J. gen. Virol. (I979), 44, 311-322
Printed in Great Britain
31 I
Sequence Studies of Poliovirus RNA.
IV. Nucleotide Sequence Complexities of Poliovirus Type 1, Type 2
and Two Type 1 Defective Interfering Particles RNAs, and
Fingerprint of the Poliovirus Type 3 Genome
By Y U A N F O N LEE, * N A O M I K I T A M U R A , A K I O N O M O T O t
AND E C K A R D W I M M E R
Department of Microbiology, School of Basic Health Sciences, State University of
New York at Stony Brook, Stony Brook, L.I., New York 11794, U.S.A.
(Accepted I2 January 1979)
SUMMARY
The 8uP-labelled genomes of poliovirus type I, 2 and 3 have been digested with
RNase T1 and the products separated by two-dimensional gel electrophoresis.
All three fingerprints differ in the separation pattern of the large oligonucleotides.
The molar yields of the large RNase Tl-resistant oligonucleotides of type I and type
2 RNA of poliovirus RNA are close to one. By comparing the yields of these oligonucleotides to the amount of RNA from which they originated, the chain length
of type I poliovirus RNA was found to be 785I~567 nucleotides (tool. wt.
2.66~o.19× Io 6) and that of poliovirus type 2, 8181~578 nucleotides (mol. wt.
2:77~o'19× t@). The chain length of two defective interfering particle (DI)
RNAs of poliovirus type I were determined to be 7o42±999 nucleotides for
DI(0 and 6639~674 nucleotides for DI(2).
INTRODUCTION
Poliovirus, a member of the Picornaviridae, contains one molecule of single-stranded
RNA in which all genetic information is stored (for a review, see Levintow, I974). Three
serologically distinct types of poliovirus (type I, type 2 and type 3) have been recognized
(for a review, see Rueckert, I976) which we shall abbreviate as PVI, PV2 and PV3, respectively. Their genome RNAs are of positive polarity and function as mRNA after entry
into the host cell cytoplasm. Virion RNA of PVI contains 3'-terminal poly(A) (Yogo &
Wimmer, I972) and is covalently linked to a small basic protein 'VPg' at the 5' end (Lee
et al. I976; I977; Nomoto et al. t977). Virion RNA of PV2 has also been shown to carry
a 5'-terrninal protein (A. Babich, A. Nomoto & E. Wimmer, unpublished data).
Except for terminal sequences (Nomoto et al I977; Flanegan et aL I977; Porter et al.
I978) the primary structure of poliovirus RNA is unknown. As part of our programme to
determine longer internal sequences of poliovirus RNA we have analysed the yield at
which large oligonucleotides are produced by exhaustive digestion of PVI and PV2 3zp.
RNAs with RNase TI. This information may reveal redundant sequences and is also
important for the mapping of oligonucleotides (Nomoto et al. I979). We have, in addition,
determined the sequence complexity of PV1 and PV2 RNAs and of the genomes of two
* Present address: 3837 Simpson Stuart Road, Life Science Department, Bishop College, Dallas, Texas
75241, U.S.A.
t Present address: Department of Public Health, Schoolof PharmaceuticalSciences,Kitasato University,
5-9-I Shirokane, Minato-ku, Tokyo, Japan.
To whomcorrespondenceshould be addressed.
Downloaded from www.microbiologyresearch.org by
o22-1317/79/oooo-3562 $o2.oo~ 1979 SGMIP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
312
Y. F. LEE A N D O T H E R S
isolates of defective interfering particles [DI(i) and DI(2)] of PVt. DI particles of poliovirus
are deletion mutants which have been found in laboratory stocks [ D I 0 ) ] of wild-type
(standard) virus or have been generated by serial passage of plaque-purified virus at high
rn.o.i. [DI(2) and DI(3); Cole et al. 1971 ; Nomoto et al 1979].
Two-dimensional gel electrophoresis has been used previously for the separation of
large fragments of virus RNA (De Wachter & Fiers, ~972). We have modified this procedure
to accommodate the separation of complete RNase TJ or RNase A digests (Lee & Wimmer,
I976 ). The fingerprints that are obtained by autoradiography of the slab gels are diagnostic
for a species of RNA and its purity (Lee & Wimmer, I976). We show here the fingerprint
of PV3 RNA and, for comparison, the fingerprints of PV1 and PV2 RNA. Although
extensive sequence homology has been observed between the RNAs of the three types of
poliovirus (Young, I973a, b), the fingerprints of all three poliovirus RNAs differ strikingly
in the separation pattern of the large, RNase Tl-resistant oligonucleotides.
METHODS
Cells and viruses. PVI (Mahoney) and PV2 (vaccine strain P217ch2ab) were propagated
in suspension of strain $3 HeLa ceils in minimum essential medium (FI4 of Gibco Co.),
5 ~ calf serum, as previously described (Dorsch-H~isler et al. 1975). PV3 (Leon strain P342)
was grown in suspended HeLa cells as described for the other two poliovirus strains except
that 3 h after infection the cells were diluted to 1.5 x Io 6 cells/ml and the incubation time
was extended to I8 h at 37 °C. The cell suspension was then centrifuged at low speed, the
pellet suspended in hypotonic buffer and a cytoplasmic extract prepared (Dorsch-H~isler
et al. 2975). The cytoplasmic extract and the fluid of the suspension were combined and
centrifuged at 78ooo g for 3 h. The pellet was suspended in medium and used as virus stock.
Viruses were titrated by a HeLa cell suspension assay (Detjen et al. 1978 ).
Labelling o f the virus. HeLa cells were suspended in MEM (Gibco FI4) at I2× lO6
cells/ml and infected at room temperature with 3o to 5o p.f.u. ( P V 0 or I5 to 25 p.f.u.
(PV2) of virus. After 3° rain medium and calf serum were added to give a final concentration
of 6× IO~ cells/ml, 5 ~ serum. The culture was incubated for I h at 37 °C. The cells were
then sedimented, washed once with phosphate-free medium (all other ingredients were as in
MEM FI4), resuspended in cold phosphate-free medium to 6× lO6 cells/ml with actinomycin D (5 #g/ml) and incubated for 1 h at 4 °C (Wimmer, I972). Dialysed calf serum (to
5 ~ ) and carrier-free phosphorus-32 (25o #Ci/ml) were added and the culture was incubated
for 5 h at 37 °C. Cells were then pelleted and the culture fluid was discarded. Intracellular
virus was isolated as previously described (Cole et al. I971; Dorsch-H~isler et al. 1975).
The yield of highly purified virus from 1.8 × io 9 cells was 4 to 8 × lO8 ct/min; and occasionaiiy 1.5 × IO6 ct/min in the case of PVI (I.5 to 3"5 × I@ ct/min/#g virus). When PV3 was
adsorbed to HeLa cells (i o to 15 p.f.u./cell) the incubation was modified as follows: infected
cells were incubated for I h at 37 °C before the change to phosphate free medium. Two h
after the addition of 32p, complete medium containing 5 ~ calf serum was added to a concentration of 1"5 × IO6 cells/ml and the culture incubated for I8 h at 37 °C. Extra- and intracellular virus was then harvested as described above. Since zonal centrifugation through
sucrose gradients in the presence of o. 15 M-NaC1, o'5 ~o SDS, often resulted in aggregation
of PV2 and PV 3 (Lee & Wimmer, I976) these viruses were purified by isopycnic centrifugation in CsC1 as follows: virus pellets were suspended in o'oi M-NaCI, O.Ol M-tris-HCt,
pH 7"5, o.oo2 M-EDTA, I ~o Brij 58, containing CsCI (1.33 g/ml) and centrifuged in a
Spinco type 65 rotor at 33ooo rev/min at 4 °C for a minimum of 16 h. After fractionation
of the gradient the virus containing solution was desalted by gel filtration on a column
(I × I5 cm) of Sephadex G25, which was equilibrated with o.I buffer (o.I M-NaCI, o.o~
M-tris-HCl, pH 7"5, o.oor M-EDTA).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
Sequence complexities o f polio R N A s
313
DI(1) and DI(2) particles of PVI (Mahoney) were labelled and purified as described
(Nomoto et al. I979).
Isolation of 3~P-RNA. This was done either by lysis of virions with detergent (SDS) at
pH 3"5 (Mandel, I964) or by treatment with phenol/chloroform as follows: virus was
dissolved in o-I buffer and mixed with I vol. each of phenol and chloroform/isopentanol
(14: 1)). SDS was added to a final concentration of o'2~o. The solutions were mixed for IO
rain at room temperature (using a rotator from Kraft Apparatus, Inc.) and centrifuged in a
SorvaU HB 4 rotor for I rain at 1ooo0 rev/min. Organic phase (phenol l) and aqueous phase
(aqu IA) were separated. Aqu I A was extracted twice more with fresh phenol/chloroform/
SDS yielding aqu I B, aqu I C, phenol II, phenol III, respectively. Phenol III was discarded.
Phenol I and phenol II were combined and extracted with I/4 vol. of o-I buffer, which was
adjusted to pH 9. The resulting organic phase was discarded; the aqueous phase aqu II of
this extraction was mixed with aqu I C and 2 vol. ethanol. The precipitated RNA was
separated from the aqueous ethanol and dissolved in o.I buffer and further purified by
sucrose gradient centrifugation (Wimmer, ~972; Yogo & Wimmer, I972 ). The yield of the
RNA varied between 7° to 9o~o of the input virus. PVI, if purified by sucrose density
gradient centrifugation, was usually treated with phenol-chloroform directly from the
appropriate fractions. The RNA, after digestion with alkali or RNase Tz, appeared to be
uniformly labelled as the base ratio of labelled nucleotide 3'-phosphates was similar to that
obtained by spectroscopic analysis (Schaffer et al I96o). However, we and others have
recently observed that a different base ratio was obtained when the same ~"P-RNA was
digested with snake venom 3'-exonuclease or Penicillium nuclease (Flanegan et al. ~977;
Nomoto et al. 1977). This result suggests that the virus RNAs are not uniformly labelled with
~P (see Results).
Preparation of fingerprints and analysis of oligonueleotides. Digestion of the 32P-RNA,
separation of the products by two-dimensional (/D) gel electrophoresis, autoradiography,
elution of oligonucleotides from the polyacrytamide gels and determination of the base
composition of oligonucleotides was as described by Lee & Wimmer (1976). The oligonucleotides were labelled at the 5' end with 32p, hydrolysed with Na2CO3 and analysed by gel electrophoresis as described by Donis-Keller et al. (I977). Although the fingerprints shown in
this paper were prepared exactly as described by Lee & Wimmer (I976), the following
modifications of the 2D-gel electrophoresis have now been introduced: (i) the connecting
gel is polyacrylamide, not agarose, and has the composition of the first dimension gel in
o'o5 M-tris-borate, pH 8-2. Immediately prior to applying the I5 ml connecting gel solution,
the Ist dimension gel, while placed between the 2nd dimension glass plates, is washed 2 to 3
times (25 min) with 2nd dimension buffer (see below). This elutes urea from the edges of the
Ist dimension gel and eliminates connecting problems that were occasionally encountered;
(ii) the buffer used for preparing the 2nd dimension gel and for the electrophoresis is
o"o5 M-tris-borate, pH 8-2, instead of tris-citrate. This has the advantage of producing very
little heat during electrophoresis at 800 V which permits the and dimension to be run at room
temperature without cooling. Consequently, the 2nd dimension gel electrophoresis is carried
out such that the glass plates lean against a gel apparatus similar to that used in rapid
nucleic acid sequencing (Donis-Keller et al I977). The gel is connected with the upper
chamber buffer by a paper wick (Whatman 3MM) that has been covered on both sides
with commercial polyethylene film (for example, 'saran wrap') to prevent evaporation
of the buffer; (iii)the glass plates used are larger: 1st dimension I5×45cm, 2nd
dimension: 32"5×4ocm. The fingerprints thus obtained show improved separation
particularly in the region of medium-sized oligonucleotides with chain lengths of I2 to
I8 nucleotides.
Determination of the yields of large RNase TI-oligonueleotides. The migration of the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
314
Y. F. LEE A N D O T H E R S
Fig. 1. Separation of RNase TI-digests of virion 3zP-RNA of (a) poliovirus, type I, (b) poliovirus,
type z and (c) poliovirus, type 3, by two-dimensional gel electrophoresis.
RNase TI digestion products after electrophoresis in slab gels was determined by autoradiography. Gel pieces containing individual large oligonucleotides were cut out with
surgical blades and placed into 5 ml shell vials (Kimble). The gel was pulverized to a powder
with a flame sealed plastic pipette tip (Eppendorf) which was fastened on to a glass rod.
With the pipette tip remaining in the glass vial, I ml concentrated ammonium hydroxide
was added and the mixture left in a hood at room temperature until o'5 ml of the liquid had
evaporated (overnight). The suspension was then mixed with 4 ml scintillation fluid (Biofluor, New England Nuclear) and the amount of radioisotope determined. The remainder
of the gel which contained labelled material was cut into pieces similar in size to those of the
large oligonucleotides and analysed for radioactivity as described above.
RESULTS
Fingerprints of poliovirus RNAs
The separation of RNase TI digests of three poliovirus RNAs (PVI, PVz and PV3) by
two-dimensional gel electrophoresis is shown in Fig. I. As expected, the 'fingerprints' are
very similar in the area of short oligonucleotides (less than Io bases) which run furthest in
the end dimension whereas little if any similarity can be observed in the lower portion of the
gel which contains large G-terminated oligonucleotides. The fingerprints reveal that
polypyrimidine tracts such as are found in genome RNAs of cardioviruses and aphthoviruses are absent. This result confirms previous data by Brown et al. (~974) and Porter
et aL (1974). RNAs of all three virion types, however, contain poly(A), which is heterogeneous in length (Yogo & Wimmer, 1972) and is present as a smear in the lower left part
of the fingerprint (Lee & Wimmer, 1976). The identity of the poly(A) was confirmed by a
determination of the base composition of the eluted homopolymer (data not shown).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
Sequence complexities o f polio R N A s
3I 5
40
261
I/,
37
t9~
i8 ¸
~6~
14
:~ (3 '.
e
6;
J~
4:
5"
t
3~
I
(b)
Fig. z. Numbering of large RNase TI-resistant oligonucleotides of (a) poliovirus, type I, RNA, and
(b) poliovirus, type z, RNA. Note that the oligonucleotides have been numbered roughly according
to size, which differs from the arbitrary numbering of Lee & Wimmer (1976).
Base composition of large oligonucleotides
Large oligonucleotides of PVI and PV2 (see Fig. 2) w e r e eluted from the slab gels and
their base composition was determined by secondary digestion with RNase A followed by
electrophoresis on DEAE-paper at pH 3"5 (Brownlee, I97Z). This analysis, however, was
complicated by the finding that the virus 3~-P-RNAs used in this study were not uniformly
labelled with 3~p under the conditions of growth (Nomoto et al I977). The deviation from
uniform labelling can be estimated for each base when the base composition of unlabelled
RNA (Schaffer et al. i96o) is compared with the base composition obtained from the ratio
of the four nucleoside 5'-phosphates of the 3zP-RNA. As can be seen in Table I, label in
3~P-pG or 82P-pC is lower, and that in 32P-pU is higher than the expected value, an observation suggesting that the precursor pools for RNA synthesis did not equilibrate during the
labelling periods. This effect is also seen when the cooling period of the cells is omitted during
virus growth or when the cells are incubated for 4 h at 6 °C after the addition of z2p (Scholtissek, I967). Digestion of azP-RNA with RNase T2, on the other hand, yields nucleoside
3'-phosphates with a base ratio close to that expected (Table t). Since the 3'-phosphates
receive the phosphate from their nearest neighbour, the imbalance in labelling presumably
averages out.
The apparent base composition of oligonucleotides of PVI and PV2 RNA as determined
by digestion of the RNase TI-resistant oligonucleotides with RNase A and separation of the
products by electrophoresis on DEAE-paper (Brownlee, ]972) is shown in Tables 2 and 3.
Since the RNA is non-uniformly labelled, the number of single uridine or cytidine nucleotides
determined by this method cannot be corrected (Nomoto et al. 5977) because the nearest
neighbour of a particular pyrimidine nucleoside is unknown. Therefore, the values shown
in Tables 2 and 3 are only an estimate of the total number of pyrimidine nucleotides (nY).
The nature of RNase A-resistant oligonucleotides (Tables 2 and 3; AnYp or AnGp), on the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
316
Y. F. LEE AND OTHERS
T a b l e I. Base composition of poliovirus RNAs determined after different degradation
procedures of the RNA
Unlabelled RNA
:
-
-
-
-
-
,
Acid hydrolysis?
a2p-RNA*
¢~
RNase T2:~
Nuclease PI :~
PVI RNA
Cp:
Ap :
Gp:
Up:
2I'8
28"5
24"8
25"0
24'3
30' I
22' 5
23"1
pC:
pA :
pG:
pO:
21.2
30"5
I2'7
35-6
PV2 RNA
Cp :
Ap :
Gp:
Up;
22"5
28"8
23"3
25"5
25'0
29"5
22"5
22"8
pC :
pA :
pG:
pU:
I7'5
36'4
4"3
4t.I
Snake venom
exonuctease~
20"9
31'o
12'5
35"6
T2/PI
n.d.§
I:43
o'8I
5"23
0"55
1"15
0"99
I'77
0-65
* Prepared as described in Methods.
? Schaffer et al. (196o).
For conditions of digestion see Nomoto et aL (I977).
§ n.d., Not done.
T a b l e 2. Base composition of oligonucleotides and sequence complexity of P VI RNA
RNase T1
Sequence complexity
oligonucleotide
Chain
of genome
no.
Base composition*
length Molar yieldt
RNA;~
I
IAaU, IA3C, IA2G, 2AU, 2AC, xSY
37
0"90+0"03
8517, 855I
2
2AaU, IAG, 4AU, 3AC, I4Y
36
0-87+-0"09
8779, 8689
3
IA~C, 1A3U, ~A2U, IA2C, 2AU, 2AC, 7Y, IG
3I
0"90+-0"07
8473, 8484
4
IAaC, 5AC, IIY, IG
26
1.o2+-o-o4
7Ioo, 7863
5
IAsU, 2AU, 5AC, 7Y, IG
26
t'o7+-o-o7
8046, 726I
6
2A3U, IAU, 4AC, 6Y, 1G
25
0"99+-0"06
8593, 8II4
8
IA3U, IAG, 4AU, xAC, IoY
26
I.O4+-o-o8
7973, 8046
lO
IAaG, tA3U, IA~U, IAU, 2AC, 6Y
23
o"94+-o"I2
7576, 6706
I2
IA~C, IA3C, IA2C, IAC, 7Y, IG
23
vo2+o-o6
7313, 7463
13
2A2U, IAzC, IAC, toY, 1G
22
1'o7+-o'o7
7832, 7250
14
IA3C, IAzC, IAG, IAU, 3AC, 5Y
22
I'o5+-o-o3
7358, 7349
15
IA~G, 3AC, I4Y
23
VlO+-O'08
6958. 720"/
16
IA3U, IA~U, HY, IG
I9
I'O3+-o'Io
7683. 7837
I9
2AU, 14Y, IG
19
t-114-o-I9
Average 785.1 :t:567
* RNase TI-oligonucleotides (see Fig. 2) were digested with RNase A and the apparent base composition
determined after separation of nucleotides on DEAE-paper. Y = pyrimidine nucteotides. Results are from
4 experiments.
t Molar yields of RNase Tl oligonucleotides and complexity determined as described in the text. Standard
deviation of 7 experiments for molar yields.
Sequence complexity of two experiments. The average is given with the standard deviation of all sequence
complexities determined.
other h a n d , can be derived fairly accurately f r o m their p o s i t i o n in the p a p e r e l e c t r o p h e r o g r a m (Brownlee, I972). The quantification o f some o f the dinucleotides ( A - Y p ) , however,
m a y have to be corrected by sequence analysis (see below). In a n y case, s e c o n d a r y digestion
with R N a s e A o f the R N a s e - T [ resistant oligonucleotides o f p o l i o v i r u s R N A labelled with
asp in vivo is n o t suitable for the d e t e r m i n a t i o n o f the chain length o f these oligonucleotides.
To o v e r c o m e this difficulty, we m a d e use o f the p r o c e d u r e o f D o n i s - K e l l e r et aL (I977) a n d
s e p a r a t e d i n c o m p l e t e alkali digests o f 5'-3~p-labelled oligonucleotides by gel electrophoresis.
A u t o r a d i o g r a p h y o f the gels reveals the correct chain length o f the oligonucleotides by
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
Sequence complexities of polio R N A s
317
Table 3. Base composition of oligonucleotides and sequence complexity of PV2 RNA
RNase T[
oligonucleotide
no.
2
3
4
5
8
9
to
ii
~2
I3
I7
28
35
36
37
Base composition*
3A2U, 2AU, 4AC, 7Y, IG
IA~C, IA4U, IA~U, 2AU, IAC, IG
IA2U, 3AU, 3AC, IG
IAaU, IA3C, IA~U, 2AU, 1AC, 9Y, IG
tA4C, 2A2U, ]AeC, IAU, 3AC, IY, lG
IA3G, IA3U, IA2U, [AU, 3AC, 5Y
lAG, 3AU, IAC, 12Y
2A2U, IA2C, IAU, IAC, 9Y, IG
1A2C, IAC, 4AC, 7Y, IG
IAsC, IA~C, 2AU, IAC, 4Y, IG
IA2U, zA3C, 3AU, 5Y, iG
~A~U, 2AU, IAC, 9Y, IG
zAU, [AC, 9Y, IG
zAU, IAC; 9Y, IG
3AU, 8Y, IG
Chain
length
29
32
27
27
24
24
22
23
21
22
2i
I9
i6
16
15
Sequence complexity
of genome
Molar yield
RNA'~
Ho4-o'o3
76054-592
r.o44-o'o2
78854-536
l.oi 4-o,oi
8o814-416
I.o34-o.o6
7920+465
0-954-o-05 86204-773
0.904-0-08 94o44-I33o
o-794-o.oi
io5634-594:~
.o934-0"07 87584-Io74
Po54-o-o3 77934-314
1.o24-o-o5 79724-289
1"o44-o'o3 78534-358
0"934-0"o4 8816:kI93
1.174-o.o7 71694-769
i.o34-o.ix
81764-546
0.964-o-o4 84554-212
Average
81814-578
* See Table 2; results from four experiments,
t See Table 2; sequence complexity is result of three experiments.
:~ This value was omitted for the determination of the average complexity.
counting the number of visible bands (Donis-Keller et al. 1977). This is illustrated in Fig. 3
for oligonucleotide # I9 of PV~. The chain lengths thus obtained for this and other oligonucleotides of PVi and PV2 are listed in Tables 2 and 3.
So far, we have confirmed the base composition shown in Table z only for oligonucleotide
# 3 . This was accompfished by incomplete RNase U2 digestion followed by gel electrophoresis of the products (Donis-Keller et al. r977). The sequence thus obtained, A-A-A-AY-Y-A-A-A-Y-Y-Y-Y-A-A-Y-Y-A-Y-A-Y-A-Y-Y-A-A-Y-Y-A-Y-Gp, is in agreement
with the base composition shown in Table 2 (A. Diamond, N. Kitamura & E. Wimmer,
unpublished results). Sequence analysis of all large RNase TI-resistant oligonucleotides of
PVI R N A is now in progress.
Molecular weights of PVI and PV2 RNAs and of PVI DI RNAs
The tool. wt. of an R N A can be determined by comparing the yield of a fragment of
known tool. wt. to the mass of the R N A from which the fragment originated. A prerequisite
for this procedure is that the yield of the R N A fragment can be determined without loss
during separation procedures. Separation of RNase T I digests by 2D-polyacrylamide gel
electrophoresis fulfils this requirement (Billeter et al. I974; Lee et al. I975). The accuracy
of the mol. wt. determination increases if the yields of several Ti-oligonucleotides are
simultaneously established.
Oligonucleotides used for this study were first analysed for the molarity at which they
occur in the genome. This was carried out as follows: the total yield in counts/min of all
large oligonucleotides listed in Table z was divided by the total number of nucleotides in all
large oligonucleotides (determined by adding up the chain lengths). The value obtained is
the average specific radioactivity per nucleotide in that particular population of oligonucleotides (Table 2). We assume that this average specific radioactivity per nucleotide is
very similar to the average specific activity of nucleotides in the total R N A chain because,
based upon the physical m a p of the RNase Tl-resistant otigonucleotides ( N o m o t o et al.
I979), the oligonucleotides listed in Table 2 originate randomly from the virus genome.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
318
Y. F. LEE A N D O T H E R S
17
16~ - - 15
14
13 f
12 ~'~
11
Q
l0 ~
, ~
9 ~ Q
8 ~ Q
7 ~ i
5
19
18
4
~
17
3
16
15
2
13
..... :
~
12
11
1
~
,*--
8
7
6
Fig. 3. Separation of an incomplete digest with Na2CO3, pH 9, of oligonucleotide no. 19 of PVI.
For the conditions of digestion and polyacrylamide gel electrophoresis see Donis-Keller et al.
(I977).
Using the average specific radioactivity per nucleotide established independently for each
32P-RNA preparation, the molar yields of several large oligonucleotides were determined.
The results are shown in Tables z and 3 and indicate that each fragment is unique in the
genome from which it originated. The molar yields of another group of Io large RNase
TI-resistant oligonucleotides, not listed in Tables 2 and 3, were also close to one (data not
shown).
The chain length of the RNA can then be determined by the simple relationship - ct/min
[oligonucleotide]: chain length [oligonucleotide] = ct/min [RNA]: chain length [RNA]
(Beemon et al. I974; Billeter et al. t974; Lee et aL I975). Our procedure to measure the
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
Sequence complexities of polio R N A s
319
Table 4. Sequence complexity of PVI DI(I) and D/(2) RNAs
RNase TI*
oligonucleotide
no.
Sequence complexityy
DI(I)
I
2
3
4
5
8
12
I3
t4
15
16
I9
Average
DI(2)
7878, 7800
8852, 8981
6989, 7306
7366, 6870
6177, 6196
6196, 6378
7312, 79II
583° , 617o
8624, 826I
69ol, 6o62
6203, 6357
5887, 6157
7512,
7832
6737,
6533,
5729,
6436,
7O85
7107
5728
6379
6085, 5859
745I, 7995
61o8, 6579
5966, 619o
6607, 6853
7042+ 999
6639 4- 674
* Same oligonucleotides as in Table 2.
t Result of two experiments.
ct/min [RNA] is to count all radioactivity in the gel (see Methods) since all labelled materials
applied to the first dimension gel can be recovered (Lee & Wimmer, 1976). Thus, internal
standards (Billeter et al. I974) to correct for losses are unnecessary.
The chain length of PVI RNA was calculated to be 785Idz567 nucleotides and that of
PV2 RNA 8181-4-578 nucleotides. Based on an average mol. wt. of the nucleotides in
PVI RNA of 339"5 and in PV2 RNA of 338"7 the mol. wt. of PVI RNA is 2"66+o'I9 × lO6
and that of PV2 RNA is 2.77:~o.i 9 × IOG.
A similar analysis was carried out for the genome RNAs of PVI D I 0 ) and DI(2). The
yield of oligonucleotides, however, was not determined since we assume that the primary
structure of DI RNA is identical with that of standard (wild type) PVI RNA with the
exception of a deletion of the 5' terminal half of the RNA (Nomoto et a! 1979). This assumption is strongly supported by the following observations: (i) fingerprints of DI(I) and DI(2)
RNAs are identical with that shown in Fig. 2(a) except for four large oligonucleotides
(No. 6, 25, 38 and 47) which are absent (Nomoto et aL I979); and (ii) annealing of virion
RNA of DI(2) particles with denatured double-stranded RNA of PVI followed by inspection of the products in the electron microscope yielded perfect hybrid heteroduplex
molecules with a single loop approx. 20 ~ from one end of the molecules (Nomoto et al.
1979). As shown in Table 4, the calculated sequence complexity of the two DI RNAs is
smaller [DI(I): 7042±999 nucleotides; DI(2): 6639 :~674 nucleotides] than that of standard
PVI RNA. This result is consistent with the fact that the DI RNAs are shorter than standard
PVI RNA.
DISCUSSION
The genome RNAs of the three types of poliovirus have been described as between 25 to
5OYo homologous in sequence (Young et al. I968; Young, I973a, b). These values were
obtained by hybridization of double-stranded RNA to excess heterologous single-stranded
RNA. Based upon the size measurements of ribonuclease resistant double-stranded regions
in heterologous hybrids, it has been suggested that segments of sequence homology between
PVI and PV 3 RNAs are at the most 380 nucleotides long, those between PVI and PV2
RNAs being even smaller (Young, I973a). The RNA fingerprints of the three serotypes
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
320
Y. F, LEE AND OTHERS
do not indicate any sequence homologies between PVI and PV2 (Fig. I) since large oligonucleotides of identical base composition have not been detected. However, I2 to I6 large
oligonucleotides represent only 5 to 8% of the virus genome and smaller oligonucleotides
shared by the different virus RNAs may be found in the upper portion of the gel. On the
other hand, the extent of sequence homology, which has been reported previously, may be
an overestimate due to the conditions of hybridization (Young et aL I968). The dissimilarity
of fingerprints of closely related picornavirus genomes has also been observed recently by
Frisby et aL (I976).
Various methods have been applied previously to determine the tool. wt. of picornavirus
RNA: sedimentation velocity (Montagnier & Sanders, I963; Anderer & Restle, I964;
Scraba et al. 1967; Granboulan & Girard, I969; Tannock et al. 197o), electron microscopy
(Granboulan & Girard, I969), gel electrophoresis (Tannock et al. I97O; Yogo & Wimmer,
I975) and nuclease digestion (Todd & Martin, I975). An average tool. weight of z.5+o'I
× ~o6 is now generally accepted (Rueckert, 1976). The tool. wt. obtained by fingerprint
analyses which we report here (2"66~o.t9 for PVI RNA, 2"77±o"I9 for PV2 RNA) are in
agreement with this value.
One problem originally encountered in this study had been the difference in the specific
radioactivities of the phosphates in the polynucleotide chain when virus was grown in
phosphate-free medium in the presence of carrier-free 32p. This phenomenon makes
sequence analysis of the virus RNA by the classical methods (Brownlee, I972 ) all but impossible. The development of rapid sequence methods of terminally labelled RNA fragments (Lockard & Rajbhandary, 1976; Donis-Keller et aL 1977; Simoncits et a[. I977),
however, has solved this problem. We have observed non-uniform labelling of poliovirus
RNAs as well as of RNA of encephalomyocarditis virus grown in L-cells or HeLa cells
(Golini et al. I978). Both picornaviruses replicate rapidly in the cytoplasm of the host cell.
One reason for the non-uniform labelling might be that the equilibria between phosphate
and nucleotide pools in the host cell form only very slowly. Addition of 32p to host cells
several hours before infection might be necessary to balance the specific radioactivities of
the nucleotide triphosphates.
We have shown previously that DI particles of PVt (Mahoney) are mutants whose
genomes have an internal deletion approx. 20~o from the 5' end (Nomoto et al. r979).
Three stocks of PVI DI particles [DI(Q, DI(z) and DI(3)] have been isolated (Cole et al.
197I). These DI particles differ in that the size of their deletions increases from DI(I) to
DI(3 ) (Cole et al. I971 ; Nomoto et al. I979) although the deletion of all three DI particles
is located in the same region of the genome (Nomoto et al. I979). On the basis of their
mobilities in polyacrylamide or agarose and in vitro protein synthesis, the size of the
deletion in DI RNA has been estimated to be IO to i5 ~/o of the standard virus genome. An
analysis of heteroduplexes between DI(2) RNA and standard virus minus strand RNA has
led to the conclusion that the deletion is approx. 8o0 nucleotides long (Nomoto et al. I979).
The chain lengths of DI(I) and DI(2) RNA reported here suggest that IO~o and I 5 ~ ,
respectively, of the standard virus genome are deleted. The variation in the extent of the
deletion clearly reflects uncertainties intrinsic to the methods used to determine them.
Only a few of the oligonucleotides generated by RNase Tr were subject to analysis in this
study. The unimolar yields of these oligonucleotides nevertheless suggest that there exist
no large redundant sequences in these RNAs since, in the case of PVI, these oligonucleotides
map randomly over the entire genome (Nomoto et al. I979).
N o t e added in proof. We (N. Kitamura and E. Wimmer, unpublished d~trL) h~tve determined the sequence of oligonucleotide no. I6 and found it to be identical to nucleotide
sequence 5-23 (beginning at the 3' end of PVI RNA) reported by Porter et al. (I978, Nature,
L o n d o n 276, 298-3oi ) .
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
Sequence complexities of polio RNAs
32I
We thank Nancy Reich for criticism of the manuscript and excellent technical assistance, and Karl Lonberg-Holm for samples of poliovirus, type 2 and type 3. This investigation was supported in part by Grant CA-16879, awarded by the National Cancer Institute,
by Grant AI-~ 5122, awarded by the National Institute of Allergy and Infectious Diseases,
Department of Health, Education and Welfare; by Grant BMS 75-o5378 awarded by the
National Science Foundation; and by a Postdoctoral Fellowship CA-olI8O of the National
Institutes of Health to Y . F . L.
REFERENCES
ANDERER, F. A. & RESTLE, H. (I964). Untersuchungen iiber ein attenuiertes Poliomyelitis Virus, Type II.
Reindarstellung und physikalisch-chemische Eigenshaffen des virus. Zeitschrift fiir Naturforsehung
I g b , IO26-IO3t.
BEEMON,K., DUESBERG,P.
& VOGT, P.
(1974). Evidence for crossing-over betweenavian tumor viruses based
on analysis o f viral R N A s . Proceedings of the National Academy of Sciences of the United States of
America 7 x, 4254-4258.
BILLETER, M. A., PARSONS,J. T. & COFFIN, J. M. (I974). The nucleotide sequence complexity o f avian tumor virus
R N A . Proceedings of the National Academy of Sciences of the United States of America 71, 3560-3564 .
BROWN, F., NEWMAN, J., STOTT, J., PORTER, A., FRISBY, D., NEWTON, C., CAREY, N. & FELLNER, P. (1974). Poly(C)
in animal viral R N A s . Nature, London 251, 342-344.
BROWNLEE, G. G. (I972). In Determination of Sequences in RNA. New York; American Elsevier Publishing
Co., Inc.
COLE, C. N., SMOLER, D., WIMMER, E. & BALTIMORE, D. (1970. Defective interfering particles o f poliovirus I.
Isolation and physical properties. Journal of Virology 7, 478-485.
DETJEN, B. M., LUCAS, J. & WIMMER, E. (I978). Polio single stranded R N A and double stranded R N A : differential infectivity in enucleated cells. Journal of Virology 27, 582-586.
DE WACHTER, R. & FIERS, W. (1972). Preparative two-dimensional polyacrylamide gel electrophoresis of
a2P-labelled R N A . Analytical Biochemistry 49, 184-197.
DONIS-KELLER, H., MAXAM,A. & GILBERT, W. 0977). Mapping adenines, guanines, and pyrimidines in RNA.
Nucleic Acids Research 4, 2527-2538.
DORSCH-H.~SLER, K., YOGO, Y. & WIMMER, E. 0975)- Replication o f picornaviruses. I. Evidence from in vitro
R N A synthesis that poly(A) of the polio-virus genome is genetically coded. Journal of Virology 16,
I5t2-1527.
FLANEGAN, J. B., PETTERSSON, R. F., AMBROS,V., HEWLETT, M. J. & BALTIMORE, D. 0977). Covalent linkage o f a
protein to a defined nucleotide sequence at the 5"-terminus of virion and replicative intermediate
R N A s of poliovirus. Proceedings of the National Academy of Sciences of the United States of America
74, 961-965.
FR1SBY, D. P., NEWTON, C., CAREY, N. H., FELLNER, P., NEWMAN, J. F. E., HARRIS, T. J. R. & BROWN, F. (1976).
Oligonucleotide mapping o f picornavirus R N A s by two-dimensional electrophoresis. Virology 7I,
379-388.
GOLINI, F., NOMOTO, A. & WIMMER, E. (1978). The genome linked protein o f picornaviruses. IV. Difference in
the VPg's of encephalomyocarditis virus and poliovirus as evidence that the genome-linked proteins
are virus-coded. Virology 89, 1 I2-I 18.
GRANBOULAN, N. & GIRARD, M. (I969). Molecular weight o f poliovirus ribonucleic acid. Journal of Virology 4,
475-479.
LEE, Y. F. & WIMMER, E. 0976). 'Fingerprinting' high molecular weight R N A by two-dimensional gel electrophoresis: application to poliovirus R N A . Nucleic Acids Research 3, 1647-1658.
LEE, Y. F., HOPKINS, T. J., WIMMER, E. & LAI, M. M.-C. (1975). Fingerprinting of Rous sarcoma virus 35S R N A
and o f poliovirus R N A by two-dimensional get electrophoresis. Abstracts of the 75th Meeting of the
American Society for Microbiology, p. 214.
LEE, Y. F., NOMOTO, A. & WIMMER, E. 0976). The genome o f poliovirus is an exceptional eukaryotic m R N A .
Progress in Nucleic Acid Research and Molecular Biology x9, 89-95.
LEE, Y. F., NOMOTO, A., DETJEN, B. M. & WIMMER, E. (I977). A protein covalently linked to poliovirus genome
R N A . Proceedings of the National Academy of Sciences of the United States of America 74, 59-63.
LEVINTOW, L. (I974). The reproduction o f picornaviruses. In Comprehensive Virology, vol. 2, pp. IO9-I69.
Edited by H. Fraenkel-Conrat and R. R. Wagner. New York: Plenum Press.
LOCKARD, R. E. & RAJBHANDARY,U. L. (I976). Nucleotide sequences at the 5' termini of rabbit ~z and fl globin
m R N A . Cell 9, 747-760.
MANDEL, B. (I964). The extraction o f ribonucleic acid from poliovirus by treatment with sodium dodecyl
sulfate, l'Trology 22, 36o-367.
MONTAGNIER, L. & SANDERS, F. K. 0973). Replicative form of encephalomyocarditis virus ribonucleic acid.
Nature, London x99, 664-667.
NOMOTO, A., DETJEN, B. M., POZZATTI, R. & WIMMER, E. (I977). The location of the polio genome protein in
viral R N A s and its implication for R N A replication. Nature, London 268, 2o8-213.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39
322
Y.F.
LEE A N D O T H E R S
NOMOTO, A., JACOBSON, A., LEE, Y. F., DUNN, J. J. & WIMMER, E. (1979). Defective interfering particles of
poliovirus: mapping of the deletion and evidence that the deletion in the genome of DI(1), (2) and (3)
is located in the same region. Journal of Molecular Biology (in the press).
PORTER, A., CAREY, N. & FELLNER, V. (I974). Presence of large poly(rC) tract within the RNA of encephalomyocarditis virus. Nature, London 248, 675-678.
PORTER, A. G., FELLNER, P., BLACK, D. N., ROWLANDS, D. J., HARRIS, T. J. R. & BROWN, F. (I978). 3'-Terminal
nucleotide sequences in the genome RNA of picornaviruses, Nature. London 276, 298-3oi.
RUECKERT, R. R. (I976). On the structure and morphogenesis of picornaviruses. In Comprehensive Virology,
vol. 6, pp. I31-2J 3. Edited by Fraenkel-Conrat and R. R, Wagner. New York: Plenum Press.
SCHAEFER, r. L., MOORE, n. ~. & SCHWERD'r, C. E. (I960). Base composition of the ribonucleic acids of the
three types of poliovirus. Virology xo, 53o-537.
sCHOLTISSEK, C. (I967). Nucleotide metabolism in tissue culture cells at low temperatures. I. Phosphorylation
of nucleosides and deoxynucleosides in vivo. Biochimiea et Biophysiea Acta x45, 228-237.
SCRABA, D., KAY, C. M. & COLTER, J. S. (1967). Physio-chemical studies of three variants of Mengo virus and
their constituent ribonucleic acids. Journal of Molecular Biology 26, 67-79.
SIMONCITS, A., BROWNLEE, G. G., BROWN, R. S., RUBIN, J. R. & GUILLEY, H. (I977). New rapid gel sequencing
method for RNA. Nature, London 269, 833-836.
TANNOCK, G. A., GIBBS, A. J. & COOPER, P. D. (I970). A reexamination of the molecular weight of poliovirus
RNA. Biochemical and Biophysical Research Communications 38, 298-304.
TODD, D. & MART1N, S.J. (1975). Determination of the molecular weight of bovine entcrovirus RNA by
nuclease digestion. Journal of General Virology 26, 121-129.
WIMMER, E. (1972). Sequence studies of poliovirus RNA. I. Characterization of the 5"-terminus. Journal of
Molecular Biology 68, 537-540.
YOGO, Y. & WIMMER, E. (I972). Polyadenylic acid at the 3'-terminus of poliovirus RNA. Proceedings of the
National Academy of Sciences of the United States of America 69, 1877-I 882.
YOGO, Y. & WIMMER,E. (1975)- Sequence studies of poliovirus RNA. lII. Polyuridylic acid and polyadenylic
acid as components of the purified poliovirus replicative intermediate. Journal of Molecular Biology 92,
467-477.
YOUNG, N. A. (I973a). Size of gene sequences shared by polioviruses, types I, 2 and 3. Virology 56, 4oo-4o3.
YOUNG, N. A. (I973b). Polioviruses, coxsackiviruses, and echoviruses: comparison of the genomes by RNA
hybridization. Journal of Virology xx, 822-839.
VOtiNG, N. A., HOVER,B. H. & MARTIN,N. A. (1968). Polynucleotide sequence homologies among polioviruses.
Proceedings of the National Academy of Sciences of the United States of America 6x, 548-555,
(Received 29 November I978)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 01:48:39