Journal of General Virology (1990), 71, 43-52.
43
Printed in Great Britain
Localization of genomic regions specific for the attenuated, mouse-adapted
poliovirus type 2 strain W-2
Daniel C. Pevear, t Chad K. Oh,~ Laura L. Cunningham, Miriam Calenoff and Burk Jubelt*§
The Les Turner A L S Research Laboratory, Department of Neurology, Northwestern University Medical School,
303 E. Chicago Avenue, Chicago, lllinois 60611, U.S.A.
In order to begin to elucidate the genomic basis of the
attenuation of mouse-adapted, poliovirus type 2 strain
W-2 (PV2/W-2), we have cloned and sequenced the
virus and compared it with the virulent, mouse-adapted
PV2/Lansing strain. In addition, we have performed
computer-generated comparisons of PV2/W-2 to the
non-mouse-adapted, attenuated PV2/Sabin strain to
determine whether mutational patterns occur that
result in attenuation. The PV2/W-2 genome is 7434
nucleotides in length, which is three bases shorter than
PV2/Lansing. The 5' non-coding region of PV2/W-2 is
747 nucleotides in length (compared to 744 in
PV2/Lansing) and shares 98.8% identity with
PV2/Lansing and 82-3% identity with PV2/Sabin.
Overall, the PV2/W-2 polyprotein (2205 amino acids)
is two amino acids shorter than that of either
PV2/Lansing or PV2/Sabin (2207 amino acids). These
contiguous deletions fall within the P3-D region
(polymerase). Within these 2205 amino acid residues
only 26 differences were observed between PV2/W-2
and PV2/Lansing (98-8% identity), whereas 92 occurred between PV2/W-2 and PV2/Sabin (95-8%
identity). The 3' non-coding region of PV2/W-2 is 72
nucleotides in length and shares 100% identity with
PV2/Lansing and 98.6% identity with PV2/Sabin.
Amino acid changes in the capsid protein region
occurred in neutralization sites 1 and 3, areas previously shown to be important for pathogenicity. The
cleavage site between non-structural proteins P2-C/P3A consisted of a glutamine-serine pair, in contrast to
other sequenced polioviruses which have a glutamineglycine dipeptide.
Introduction
virion genome relate to the occurrence of late paralysis
(Rozhon et al., 1984).
In contrast, the Lansing strain of poliovirus type 2
(PV2/Lansing) (Armstrong, 1939) displays a virulent
phenotype in mice (Sabin, 1950) characteristic of human
poliomyelitis (Jubelt et al., 1980a). PV2/W-2 and
PV2/Lansing are the only mouse-adapted poliovirus
strains that have been well characterized biologically
and, in addition, PV2/Lansing has been cloned and
sequenced (Racaniello, 1984; La Monica et al., 1986).
Recombination studies have revealed that the PV2/
Lansing capsid region is necessary for mouse intracerebral neurovirulence when compared with the nonmouse-adapted PV1/Mahoney strain (La Monica et al.,
1986; Martin et al., 1988; Murray et al., 1988). Rather
than using host range to analyse the molecular basis for
virulence and attenuation, our general strategy is to
compare the two mouse-adapted PV2 strains, Lansing
and W-2. In the present paper we have determined the
complete nucleotide sequence of PV2/W-2 and compared this sequence with that of PV2/Lansing and PV2/
Sabin (a non-mouse-adapted, attenuated virus). These
analyses have identified a limited number of nucleotide
Determination of genomic regions responsible for
various biological phenotypes (e.g. virulence and host
range) remains a central question in many virus-host
systems. The W-2 strain of poliovirus type 2 (PV2/W-2)
represents a particularly attractive virus for such studies
in that the virus has been adapted for growth in murine
tissues (Koprowski et al., 1951, 1952) and exhibits the
characteristics of attenuated poliovirus strains, including
temperature sensitivity (Carp et al., 1963) and a
decreased rate of dissemination within the central
nervous system (CNS) (Jubelt & Meagher, 1984a, b).
PV2/W-2 rarely causes paralysis in mice (Jubelt &
Meagher, 1984a), but can persist in the CNS of immunosuppressed mice, where accumulated mutations in the
t Present address: Stealing Drug Incorporated, Rensselaer, New
York, U.S.A.
:~Present address: Department of Pediatrics, Rush-PresbyterianSt Luke's Hospital, Chicago, Illinois, U.S.A.
§ Present address: Department of Neurology, SUNY Health Science
Center, 750 East Adams St, Syracuse, New York 13210, U.S.A.
0000-9151 © 1990 SGM
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44
D. C. Pevear and others
Bgl I I
and amino acid differences between the attenuated and
virulent mouse-adapted PV2 strains and suggest future
experiments to m a p definitively the genomic regions
controlling virulence.
748
l
PI
33851
I
Apa I
P2
51110~
P3
5'
7363
i(A)~
>p1308
Methods
Materials. The following reagents were purchased: reverse tran-
scriptase (Seikagaku), actinomycin D (Merck), RNasin (PromegaBiotech), [ct-thio-3sS]dATP and [ct-32p]dCTP (Amersham), deoxynucleoside and dideoxynucleoside triphosphates (Boehringer), T4
DNA ligase, oligo(dT)12_,s, large (Klenow) fragment of Escherichia
coli DNA polymerase I, E. coli DNA polymerase I (holoenzyme),T4
DNA polymerase, RNase H, terminal deoxynucleotidyltransferase,
and CycloneI Biosystem(IBI), restriction endonucleasesand universal
sequencing primer (New England Biolabs).
Virus and cell culture. PV2/W-2 was obtained from the American
Type Culture Collection and was twice plaque-purified in RD cells
(Jubelt & Meagher, 1984a).
Virion RNA. Virus was grown in RD cells and purified in a 20 to 70~
sucrose gradient (Jubelt & Lipton, 1987), followedby isopycnic centrifugation in caesium sulphate (Ziola & Scraba, 1974). Virion RNA
was extracted from 1 mg of purified virions by phenol-chloroform
extraction (Pevear et al., 1987).
Preparalion of the cDNA clones. Viral RNA was transcribed into
cDNA for cloning into pBR322 using the replacement synthesis
procedure of Kandolf & Hofschneider (1985) with some modifications,
as described previously (Pevear et al., 1988).
Restriction endonuclease digestion. Recombinant plasmid DNAs were
digested with restriction endonucleases according to the manufacturers' instructions. The restriction fragments were sized by agarose
gel electrophoresis.
DNA sequencing. Overlapping recombinant cDNA clones p 1308 and
p1345 were used to determine the complete nucleotide sequence of
PV2/W-2 by the dideoxynucleotide sequencing procedure of Sanger et
al. (1977). Subclones in MI3 mpl8 and mpl9 were generated by the
unidirectional deletion of large clones(Dale et al., 1985). Each base was
sequenced an average of 3.5 times to generate unambiguous data and
about 50~ of the sequence was confirmedon both DNA strands. Every
base was read at least twice to eliminate user entry errors.
Computer analysis of the DNA sequence. All DNA sequence
manipulations were performed with the UWGCG and Staden
programs of the program library of the University of Wisconsin
Genetics Computer Group (Staden, 1980; Devereuxet al., 1984)on the
Northwestern University VAX 11/780 super.minicomputer.
Results
Analysis o f c D N A clones
Synthesized oligo(dC)-tailed ds c D N A to PV2/W-2
R N A was inserted into the P s t I site of oligo(dG)-tailed
pBR322 for transformation of E. coli DH1 cells by the
method of H a n a h a n (1983). Tetracyclin-resistant, ampicillin-sensitive colonies were screened for insert size by
rapid disruption (Maniatis et al., 1982) followed by
agarose gel electrophoresis. Restriction enzyme analysis
p1345>
Fig. 1. A composite partial restriction endonuclease map of PV2/W-2
cDNA clones indicating the position of the unique BglII and ApaI
sites. The organization of the PV2/W-2 genome is shown on the top
line. The positions of the virus-derived inserts from the two clones are
illustrated and arrowheads indicate the orientation of the insert relative
to the EcoRI site in pBR322.
of the larger clones identified two c D N A s , which
together appeared to span the entire PV2/W-2 genome
(Fig. 1). However, upon sequencing, c D N A clone p1308
was found to lack the bases corresponding to the first two
uridine residues at the 5' end of the viral R N A genome.
Clones p1308 and p1345 were joined in pBR322 at the
unique A p a I site. A unique B g l l I restriction endonuclease site at position 3795 and the P s t I sites introduced into
the c D N A during cloning into pBR322 were used for
subcloning in M13 m p l 8 and m p l 9 for generation of
single-stranded sequencing templates.
Nucleotide sequence and g e n o m e organization
By alignment with the reported sequence of PV2/Lansing (La Monica et al., 1986), as well as a number of other
poliovirus strains, it was clear that p1308 lacked only the
first two thymidine residues at the 5' end of the viral
genomic c D N A . In order to generate a numbering
sequence for the PV2/W-2 genome that was consistent
with that of PV2/Lansing, we have included these
residues in Fig. 2. Overall, the organization of the PV2/
W-2 genome was identical to that of PV2/Lansing and
was typical of all poliovirus serotypes (Rueckert &
Wimmer, 1984). The 5' non-coding region of the PV2/W2 genome was 747 nucleotides in length, making it three
bases longer than that of PV2/Lansing (Fig. 2). By
computer-generated alignment of the 5' non-coding
regions, the three additional guanosine nucleotides in the
PV2/W-2 genome were inserted after positions 351,352
and 366 (Table 1). O f the 744 nucleotides which could be
aligned, 735 bases were identical between the two viruses
(98.8~ identity; Table 1). Although the 5' non-coding
regions of PV2/W-2 and PV2/Sabin were identical in
length, the computer algorithm introduced a total of
eight gaps into the sequences to optimize alignment with
the reported sequence of PV2/Sabin (Toyoda et al.,
1984). Furthermore, of the remaining 739 nucleotides
which did align, 132 differed between these two PV2
strains (82.1 ~ identity; data not shown).
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Sequencing of attenuated poliovirus 2 strain W-2
Table 1. Nucleotide differences between PV2/W-2 and
PV2/Lansing in the 5' non-coding region
Nucleotide*
position
PV2/W-2
PV2/Lansing
63
120
136
351/3521"
352/353t
366/367t
599
606
692
728
735
738
C
C
A
G
G
G
U
A
C
G
U
U
U
U
G
C
U
U
A
C
C
The polyprotein coding region of PV2/W-2 began at
nucleotide position 748 and extended to position 7362,
making it six nucleotides shorter than both PV2/Lansing
and PV2/Sabin (Fig. 2). In both cases, the deletions in
PV2/W-2 occurred as contiguous nucleotides between
PV2/W-2 positions 6672 and 6673 (region P3-D of the
genome). The 3' non-coding regions of all three virus
strains were 72 nucleotides in length and displayed striking identity with one another. PV2/W-2 and PV2/
Lansing shared 100% nucleotide identity in their 3' n o n coding regions, whereas PV2/Sabin had a single mismatch at PV2/W-2 position 7399 (an A in PV2/W-2 to a
G in PV2/Sabin). Including gaps introduced into the
alignments by the computer algorithm, only 100 nucleotide differences occurred in the entire genome between
PV2/W-2 and PV2/Lansing (98.7 % identity). The length
* Based on the PV2/Lansing nucleotide sequence.
t Represents insertions between these PV2/Lansing nucleotides.
T a b l e 2.
45
Amino acid differences between PV2/W-2, P V2/Lansing and~or P V2/Sabin based upon P V2/Lansing amino acid
positions
Protein
Position
W-2
Lansing
Sabin
VP4
67
T
*
M
VP2
11
43
45
105
115
137
141
186
239
D
K
s
S
S
A
L
A
A
V
59
62
73
75
140
143
235
S
K
N
A
K
E
V
2
5
9
13
15
19
21
23
24
26
29
30
31
32
41
69
99
103
143
170
L
L
V
V
R
T
L
P
V
N
D
T
R
S
T
K
R
K
T
R
VP3
VP1
G
T
F
R
T
G
F
T
F
V
V
Protein
P2-A
N
E
K
A
A
Q
K
W
R
S
T
E
K
A
I
M
A
I
K
V
P
S
T
S
G
H
K
P
I
R
K
R
I
W
P2-B
P2-C
Position
W-2
Lansing
252
280
289
291
295
299
K
Y
G
T
E
I
31
32
33
36
42
45
47
49
56
58
76
144
I
N
I
I
V
K
Q
I
N
H
V
V
2
23
25
75
94
95
96
V
N
I
A
I
M
R
I
D
V
I
A
I
E
9
30
33
34
61
88
96
246
250
274
311
319
T
D
K
E
A
R
V
M
I
Q
I
S
A
S
Q
D
S
K
H
V
V
P
A
G
T
V
T
E
K
Sabin
Protein
R
F
P3-A
T
V
S
V
N
A
R
L
T
S
N
I
E
P3-C
H
V
N
* A blank space m e a n s that the amino acid is the same as that of W-2.
t A dash indicates a deletion of that a m i n o acid.
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P3-D
Position
W-2
Lansing
Sabin
1
12
15
26
44
45
S
I
T
H
A
D
G
G
V
S
Q
V
N
16
21
31
46
48
56
75
128
182
L
I
H
T
V
V
V
G
S
V
S
Y
S
A
I
I
7
16
46
51
69
90
113
142
230
231
388
424
R
V
S
K
E
I
S
Q
-t
V
S
K
I
N
R
D
F
T
R
F
A
I
N
Q
V
F
A
N
46
D. C. Pevear and others
1
UUAAAACAGCUCUGGGGUUGUUCCCACCCCAGAGGCCCACGUGGCGGC•AGUACACUGGUAUCGCGGUACCUUUGUACGCCUGUUUUAUACUCCCUU•C••CGUAACUUAGAAGCACAAC
121
GUCCAAG~-~CAAUAGAAGGGGGUACAAACCAGUACCACCACGAACAAGCACUUCUGUUCCCCCGGUGAGGCUGUAUAGGCUGUUUCCACGGCUAAAAGCGGCUGAUCCGUUAUCCGCUCA
241
UGUACUUCGAGAAGCCUAGUAUCACCUUGGAAUCUUCGAUGCGUUGCGCUCAACACUCAACCCCAGAGUGUAGCUUAGGUCGAUGAGUCUGGACGUUCCUCACCGGCGACGGUGGUCCAG
381
GCUGCGUUGGCGGC•UACCUGUGGCCCARAGC•ACAGGACGCUAGUUGUGAACAAGGUGUGAAGAGCCUAUUGAGCUACCUGAGAGUCCUCCGGCCCCUGAAUGCGGCUAAUCCUAACCA
481
CGGAGCAGGCAGUGGCAAUCCAGCGACCAGCCUGUCGUAACGCGCRAGUUCGUGGCGGAACCGACUACUUUGGGUGUCCGUGUUUCCUUUUAUUUUUACAAUGGCUGCUUAUGGUGACAA
601
UUAUUGAUAGUUAUCAU~GCAAAU~GGAUUGGCCAUCCGGUGAGAAUUUGAUUAUUAAAUUACUCUCUUGUUGGGAUUGCUCCUUUGAAAUCCUGUGCACUCACACCUAUUGGAAUUA
721
CCUCAUUGUUGAGAUAUUAUUACCACU
(start)
A U G G G C G C C C A A G U C U C A U C A C A G A A A G U U G G A GCC C A U G A G A A U U C A A A C A G A G C U U A U G G U G G A UCC
M
817
G
A
Q
V
S
S
Q
K
V
G
A
H
E
N
S
N
R
A
Y
G
G
S
A C C A U U A A U U A C A C U A C U A U U A A U U A U U A C A G G G A U U C U G C G A G C A A U G C C G C U A G U A A G C A G G A C U U U G C A C A A G A C C C A U C C A A G UUC
T
I
N
Y
T
T
I
N
Y
Y
R
D
S
A
S
N
A
A
S
K
Q
D
F
A
Q
D
P
S
K
F
(IA/IB)
907
A C U G A A C C C A U U A A A G A U G U U C U C A U U A A G A C C G C U C C C A C G C U A A A C U C U CCU A A U A U C G A G G C G U G U G G G U A U A G C GAC A G A G U G A U G
T
997
I
K
T
A
P
T
L
N
S
P
N
I
E
A
C
G
Y
S
D
R
V
M
L
T
L
E
A
N
G
N
S
T
I
T
T
Q
E
A
A
N
S
V
V
A
Y
G
R
W
P
E
Y
I
K
D
C C U G U G G A C C A G C C A A C U G A G C C G G A C G U U GCC G C A U G C A G G U U U U A C A C A C U A G A C A C U G U U A C U U G G C G C A A G GAG
P
V
D
Q
P
T
E
P
D
V
A
A
C
R
F
Y
T
L
D
T
V
T
W
R
K
E
R
G
W
W
W
K
L
P
D
A
L
K
D
M
G
L
F
G
Q
N
M
F
Y
H
Y
L
G
R
A
Y
T
V
H
V
Q
C
N
A
S
K
F
H
Q
G
A
L
G
V
F
A
V
P
E
M
C
L
A
G
S
A
T
H
M
L
T
K
Y
E
N
A
N
P
G
E
K
G
G
E
F
K
G
S
F
T
L
D
T
A
T
N
P
A
R
N
F
C
P
V
D
Y
L
F
G
S
G
V
L
A
G
N
A
F
V
Y
P
H
C A G A U A A U U A A U C U G C G C A C C A A C A A C U G U GCC A C G U U G GUG C U G C C A U A C G U U A A U U C A C U U U C C A U A G A C A G C A U G A C A A A A CAC AAC
Q
1627
L
A A C G C U A C C A A C C C U G C A CGC A A C U U U U G U C C C G U U G A U U A U CUC U U C G G G A G C C G A G U A C U G G C G G G A A A U G C G U U U G U U UAC C C A CAU
N
1537
V
GAC AGU GCA ACC CAC AUG CUU ACA AAA UAU GAG AAU GCA AAU CCG GGU GAG AAA GGG GGU GAG UUC AAA GGG AGU UUU ACU CUG GAU ACU
D
1447
D
A G C U A U A C U G U G C A C G U A C A G U G U A A U G C U U C A A A G U U U CAC CAG G G C G C C C U C G G G G U A U U U G C A G U U C C A G A A A U G U G C CUG G C A GGC
S
1357
K
U C C A G A G G G U G G U G G U G G A A A C U G C C U G A U G C G C U A A A G G A U A U G G G A U U A U U C G G C C A G A A C A U G U U C U A C CAC U A C C U C GGG A G G GCU
S
1267
I
UCA GAA GCAAAU
S
1177
P
C A A C U A A C C C U A G G C A A U U C U A C C A U U A C C A C A C A G G A G G C G GCC A A U U C U G U C G U U G C A U A C G G C C G G U G G C C C G A G UAC A U C A A G GAU
O
1087
E
I
I
N
L
R
T
N
N
C
A
T
L
V
L
P
Y
V
N
S
L
S
I
D
S
M
T
K
H
N
A A U U G G G G A A U U G C U A U C C U U C C G C U G G C A C C A C U U G A C U U U GCC A C C G A G U C C U C C A C U GAG A U A C C C A U U A C U C U A A C U A U U GCC CCU
N
W
G
I
A
I
L
P
L
A
P
L
D
F
A
T
E
S
S
T
E
I
P
I
T
L
T
I
A
P
(IB/IC)
1717
A U G U G U U G U G A A U U C A A U G G G U U G C G C A A C A U C A C U G U A C C C A G A A C U C A A G G G U U G C C A G U C U U A A A C A C U C C A G G A A G C A A C CAG UAC
M
1807
L
R
N
I
T
V
P
R
T
Q
G
L
P
V
L
N
T
P
G
S
N
Q
Y
T
A
D
N
Y
Q
S
P
C
A
I
P
E
F
D
V
T
P
P
I
D
I
P
G
E
V
R
N
M
E
L
A
E
I
D
T
M
I
P
L
N
L
T
S
Q
R
K
N
T
M
D
M
Y
R
V
K
L
N
A
A
H
S
D
T
P
I
L
C
L
S
L
S
P
A
S
D
P
R
L
A
H
T
M
L
G
E
I
N
Y
Y
T
H
W
A
G
S
L
K
F
T
F
L
F
C
G
S
M
M
A
T
G
K
L
L
V
S
A
P
P
G
A
K
A
P
E
S
R
K
E
A
M
L
G
T
H
V
I
W
D
I
G
L
Q
S
S
U G C A C U A U G G U G G U A C C U U G G A U U A G C A A C A C C A C A U A C A G A C A A A C U A U C A A C G A U A G C U U C A C A G A A G G A GGG U A U A U C A G U A U G UUU
C
2347
G
U A U G C A C C U C C U G G U G C G A A A G C C C C U G A A A G C C G C A A A G A A G C G A U G C U C GGC A C C C A C G U G A U C U G G GAC A U C G G G U U A CAG U C A U C A
Y
2257
N
C U G A A C U A C U A C A C A C A C U G G G C A G G G U C U U U G A A G U U C A C A UUU CUC U U C U G C G G C U C A A U G A U G G C C A C U G G U A A A U U G C U A GUG UCC
L
2167
F
G A U G C G G C U C A C U C U G A C A C A C C A A U A U U G U G U C U U U C A C U G UCU C C A G C A U C A G A U C C U A G G C U A G C A C A C A C U A U G C U A GGU G A A A U A
D
2077
E
A U G G A A U U G G C A G A G A U A G A C A C U A U G A U A C C U C U C A A U C U G A C G A G C C A G CGC A A G A A C A C C A U G G A U A U G U A C A G A G U C G A A C U G A A U
M
1987
C
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1897
C
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Sequencing of attenuated poliovirus 2 strain I41-2
47
(IC/ID)
2437
CUG UUG CGU GAC ACG ACG CAC AUA AGC CAA GAG GUU AUG CCA CAA GGA UUG GGU GAU UUA AUU GAA GGG GUU GUU GAG GGA GUC ACG AGA
L
2527
P
L
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E
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M
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D
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ACA CCU GUC AAC AAC UUG CCU GAU ACA CGA UCU AGC GGC CCA GCC CAC UCU AAG GAA ACA CCA GCG CUG ACA
T
P
V
N
N
L
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D
3247
T
S
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V
3157
L
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AUC CCU GGC AAG CGG AAU GAU UAC ACA UGG CAA ACG UCA UCU AAC CCA UCA GUG UUU UAC ACU UAC GGG GCA CCU CCA GCU AGA AUA UCA
I
3067
A
H
UUU GUG GUU ACA UCC AAU UAU ACC GAU GCA AAC AAU GGG CAC GCA CUG AAU CAA GUU UAC CAG AUA AUG UAC AUA CCA CCU GGG GCA CCG
F
2977
T
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S
2887
T
ACG GUU GAG UCU UUC UUC GCA AGA GGA GCU UGU GUG GCC AUU AUU GAA GUG GAU AAU GAU GCU CCA ACA AGG CGU GCC AGU AAA UUA UUU
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2797
D
GCC GUA GAG ACA GGG GCC ACC AAC CCA UUG GUG CCU UCA GAC ACG GUA CAA ACU CGU CAC GUC AUC CAA AAG CGG ACG CGG UCG GAG UCU
A
2707
R
AAU GCU UUG ACA CCA CUG
N
2617
L
S
L
Y
G
A
A
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L
N
D
F
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A
V
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A A A A U C A G A G U G U A C A U G A A A C C A A A G C A C GUC A G A G U G U G G U G U C C G C G A CCC C C U C G A G C A G U C C C A U A C U A C G G A C C A G G G G U U G A C
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(ID/2A)
3337
UAC AAG GAU GGA CUA ACC CCA CUG CCA GAG AAA GGC UUG AUA ACC UAU GGU UUU GGC CAC CAA AAU AAG GCA GUG UAC ACG GCA GGU UAC
Y
3427
L
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F
3697
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K
3607
D
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K
3517
K
V
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S
P
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(2A/2B)
3787
GAC AUC AGA GAU CUG UAC GCA UAC GAG GUG GAG GCU AUG GAG CAG GGA GUC UCC AAC UAU AUU GAG UCC CUU GGG GCU GCA DUU GGG AGU
D
3877
R
D
L
Y
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E
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M
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G
3967
I
F
T
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Q
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I
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A A A A U A A U U U C A U C C C U U G U U A U C A U C A C C A G A A A C U A U G A A GAC A C G A C C A C A G U G C U G G C U A C U C U U G C U C U C C U C G G U U G U G A U G C G
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(28/2C}
4057
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4147
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S
4417
Q
CAG GCU AGA GAC AAG CUA GAG UUU GUU ACC AAA CUG AAG CAA CUA GAA AUG UUG GAG AAC CAA AUU GCA ACC AUU CAU CAA UCG UGC CCA
Q
4327
W
A C A G A G G C A U G C A A U G C A G C C A A G G G A U U G G A A U G G G U G U C U A A U A A A A U A U C C A A A U U U A U U GAC U G G C U C A A A G A G A A G A U C A U U C C A
T
4237
P
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On: Sun, 18 Jun 2017 11:01:14
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48
4507
D. C. Pevear and others
G G U A G C C C A GGC A C G G G C A A G U C A G U U G C C A C C A A U U U A A U U GCC A G A G C A A U A G C A G A G A A G G A G A A C A C C U C C A C A U A C U C A C U A C C A
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4597
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Q
4957
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L
4867
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CUG UUU UGU CAG AUG GUC UCU ACU GUA GAA UUC AUA CCA CCA AUG GCU UCG CUA GAA GAA AAG GGU AUU UUG UUC ACA UCU AAU UAC GUU
L
4777
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C C A G A U C C C U C C C A U U U C G A U G G G U A C A A G C A A C A A G G U G U G G U G A U C A U G GAU G A U H U G A A U C A G A A H C C A G A U G G A G C A G A C A U G A A G
P
4687
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(2C/3A)
5047
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M
5137
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5227
I
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K
G
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S
Q
V
Q
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I
N
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(3A/3B)
5317
G C C G C A G U U GCU G G A G U G G U G U A U G U G A U G U A C A A A C U C U U U G C A G G G C A U C A A G G A G C G U A U A C A G G G C U U C C C A A C A A G A G A C C C A A U
A
A
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(3B/3C)
5407
G U C C C C A C C A U C A G G A C U G C C A A G G U U C A G GGC C C A G G A U U U GAC U A C G C A G U G G C A A U G G C C A A A A G A A A C A U H C U U A C G G C A A C U A C C
V
5497
T
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A A C A U G U A U GUU C C U G U C G G U G C U G U G A C U G A A C A G G G G U A U CUU A A U CUC GGU G G A C G C C A A A C U G C U C G U A C U U U A A U G U A C A A C UUU
N
5857
R
G A G A A G U U C A G G G A C A U C A G A C C A C A C A U C CCC A C U C A A A U C A C U G A G A C G A A U G A U G G A G U U U U A A U U G U G A A C A C u A G U A A G UAC CCC
E
5767
I
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G
5677
T
A U U A A G G G A G A G U U C A C A A U G C U C G G A G U G C A U G A U A A U G U G GCC A U U C U A C C A A C C C A C G C A U C A C C G G G U G A A A C A A U A G U C A U U GAU
I
5587
P
M
Y
V
P
V
G
A
V
T
E
Q
G
Y
L
N
L
G
G
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R
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N
F
C C A A C G A G A G C A G G U C A A U G U G G C G G A G U U A U C A C C U G C A C U GGC A A G G U C A U C G G G A U G C A U G U U G G U G G G A A C G G U U C A C A U G G G UUC
P
T
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S
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(3C/3D)
5947
G C A G C A G C C CUG A A G C G A U C C U A U U U C A C U C A G A G U C A A G G U GAA A U C C A G U G G A U G A G A C C A U C A A A A G A A G U G G G C U A C C C C G U U A U U
A
6037
S
Y
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T
6487
R
G A A G C U C U A G A C C U C A G U A C C A G U G C U G G G U A U C C C U A U G U G GCA A U G G G G A A G A A G A A A A G A GAC A U U U U G A A U A A G C A A A C C A G A GAU
E
6397
K
G U C G A U CAU UAC G C A G G C C A G CUC A U G U C A C U A GAC A U C A A C A C A G A A C A A A U G U G C C U U G A G G A U G C A A U G U A U G G C A C U G A C G G U CUC
V
6307
L
C C C A G A U U G A A G A C A G A U U U U G A A G A G G C U A U C U U U U C C A A G UAC G U G G G A A A U A A G A U U A C U G A A G U A G A U G A G U A C A U G A A A G A A G C U
P
6217
A
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49
Sequencing of attenuated poliovirus 2 strain W-2
6577
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Fig. 2. The completenucleotidesequenceof the RNA genomeof PV2/W-2. Cleavagesites within the polyprotein-codingregion are
assigned based on the original poliovirusprotein sequences(Kitamura et al., 1981). The systematicnomenclature for picornavirus
proteins is used (Rueckea & Wimmer, 1984).
of the PV2/W-2 genome was 7434 nucleotides to the
guanosine residue preceding the polyadenylation tract at
the 3' end of the R N A (Fig. 2).
Deduced amino acid sequence o f the polyprotein coding
region
The deduced amino acid sequence of PV2/W-2 was 2205
residues in length, making it two amino acids shorter
than PV2/Lansing and PV2/Sabin. As shown in Table 2
only 26 other amino acids differed between PV2/W-2
and PV2/Lansing (98-7~ amino acid identity). Based on
the high degree of amino acid identity between PV2/W-2
and PV2/Lansing, the location of their polyprotein
cleavage sites is likely to be identical. Although this
appears to be the case, the cleavage between P2-C and
P3-A, which is carried out by the P3-C protease
(Hanecak et al., 1982; Pallansch et al., 1984), occurred at
a glutamine-serine pair in PV2/W-2. This cleavage
occurs at a glutamine-glycine pair in all other sequenced
polioviruses (Semler et al., 1981; Emini et aL, 1982;
Pallansch et al., 1984; Stanway et al., 1984; Toyoda et al.,
1984; La Monica et al., 1986). Of the 28 amino acid
differences between PV2/W-2 and PV2/Lansing, 16
occurred in the capsid protein-coding region (P1) and 12
in the non-structural protein-coding regions (P2 and P3).
As previously mentioned the amino acid deletions in
PV2/W-2 occurred in P3-D (polymerase) at PV2/
Lansing amino acid positions 230 and 231 (Table 2).
As expected from the relatively low degree of
nucleotide identity observed in the 5" non-coding regions
of PV2/W-2 and PV2/Sabin, a total of 92 amino acid
differences were found in the polyproteins in addition to
the amino acid deletions in P3-D of PV2/W-2 (95.7~
amino acid identity; Table 2). Twenty-three of these
differences were located in the major surface capsid
protein P1-D (VP1), with 14 falling within 32 amino acid
residues of the N terminus of the protein.
Discussion
Determination of genomic regions responsible for
various biological phenotypes remains a central question
in many virus-host systems. For picornaviruses, the
system which has been most extensively studied involves
the polioviruses. La Monica et al. (1986) have mapped
regions required for neurovirulence within the mouseadapted PV2/Lansing genome using recombinants of
PV2/Lansing and the non-mouse-adapted PV1/
Mahoney. They found that a contribution to neurovirulence and attenuation was made not only from the capsid
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50
D. C. Pevear and others
coding region, but also from regions of the genome
involved in controlling R N A replication and translation.
More recent studies also demonstrate that attenuating
determinants may occur in non-structural protein-coding
regions (Kawamura et al., 1989; Moss et al., 1989). These
studies suggest that the ability to replicate in critical
tissues (i.e. the host range of the virus) is not in itself
sufficient for pathogenesis.
The attenuated PV2/W-2 strain provides an interesting alternative for studying neurovirulence in the
apparent absence of a clear contribution from host range
limitations. As shown in this report, PV2/Lansing and
PV2/W-2 are very closely related genotypically, although
PV2/W-2 was not generated from PV2/Lansing (Carp et
al., 1963). There were only 12 non-identical nucleotides
occurring in the non-coding regions of the genomes and
28 amino acid differences in the polyproteins (Tables 1
and 2). All 12 of the nucleotide changes in the non-coding
regions occurred in the 5' untranslated 747 bases. Two
specific bases in this region of the PV3 genome have
been implicated in neurovirulence, base 472 (Evans et
al., 1985) and bases 220 and 472 (La Monica et al.,
1987a). However, there were no nucleotide differences
between PV2/Lansing and PV2/W-2 within 80 bases of
either of these two sites, indicating that a direct
alignment of the PV2 and PV3 genomes would not
identify potentially important mutations for attenuation
of PV2/W-2.
Sixteen of the amino acid differences in the polyproteins occurred in sequences encoding the capsid proteins
of the virus. One of these changes was at VP1 position 99,
a Lys in PV2/Lansing to Arg in PV2/W-2, one of the
amino acids in neutralization site 1 for PV2/Lansing
(Minor et al., 1986; La Monica et al., 1987b). Antigenic
variants of PV2/Lansing with altered neurovirulence
display amino acid changes of VP1 residues 93 to 105,
with five out of 14 having changes at position 99 (La
Monica et al., 1987b). Insertion of an oligonucleotide
cartridge corresponding to amino acids 95 to 102 of PV2/
Lansing into the identical site in PV1/Mahoney (avirulent for mice) resulted in a virus which induced paralytic
disease in mice (Martin et al., 1988; Murray et al., 1988).
These studies suggest that changes in neutralization site
1 may alter receptor binding and consequently the
pathogenicity of the virus for mice.
A second change occurred at VP1 position 170, in
which a Trp in PV2/Lansing was replaced with an Arg in
PV2/W-2. Amino acids from VP1 positions 162 to 178
may contribute topographically to neutralization site 1
(Hogle et al., 1985; Minor et aL, 1986). Although PV2/
W-2 grows to levels comparable to those of PV2/Lansing
in the mouse brain after intracerebral inoculation (Jubelt
et al., 1980b; Jubelt & Meagher, 1984b), the attenuated
virus displays a limited ability to disseminate (Jubelt
& Meagher, 1984a, b). The onset of flaccid paralysis,
characteristic of human poliomyelitis infections, correlates with the involvement of anterior horn cells of the
spinal cord, as is seen early in the infectious process of
mice with PV2/Lansing and late in infection with PV2/
W-2 of immunosuppressed animals (Jubelt et al., 1980a,
b; Jubelt & Meagher, 1984a, b). Thus, the amino acid
substitutions in VP1 at positions 99 and 170 may be
influential in the inability of PV2/W-2 to spread to the
spinal cord.
Another feature which distinguishes PV2/W-2 and
PV2/Lansing is that the former virus induces a brisk
neutralizing antibody response, whereas only nonneutralizing antibodies can be identified in PV2/Lansing
infection of BALB/c and Swiss mice (Jubelt & Meagher,
1984a; Jubelt et al., 1980a; Jubelt & Lipton, 1987;
Miller, 1981). Neutralization site 1 appears to be the
immunodominant site in both PV2/Lansing and PV3/
Sabin (Minor et al., 1986). Thus, the amino acid
substitutions at VP1 residues 99 and 170 may explain the
difference in neutralizing antibody responses elicited by
these two type 2 polioviruses. Alternatively, the numerous amino acid substitutions in or near neutralization site
3, at amino acids 289, 291,295 and 299 of VP1 and 59 of
VP3, may explain the observed difference in neutralizing
antibody responses, although site 3 has not been
described as an important immunogenic site for type 2
poliovirus (Minor et aL, 1986).
A role for the non-structural proteins in virulence
would appear to be minimized by the observation that
inoculation of PV2/W-2 directly into the spinal cord
results in paralysis (Jubelt & Meagher, 1984b). However,
overall replication rates of the attenuated virus in the
brain do not provide insight into replication rates within
tissues that may be critical for spread to the spinal cord.
Of particular interest in this context are the changes in
the polymerase (P3-D), protease (P3-C) and the P2-C/
P3-A cleavage site. It is quite conceivable that the two
contiguous amino acid deletions in the polymerase might
~ffect replication rates in specific tissues. It was
surprising to find that the P2-C/P3-A cleavage site in
PV2/W-2 occurred at a glutamine-serine pair, as other
polioviruses have a glutamine-glycine pair at this
location (Semler et al., 1981; Emini et al., 1982;
Pallansch et al., 1984; Stanway et al., 1984; Toyoda et al.,
1984; La Monica et aL, 1986). Other human picornaviruses (coxsackieviruses, hepatitis A virus and rhinoviruses) also have a glutamine-glycine pair at this location
(Iizuka et al., 1987; Jenkins et aL, 1987; Lindberg et al.,
1987; Cohen et al., 1987; Duechler et al., 1987).
Interestingly, picornaviruses not typically associated
with human infections (e.g. encephalomyocarditis virus
and Theiler's viruses) have a glutamine-serine cleavage
site like PV2/W-2 (Palmenberg et al., 1984; Ohara et aL,
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Sequencing o f attenuated poliovirus 2 strain W-2
1988; Pevear et al., 1987, 1988). W h e t h e r the single
a m i n o acid change in the viral protease (P3-C) alters its
specificity for this site will be d e t e r m i n e d .
I n s u m m a r y , we have sequenced the entire g e n o m e of
the attenuated, m o u s e - a d a p t e d P V 2 / W - 2 strain a n d
identified a limited n u m b e r of regions w i t h i n the g e n o m e
w h i c h differ from the n e u r o v i r u l e n t P V 2 / L a n s i n g strain.
Similar to the coxsackievirus B3 clone ( K a n d o l f &
Hofschneider, 1985), the P V 2 / W - 2 clone is m i s s i n g the
first two t h y m i d i n e nucleotides at the 5' end. However,
unlike the coxsackievirus B3 clone, the P V 2 / W - 2 c D N A
was n o t infectious in our initial experiments. W e are
currently e n g i n e e r i n g a full-length clone of PV2/W-2,
complete with the two t h y m i d i n e residues, in order to
confirm the phenotype. Subsequently, we propose to
focus entirely o n the c o n s t r u c t i o n of r e c o m b i n a n t s
b e t w e e n the m o u s e - a d a p t e d strains in order to e l i m i n a t e
the c o n t r i b u t i o n of host range. R e c o m b i n a n t s b e t w e e n
P V 2 / W - 2 a n d P V 2 / L a n s i n g should p r o v i d e detailed
insight into g e n o m i c regions i m p o r t a n t for poliovirus
attenuation.
We acknowledge the expert assistance of Dr Ann Palmenberg for
sequence formatting and manuscript suggestions, Dr Dorothy KelleyGeraghty for manuscript suggestions, Lisa Roberts for technical
assistance and Rosemary Oritz and Louise Wayson for manuscript
preparation. This research was supported by U.S. Public Health
Service Grant NS21756.
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(Received 28 July 1989; Accepted 26 September 1989)
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