.
all the segments may imply a specific structural feature in the plus (or minus)
RNA molecules. The differential migration rates observed for reovirus complementary strands may similarly be intrinsic for each genome segment.
Comparison of base composition of the plus and minus strands of CPV genomes
The physieochemical basis that accounts for the separation of complimentary genome RNAs was studied.
Amino groups in Cytosine (C) and Adenosine (A)
in RNAs are partially protonated under the acidic condition used for the gel
electrophoresis because the 4-amino and 6-amino groups of C and A have pka's of
1.2 and 3.7, respectively.
The total negative charge of each of the RNAs
5275
Nucleic Acids Research
should cause those with greater minus charges but identical molecular weights
and secondary structure to migrate faster by gel electrophoresis. To determine
if the (-) strands of CPV RNA are AC-rich, which would retard migration or,
conversely, the (+) strands are GU-rich, which would stimulate migration, the
base composition of each mRNA species was measured. Viral mRNAs synthesized in
the presence of either a- P-UTP, -CTP, -ATP or -GTP were separated by gel
electrophoresis and nearest neighbor analysis was performed on each of the
separated, extracted mRNA species. Table 1 summarizes the base compositions of
nine CPV mRNA species.
The A+C and G+U contents in each mRNA (+ strand)
correspond respectively to the U+G and C+A contents of the related genome (-)
strand based on their mirror-Image relationship.
As seen in Table 1, the A+C
contents of all the CPV mRNAs are equal to or slightly higher than G+U, which is
equal to C+A in the (-) strands.
These results predict, inconsistent with
experimental observations, that CPV (+) strands would have the same or lower
mobilities than the (-) strands.
Therefore, the different electrophoretic
mobility of the (+) and (-) strands cannot be accounted for by differences in
base composition and by electrostatic charges.
Table 1
Base Composition of CPV mRNAs
Base
1
C
21.5
A
G
4
5
19.9
21.9
20.8
29.2
29.8
28.9
22.9
23.1
22.2
2,3
•
mRNA species
6
7
22.3
23.2
30.8
30.0
30.0
27.8
29.8
22.3
22.1
23.4
25.1
23.0
23-3
25.4
22.4
27.0
23.6
52.6
50.1
53.0
47.5
50.0
46.9
27.0
26.9
25.6
A+C
50.7
19.7
50.8
50.9
52.1
51.2
17.7
48.8
49.2
22.6
29.9
27.1
50.2
10
21.2
26.3
19.2
9
21.5
U
G+U
8
19.4
Calculated from analysis by nearest neighbor transfer.
CPV mRNAs alternately labeled by a-32P-ATP, -GTP, -CTP and -UTP were
separated into nine bands by electrophoresis as described in Materials and
Methods.
Each of the separated mRNAs was then totally digested into mononucleotides by incubation with ribonuclease T2 (50 U/ml of ribonuclease T2,
37°C, 1 hr). The resulting 3'-phosphorylated nucleotides were separated by
paper electrophoresis (Whatmaxu 3mm) in 5J acetic acid-0.5J pyridine (pH 3-5)
buffer for 1 hr at 3000 V.
P-Containing spots were detected by autoradiography, cut out and counted in a toluene-based scintillation cocktail. The %
base composition was calculated from the nearest neighbor transfer frequencies.
5276
Nucleic Acids Research
RNA secondary structure affects relative mobility of the plus and minus strands
The aldehyde reagent glyoxal (OHC-CHO) reacts with unpaired guanine residues giving rise to an adduct (25) which prevents inter- and intramolecular
base-pairing in DNA or RNA (20). In an attempt to determine if the secondary
structure of RNAs affects electrophoretic mobility, we treated viral mRNAs or
denatured genome RNAs with glyoxal and compared the electrophoretic mobilities
of
the glyoxalated
and unmodified
RNAs.
The effect of glyoxal on the
mobilities of the RNA is striking as seen in Figs. 3 and 1. The mobilities of
the mRNAs of CPV (Fig. 3, lanes A and B) and reovirus (Fig. H, lanes C and D)
decrease significantly (by about ^0%) as compared with the untreated mRNAs.
This effect appears to be general for all the mRNA species of various molecular
weights. The apparent reduction of electrophoretic mobility cannot be ascribed
to an increase in molecular weight due to binding of glyoxal molecules to
A B C
0
E
4-ORIGIN
mRNA
SPECIES
2,3 —
4 —
5—
.1
+STRAND
SPECIES
(+1VII —
6—
(+1VIII —
(+1IX —
7—
8—
9—
(+IX— I
i
Figure 3:
1.75% agarose gel electrophoresis of CPV mRNAs (lane A ) , mRNAs
treated by glyoxal (lane B), genome RNAs (lane D), and genome RNAs.treated by
glyoxal (lanes C and E ) . Genome RNAs were-labeled by addition of
P-pCp with
RNA ligase, and oRNAs were labeled by a- P-UTP during the synthesis. RNAs
were modified with glyoxal as shown in Materials and Methods and applied
directly to 1.75$ agarose - 7 M urea gels.
5277
Nucleic Acids Research
A
GENOME
RNA
SPECIES
1
I
-Ml .•MIL
+ M2J
-M3, -M2TJ
+M3J
-SI, + SI-
+ S2-
-S2, + S 3 -
D
E
F
mRNA
SPECIES
I
mlm2m3-
. I
sl-
«
s2s3s4-
•
-S3- H
S4, + S 4 -
C
B
•
]
Figure 4: 1.75$ agarose gel electrophoresis of reovirus genome RNAs (lane A ) ,
genome RNAs treated with glyoxal (lane B), reovirus mRNAs (lane C ) , mRNAs
treated by glyoxal (lane D), mRNAs made by substituting ITP for GTE (lane E ) ,
ITP-mRNA treated by glyoxal (lane F ) . Genome^BNAs were labeled by
P-pCp and
RNA ligase and mRNAs were synthesized using a- P-UTP as radioactive precursor.
RNAs were treated by glyoxal as indicated in Materials and Methods and applied
directly to 1.75$ agarose - 7 M urea gels.
guanine residues in RNAs, since this increase would be at most 4$ of total
molecular weight if all guanine
residues are modified.
Rather, it is more plausible to ascribe the decrease in mobility to an RNA
conformational change.
Perhaps mRNA3 can maintain secondary structure by
partial base-pairing even in the presence of 7 M urea and low ionic strength.
The unfolded RNA structure produced by glyoxal treatment probably causes an
increase in the partial specific volume of the RNA molecule and eventually a
greater frictional retardation in the agarose gel.
Another marked change resulting from the glyoxal treatment is the disappearance of resolution of the (+) and (-) strands.
As seen in Fig. 3 (lane C
and D, CPV) and Fig. 4 (lane A and B, reovirus), the (+) and (-) strands of the
glyoxal treated genome RNAs comigrate at the same posi tions as glyoxalated
mRNAs (Figs. 3 and 4, lane B).
These changes occur irrespective of the order of
migration of (+) and (-) strands.
5278
The results clearly indicate that the (+)
Nucleic Acids Research
and (-) strands of the virus genomes are separated mainly due to the secondary
structure inherently contained in the individual strands and maintained in the
presence of 7 M urea. Thus, on the basis of their respective electrophoretic
mobilities, the (+) strands of CPV and most of the (-) strands of reovirus
genome RNAs contain more compact structures than the corresponding strands of
counter polarity.
Secondary structure that stimulates electrophoretic migration of RNA is
guanine mediated.
Secondary structure in the single-stranded RNA affects electrophoretic
mobility as described above. Guanine seems to be the base that is important
for maintaining secondary structure, presumably by G-C base-pairing, since
guanine-specific modification by glyoxal treatment significantly decreases
mobility. In order to prove the stimulatory effect of guanine on RNA migration
by another approach, we prepared reovirus mRNAs which contain hypoxanthine in
place of guanine as described previously (21), and compared the migration rates
with those of the normal G-containing mRNAs before and after glyoxal treatment.
Hypoxanthine-substituted reovirus mRNAs (referred to as ITP-mRNA) were synthesized In vitro in reaction mixtures containing ITP substituted for GTP.
Synthesis of reovirus ITP-mRNA occurred 70$ as efficiently as that for normal
mRNA with GTP while replacement of GTP by ITP, like as that with pNHppA for ATP
(27), inhibited CPV mRNA synthesis by more than 90$. By agarose gel electrophoresis, reovirus ITP-mRNAs (Fig. 4, lane E) migrate much more slowly than
normal mRNA (lane C ) . In fact, their migration rates are almost identical to
the glyoxalated normal mRNA(lane D ) , consistent with our hypothesis that
guanine participates in the formation of secondary structure in the presence of
7 M urea. Further, glyoxal treatment of ITP-mRNAs results in little, if any,
influence on electrophoretic migration (lane F ) .
Isolation of plus and minus RNAs from CPV genome dsRNA segments.
The results described above suggest that the agarose-urea gel electrophoresis would be useful for isolation of pure plus and minus strands of each
viral genome dsRNA on a large scale. In fact, as shown in Fig. 5 several ug of
CPV genome RNA VIII and X were separated into their component strands, (-) VIII
and (+) VIII, (-) X and (+) X, respectively. Each lane in Fig. 5 shows the
separation profiles after two purifications by agarose-urea gel electrophoresis. As seen in the preparation of (-) VIII (lane 1), the minus strands tend to
be contaminated by a low amount of plus strand after the first separation.
This is due to trailing of the plus strand which migrates faster than the minu3
strand. However, after a second gel electrophoresis, in which agarose gel
5279
Nucleic Acids Research
1 2
3
4
ORIGIN—
Figure 5: Isolation of plus and minus strands of viral genome dsRNAs on a
preparative scale. About 5 Ug (M.O x 10 cpm) of each of dsRNA-VIII and
dsRNA-X was separated into the complementary strands by agarose gel electrophoresis after denaturation in 90% DMSO. The figure shows the separation
profile after second agarose gel electrophoresis (1 V/cm, 36 hr, 1t°C). Lane 1:
(-) VIII strand; lane 2: (+) VIII strand; lane 3: (-) X strand; and lane t:
(+) X strand. An arrow indicates the position of xylene cyanol.
slices containing resolved RNA were embedded in new slab gel plate, contamination of foregoing RNA was eliminated. RNA bands marked by asterisks perhaps
resulted from either inappropriately embedded agarose fragments of the first
gel or partially denatured dsRNA that comigrated with slower migrating minus
strands.
RNAs in each band were recovered by an electrophoretic elution from
gel, the method described by Yong et^ g_l. (22).
Only plus strands of CPV genome dsRNA can form a 3table translation initiation
complex.
It is considered that genome segments of CPV and other dsRNA containing
viruses consist of the strands which have the same structure as viral mRNAs
(plus strands) and their complementary strands (minus strands).
In fact,
McCrae and Joklik (28), in their effort to establish the coding assignment of
reovirus genome segments, have shown that the denatured dsRNA segments can be
translated under suitable conditions in the wheat germ protein synthesizing
system. It has been shown that duplex genome segments from reovirus (29) and
rice dwarf virus (30) are inactive as a template for protein synthesis in vitro
5280
Nucleic Acids Research
and it is likely that only the denatured plus strands which contain a 5'-cap
structure can serve as coding strands for translation.
However, the minus
strands may also have a potential to be translated or at least bind to
ribosomes.
32 •
Each of the isolated complementary strands from 3'- P-pCp labeled CPV
genomes VIII and X was tested for ability to form a protein synthesis initiation complex (80S riboscme-RNA) in wheat germ extract.
RNAs were incubated
with the extract in the presence of sparsomycin which inhibits peptide chain
elongation and the mixtures were analyzed for 80S initiation complex by glycerol gradient centrifugation as described in Materials and Methods.
trols, 5'- H-methyl-labeled reovirus mRNAs and
and -X were similarly tested.
As con-
32
P-pCp-labeled CPV dsRNA-VIII
As shown in Fig. 6 (panel A ) , reovirus mRNAs
formed initiation complexes with ribosomes and migrated in a position of 80S
(shown by arrows).
About 50$ of the input mRNAs, as measured by radioactivity,
were incorporated in the complex under this ribosome-saturated condition.
By
contrast, dsRNA-VIII (and dsRNA-X, data not shown) failed to bind ribosomes
under the same conditions and migrated near the top of gradient (panel A,
fraction 20-27).
When the plus and minus strands of genome RNA-VIII and -X were tested, it
was found that only the plus strands could form, though less efficiently than
reovirus mRNAs, the 80S ribosome-RNA complexes (panel B and C ) .
strands were, like dsRNAs, inactive in binding to ribosomes.
The minus
In (+)VIII and
(+)X strands, 20 and 10$ of the input RNA molecules were incorporated in 80S
complexes, respectively.
Ribosome bindings obtained with CPV genome plus
strands were significantly (65$) inhibited by the addition of cap analog m7GMP
(1 mM) (data not shown).
As expected from the presence of cap in the plus
strand (10) and importance of cap during initiation of translation (21!), the
binding by the plus strands is apparently cap dependent.
Also, it should be
mentioned that this is the first report of ribosome binding with RNAs labeled
at the 3'-termini.
DISCUSSION
Each viral genome segment from CPV and reovirus was labeled at the two 3'32
termini with 5'- P-pCp by M-induced RNA ligase. As observed recently in
reovirus dsRNA (26,31), the two 3'-termini in a CPV dsRNA segment were labeled
equally with pCp indicating that the 5'-cap structure protruding from the end
of the duplex does not inhibit the RNA-ligase reaction.
Recently, we demonstrated
that a mixture of ten viral mRNA species
5281
Nucleic Acids Research
0
Bottom
10
20
FRACTION
Figure 6: Ribosome binding by plus strands of CPV genome dsRNA,_ AJout 10,000
opm (0.01 ug) of fiacji of T H-methyl-labeled reoviru3 mRNA, 3 ' - P-pCp-labeled
duplex RNA, 3 ' - P-pCp-labeled plus and minus strand3 of CPV dsRNA were
analyzed for t h e i r rJbosome binding a c t i v i t y as described in Materials and
Methods. Panel A: J H-labeled reovirus mRNAs (0—0), 5 P-labeled CPV dsRNAVIII (•
1).
Panel B: plu3 strand of dsRNA-VIII (0—0), minus strand of
dsRNA-VIII (•
• ) . Panel C: plus strand of dsRNA-X (0—0), minus strand of
dsRNA-X (t
• ) . Arrows indicate the position of 80S ribosome-RNA complexes.
synthesized in v i t r o by purified CPV or reovirus can be separated into the
individual RNA species by agarose gel electrophoresis (15). In addition, as
demonstrated in the present paper, t h i s technique has proven useful in
separating the complementary strands of genome dsRNAs that have a greater
complexity than the mRNAs. The primary basis for separation of RNAs in agarose
gel e l e c t r o p h o r e s i s i s the difference in molecular s i z e .
This has been
documented in our previous report concerning separation of CPV mRNAs with ten
different molecular weights. Another important concern for the separation of
single-stranded RNAs of similar molecular weights i s conformation of RNA
5282
Nucleic Acids Research
molecules as discussed previously by McMaster and Carmichael (20).
Viral
genome RNAs treated with glyoxal, which reacts with the N1 and C2 amino group
of guanine to form an additional ring on the base and thus prevent basepairing, fail to separate into complementary strands.
Furthermore, glyoxal
treatment significantly decreases the electrophoretic mobilities of viral
mRNAs.
These observations clearly indicate that both the (+) and (-) strands
of the viral genomes retain even in 7 M urea secondary structures that are
mainly due to guanine-related base-pairing, e.g. G-C and G-U pairs. Consistent
with this suggestion, the replacement of G by I or glyoxalation of G in RNAs,
both effects that prevent G-C and G-U pairing, renders the strands inseparable
and decreases their mobilities.
Perhaps glyoxalation removes intramolecular
G-C pairings which have already been weakened by 7 M urea and results in RNAs of
a relaxed structure, presumably a linear form as observed in Sindbis genome RNA
by Hsu e_t al^. (25). The RNAs would migrate at reduced rates because of their
increased partial specific volumes.
Reovirus mRNAs with G+I substitution
apparently have reduced levels of secondary structures as reported recently by
Kozak (32).
Slight changes in the total net charge which are dependent on base composition are not important factors affecting the relative mobilities of RNAs of
similar molecular size. This is well demonstrated with the (+) and (-) strands
of CPV genome segment X.
composition [(+) X;
Both strands contain small differences in base
A = 29.8$, C = 23.2%, G = 23.3$ and U = 23.6$. (-) X; A =
23.6$, C = 23.3$, G = 23.2$ and U = 29.8$]; nevertheless their electrophoretic
mobilities are significantly different (Fig. 1, lane B).
Consistently, neither
the plus charge in the 5' cap nor a slightly higher content of A+C bases that
are protonated under the acidic conditions employed, reduces the electrophoretic mobilities of CPV (+) strands.
Previously, Heyward (33) reported that denatured DNA from E. coli phage
T1, T2, T7 and Xb2b5, and B. tiberius phage a were separated by agarose gel
electrophoresis into the complementary strands. He also found that those phage
DNAs which gave good strand separation in the gel have an asymmetric distribution of poly(G)-binding sites oetween their strands.
In the three examples
studied (T7, Xb2b5 and a ) , the strand with the greater affinity for poly(G)
migrated slower than the strand with little or no affinity for poly(G).
His
findings in DNA are consistent with our hypothesis in regard to RNA secondary
structure and electrophoretic migration, because the strand with locally clustered G-residues, which perhaps contributes more in forming secondary structure than clustered C-residues, migrates faster by gel electrophoresis.
5283
Nucleic Acids Research
In the paper of Maxam and Gilbert (31*) for DNA sequencing, one finds full
duplex DNA 64-mers which were derived from lac operon DNA by restriction enzyme
cleavages. One strand was referred to as "Fast" strand which migrated faster
in the neutral 8$ polyacrylamide gel electrophoresis than the complementary
"Slow" strand. Interestingly, the "Fast" strand contains 50$ more G than the
"Slow" strand ("Fast": G,21, C, 14, T, 12, A, 16; "Slow": G,TJ, C,21, T, 16,
A, 12). In both strands, G residues are not particularly clustered and are
distributed over the entire strands yet the G-rich strand migrates faster in
the absence of urea. These observations suggest that not only the clustered
G's but also the total mass of unclustered G's, which have greater potential
for base-pairing than C's, affect electrophoretic mobility.
In CPV, the complimentary strands of each genome dsRNA are not significantly different in guanine content as shown in Table 1. The presence of
specific loci of clustered G-residues, therefore, may account for the altered
migration rate of the fast-migrating strands. Collectively, CPV (+) strands
[and reovirus (-) strands] are of particular interest with respect to their
primary and secondary structures 3ince these strands function as either viral
tively. The plus strands of duplex, for example, serve not only as messages
for virus protein synthesis but also function as templates for replication of
genome duplex RNA. A common recognition structure for viral replicase is
expected to occur in the 3'-terminal region of each (+) strand. Whether a
hypothetical (G)-cluster in the fast-migrating strand3 is related to RNA
function or its presence is fortuitous is a subject for future work.
In addition to the insights into RNA secondary structure, the present
findings provide opportunities to test complementary strands of dsRNA in the
protein synthesis ijn vitro, nucleotide sequencing and cDNA synthesis after
appropriate tailing. As described in Results, separated plus and minus strands
of viral genome RNAs were analyzed for protein synthesis activity by binding
assay to wheat germ ribosomes. The results clearly showed that only plus
strands, as analyzed in dsRNA-VIII and dsRNA-X, could form stable 80S initiation complexes, while the minus strands were unable to do so (Fig. 6). This may
be due to the greater sensitivity of the uncapped minus strands to exonuclease
activity (35). Moreover, the binding by the plus RNAs appears to be cap
dependent since the addition of cap analog m GMP in the mixture inhibited to a
similar extent (65$) as for capped reovirus mRNAs. A relatively lower extent
of ribosome binding of the 3'-labeled RNAs is probably due to a greater
^2 •
^
distance of 3'- P-pCp than 5'- H-methyl group in mRNAs from the initiator
codon AUG which usually locates in 5'-proximal region. Cleavages which occur
5284
Nucleic Acids Research
in internal region of RNA molecules by wheat germ nuclease may prevent the
resulting labeled
3'-fragments
from binding to ribosomes, while the 5'-
terminally labeled fragments could retain the potential to form ribosome-RNA
complexes as shown previously in 5'-fragments of reovirus mRNAs (36). It is
conceivable that the minus RNA strands are formed only for replication of
genome RNAs and not for viral protein synthesis. In any case, these methods
32
which include 3'- P-labeling, strand separation and ribosome binding assay
should be useful to identify the coding strand in viral genome dsRNAs.
In another application of the method, 3'-terminal regions of each of the
32
P-pCp labeled and separated RNAs of CPV genome dsRNA-IV, V, VIII, IX and X
were sequenced
by the method of Peattie (37) in collaboration with Drs.
Kuchino and Nishimura in National Cancer Center Research Laboratory of Japan.
OH
The 3'-terminal hepta nucleotides (-GUUAGCCC/M,) in plus strands and penta
HO
nucleotides (uri2uCAUU-) were found to be common for five genome segments
analyzed
(manuscript
in preparation).
These homologous sequences may be
signals for RNA synthesis for mRNA synthesis and genome replication, respectively.
ACKNOWLEDGMENTS
We thank Dr.
Aaron J. Shatkin for helpful discussion and encouragement
during the study.
We also thank Ms. Alba J. LaFiandra for supplying purified
reovirus and reovirus genome RNA.
REFERENCES
1. Payne, C.C. and Rivers, C.F. (1976) J. Gen. Virol. 33, 71-85.
2. Gomatos, P.J. and Tamm, I. (1963) Proc. Nat. Acad. Sci. USA 49, 707-711.
3. Miura, K., Fujii, I., Fuke, M., Sakaki, T. and Kawase, S. (1969) J. Virol.
21, 1211-1222.
4. Shatkin, A.J. and Sipe, J.D. (1968) Proc. Nat. Acad. Sci. USA 61, 14621469.
5. Miura, K-I., Watanabe, K., Sugiura, M. and Shatkin, A.J. (1974) Proc.
Nat. Acad. Sci. USA 71, 3979-3983.
6. Muthukrishnan, S. and Shatkin, A.J. (1975) Virology 64, 96-105.
7. Furuichi, Y., Muthukrishnan, S. and Shatkin, A.J. (1975) Proc. Nat. Acad.
Sci. USA 72, 742-745.
8. Furuichi, Y., and Miura, K-I. (1973) Virology 55, 418-425.
9. Furuichi, Y. and Miura, K-I. (1975) Nature 253, 371-375.
10. Miura, K-I., Furuichi, Y., Shimotohno, K., Urushibara, T., Watanabe, K.
and Sugiura, M. (1975) Abstract of Colloque on "In Vitro Transcription
and Translation of Viral Genomes" held in Paris, France, pp. 153-160.
11. Borsa, J. and Graham, A.F. (1968) Biochem. Biophys. Res. Commun. 33,
859-901.
12. Lewandowski, L.J., Kalmakoff, J. and Tanada, Y. (1969) J. Virol. 4, 857865.
5285
Nucleic Acids Research
1314.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
5286
Furuichi, Y., Muthukrishnan, S., Tomasz, J. and Shatkin, A.J. (1976)
J. Biol. Chem. 251, 5043-5053.
Shatkin, A.J. (1976) Cell 9, 645-653.
Smith, R.E. and Furuichi, Y. (1980) Virology 103, 279-290.
Rosen, J.M., Woo, S.L.C., Holder, J.W., Means, A.R. and O'Malley, B.W.
(1975) Biochemistry 14, 69-78.
Laskey, R. and Mills, A.D. (1975) Eur. J. Biochem. 56, 335-341.
England, T.E. and Uhlenbeck, O.C. (1978) Nature 275, 560-561.
Miura, K-I., Watanabe, K., Sugiura, M. and Shatkin, A.J. (1974) Proc.
Nat. Aoad. Soi. USA 7J_, 2979-3983.
McMaster, G.K. and Carmichael, G.G. (1977) Proc. Nat. Acad. Sci. USA
74, 4835-4838.
Morgan, M.A. and Shatkin, A.J. (1980) Biochemistry 19, 5960-5966.
Yang, R.C-A., Lis, J. and Wu, R. (1979) in Methods in Enzymology, Wu, R.
E.d., Vol. 68, pp. 176-182 Academic Press, New York.
Furuichi, Y., Morgan, M.A. and Shatkin, A.J. (1979) J. Biol. Chem. 254,
6732-6738.
Both, G. W., Furuichi, Y., Muthukrishnan, S. and Shatkin, A.J. (1975)
Cell 6, 185-195.
Hsu, M.T., Huang, H.J. and Davidson, N. (1973) Cold Spring Harbor Symp.
Quant. Biol. 38, 943-950.
Darzynkiewicz, E. and Shatkin, A.J. ( 1980) Nucleic Acids Res. 8, 337350.
Furuichi, Y. (1976) Proc. Nat. Acad. Sci. USA 75, 1086-1090.
McCrae, M.A. and Joklik, W.K. (1978) Virology 89, 578-593.
Gomatos, P.J., Krug, R.M. and Tamm, I. (1964) J. Mol. Biol. 9, 193-207.
Miura, K-I. and Muto, A. (1964) Biochim. Biophys. Acta 108, 707-709.
Li, J.K.-K., Keene, J.D., Scheible, P.P. and Joklik, W.K. (1980)
Virology 105, 41-51.
Kozak, M. (1980) Cell 19, 79-90.
Heyward, G. (1972) Virology 49, 342-344.
Maxam, A.M. and Gilbert, W. (1977) Proc. Nat. Acad. Sci. USA 74, 560564.
Furuichi, Y., LaFiandra, Y. and Shatkin, A.J. (1977) Nature 266, 235239.
Kozak, M. and Shatkin, A.J. (1977) J. Biol. Chem. 252, 6895-6908.
Peattie, D.A. (1979) Proc. Nat. Acad. Sci. USA 76, 1760-1764.