Journal of General Virology (1990), 71, 1555-1560. Printed in Great Britain
1555
Strain-variable editing during transcription of the P gene of mumps virus
may lead to the generation of non-structural proteins NS1 (V) and NS2
G. D. Elliott, R. P. Yeo, M. A. Afzal, E. J. B. Simpson, J. A. Currau and B. K. Rima*
School o f Biology and Biochemistry, Medical Biology Centre, The Queen's University o f Belfast, Northern Ireland
B T 9 7BL, U.K.
The sequence of the P (phosphoprotein) gene o f
mumps virus has been determined. It has two open
reading frames, the first of which probably encodes the
NSI (or V) protein o f mumps virus. Expression o f the
P protein requires the insertion o f two non-templated
residues to link the two O R F s in a process analogous to
that observed in the P/V gene of simian virus type 5 to
which m u m p s virus is closely related. Strain differences
in the accuracy of insertion of non-templated G
residues in the P / V gene transcripts have been
described.
Introduction
requires the insertion of non-templated residues into the
P gene transcripts (Thomas et al., 1988). A similar
insertional mechanism has been shown to generate a 'V'like protein from the P gene of morbilliviruses (Cattaneo
et al., 1989).
Although mumps virus appears to be very closely
related to SV5 (Rima, 1989) two recent papers which
report the sequence of the P gene of mumps virus
(Takeuchi et al., 1988: Elango et al., 1989) suggest that
the generation of the non-structural proteins of mumps
virus involves internal translational start sites. We
provide here nucleotide sequence evidence that suggests
that the generation of these proteins in mumps virus is
similar to the process demonstrated for SV5. This
provides an explanation for the generation of the nonstructural proteins of mumps virus.
Non-structural proteins have been described in paramyxoviruses such as Sendal virus (Lamb & Choppin,
1978), Newcastle disease virus (NDV) (Collins et al.,
1982; Chambers & Samson, 1982), simian virus type 5
(SV5) (Peluso et al., 1977) and mumps virus (Rima et al.,
1980) as well as in the morbilliviruses (Rima & Martin,
1979; Vainionpaa, 1979; Campbell et al., 1980) and in
the pneumovirus respiratory syncytial virus (Huang &
Wertz, 1982; Venkatesan et a l , 1983). The function of
these proteins remains unclear, but the manner in which
they are generated has been established in most members
of the paramyxoviridae. In the pneumoviruses, separate
genes appear to exist that code for some of the nonstructural proteins (Collins et al., 1984). In viruses
belonging to the Sendal virus subgroup of the paramyxoviruses and in the morbilliviruses, non-structural 'C'
proteins are generated from translation of the P
(phosphoprotein) messenger R N A in different frames
(Giorgi et al., 1983; Bellini et al., 1985; Barrett et al.,
1985). In the case of Sendal virus multiple C proteins
with different starting codons have been observed
(Curran & Kolakofsky, 1988; Vidal et al., 1990 and
references therein). These non-structural proteins have
no amino acid sequence similarity to the P protein.
However, in N D V (Collins et al., 1982) and mumps virus
(Herrler & Compans 1982; Simpson et al., 1984) the nonstructural proteins appear to share peptides with the P
protein.
Recently, the non-structural V protein of SV5 has been
shown to be translated from the unaltered transcript of
the P gene, whereas the generation of the P protein
0000-9433 © 1990 SGM
Methods
Cells and viruses. Mumps virus was grown either in 8 day old
embryonatedeggs for the SBLI/E (egg-adapted)strain or in Vero cells
for the SBL1/VVeto cell-adaptedstrain, the Enders strain and the BF
strain. The SBL1 strains were a generous gift from Dr C. Orvell
(Statens Bakteriologiska Laboratoriet, Stockholm, Sweden). The
Enders and BF strains have been described before (Rima et al., 1980).
Cloning, sequencing and polymerase chain reaction. Cloning and
sequencingof cDNA was carried out as describedbefore (Elliott et al.,
1989). For the polymerasechain reaction (PCR) poly(A)÷ RNA was
extracted from infected cells as described before (Elliott et al., 1989).
Genomic RNA was obtained by extraction of RNA from purified
nucleocapsids obtained from infected Vero cells. Nucleocapsidswere
purified according to the method of Kolakofsky(1976) as modifiedby
Takeuchi et al. (1988). The extracted RNA species were reversetranscribed with specificprimers for the synthesis of the complemen-
1556
G. D. Elliot and others
___¢/
N
t ss
S S
S
2MuS13
M
,s
S
•
1o'oo
5()0 "1"
1
e--//
P
S
i
H
I,
Ef l
,"b-,1316
,~
'iMuSl8"
' -
¢/---~
pMuS817
pMuS893
,-._>
i'us 0
,/
°
pMuE630 "
C
< lInsertion site
,
)
S
> PCR fragment
Fig. 1. Subcloningand sequencing strategy for the P gene of mumps virus. Clones and their numbers used in this study, restriction
enzymesites used in the subcloningand sequencing strategyare indicated. Arrowsindicate the regionsof the clonessequenced and the
direction of sequencing. Abbreviations:E, EcoRI; H, HaeIII; S, Sau3A; C, Clal; N, nucleocapsid gene; P, phosphoprotein gene; M,
matrix gene.
tary strand in 25 gl reaction mixtures. To make the complementary
positive strand from genomic RNA we utilized a primer with the
sequence 5' GGAAAAGAGAGAATGATTA3' (nucleotides 461 to
479). To prime from mRNA we used 5' CAAGATGTTGCAGGCGAGC 3' (complementaryto nucleotides 774 to 756). After first-strand
synthesis the resulting RNA/DNA hybrid was subjected to PCR. The
reverse transcription reaction mixture was diluted with PCR buffer,
Taq DNA polymerase (0.5 units), the second oligonucleotide and
deoxynucleotides to a final volumeof 100 gl and PCR was carried out
according to the manufacturer's instructions (Perkin-Elmer Cetus).
The amplified products were cut with the appropriate restriction
enzymes (Clal and Sau3A) and ligated into BamHI- and A¢cI-cut M13
mp9 and Ml3 inp8 for sequencing by the dideoxynucleotide chain
termination method (Sanger et al., 1977).
Results
c D N A cloning o f the P gene o f mumps virus
The non-structural proteins NS 1 (23K) and NS2 (18K) of
mumps virus have been shown to share tryptic peptides
when analysed by H P L C (Herrler & Compans, 1982).
Two-dimensional fingerprinting (Simpson et al., 1984)
has shown that the pSS]methionine-labelled tryptic
peptides in NS2, NS1 and the P protein form a nested
set.
In order to elucidate how the non-structural proteins
were generated we decided to study the P gene of mumps
virus further by c D N A cloning and nucleotide sequence
determination, c D N A clones prepared from the purified
genome of SBL1/E have been generated and sequenced
as described before (Elliott et al., 1989). Fig. 1 describes
the subcloning and sequencing strategy employed for this
gene. c D N A clones generated from Enders virusinfected cell m R N A were also available in this study
(Elliott et al., 1989 and further clones generated from
such material for this study). Two mumps virus-specific
c D N A clones for almost all parts of the P gene were
generated and these have been sequenced in both
directions. Fig. 2 shows the sequence determined from
the genomic c D N A clones of the P gene of SBL1/E.
This sequence shows the presence of two open reading
frames (ORFs) covering the 5' end to the 3' end of the
gene and varies from those published earlier for SBL1/V
(Elango et al., 1989) and the Enders and Miyahara
strains (Takeuchi et al., 1988) particularly between
residues 521 and 560 as detailed in Fig. 3. It was clear
that nucleotides 531 to 536 of the sequence that we have
found to be present in genomic clones of SBL1/E were
the same as those published for SBL1/V (Elango et al.,
1989) but that nucleotides 543 to 550 were the same as
those presented for the Enders and Miyahara strains
reported by Takeuchi et al. (1988). One of the clones of an
m R N A extracted from Enders virus-infected cells had
nine G residues in the m R N A sequence after position
530 instead of the eight proposed by Takeuchi et al.
(1988) and the six G residues in the genomic c D N A
clones. Thus we decided to determine the nucleotide
sequence of a larger number of genome- and m R N A derived c D N A clones around this site.
Sequence variation around the insertion site
We synthesized oligonucleotide primers at either side of
the region of interest and carried out P C R using c D N A
reverse-transcribed from infected cell R N A oligo(dT)selected twice, or with genomic R N A extracted from
purified nucleocapsid R N A so that the generation of
genomic or m R N A c D N A clones was assured. The
amplified gene products were cut with ClaI and S a u 3 A
restriction enzymes, force-cloned into M 13 mp8 and mp9
and sequenced using the dideoxynucleotide method. A
total of 40 clones of genomic c D N A from the Enders,
SBL1 and BF strains of mumps virus were analysed and
Mumps virus P gene
20
40
60
AGGCCCGGAAAGAATTAGGTCCACGATCACAGGCACAATCATTCTGATCGTGTTTcTTTCCGGGCAAGCCATGGATCAAT
M
D
1557
80
Q
i00
120
140
160
TTATAAAACAGGATGAGACTGGTGATTTAATTGAGA•AGGAATGAATGTTGCAAATCATTTCCTATcCGCCCCCATTCAG
F
I K
Q
D
E
T
G
D
L
I
E
T
G
M
N
V
A
N
H
F
L
S
A
P
I
Q
180
200
220
GGAAcCAACTCGcTGAGCAAGGccTCAATcATCCcTGGCGTTGcACCTGTAcTCATTGGcAATccAGAGCAAAAGAACAT
G
T
N
S
L
S K
A
S
I
I
P
G
V
A
P
V
L
I
G
N
P
E
Q
K
240
N
260
280
300
TCAGcAccCTACCGCATCACATCAGGGATC•AAGTCAAAGGG•AGcGGCTcAGGGGTcAGGTCCATcATAGTCCCAcCcT
Q
H
P
T
A
S
H
Q
G
S
K
S
K
G
S
G
S
G
V
R
S
I
I V
P
I
320
P
340
360
380
400
CCGAAGcAGGCAATGGAGGGACTCAGGATCCTGAGCCCCTTTTTGCAcAAACAGGACAGGGTGGTATAGTCACAACCGTT
S
E
A
G
N
G
G
T
Q
D
P
E
P
L
F
A
Q
T
G
Q
G
G
I V
T
T
V
420
440
460
TATCAGGATCCAACcATcCAACCAACAGGTTCATCTCGAAGTGTGGAATTGGCGAAGATCGGAAAAGAGAGAATGATTAA
Y
Q
D
P
T
I Q
P
T
G
S
S
R
S
V
E
L
A
K
I
G
K
E
R
M
480
I
N
500
520
540
560
TCGATTTGTTGAGAAACCTAGAATCTCAACGCCGGTGACAGAATTTAAGAGGGGGGCCGGGAGCGGCTGCTCAAGGCCAG
G
G
R
E
R
L
L
K
A
R
R
F
V
E
K
P
R
I
S
T
P
V
T
E
F
K
R
G
A
G
S
G
C
S
R
P
G
G
P
G
A
A
A
Q
G
Q
580
600
620
640
ACAATcCAAGAGGAGGGCATAGACGGGAATGGAGCCTCAGCTGGGTCCAAGGAGAGGTCCGGGTCTTTGAGTGGTGCAAC
Q
S
K
R
R
A
*
D
N
P
R
G
G
H
R
R
E
W
S
L
S W
V
Q
G
E
V
R
V
F
E
W
C
N
T I Q E E G I D G N G A S A G S K E R S G S L S G A T P
660
680
700
NS2
NSI
P
NS2
NSI
720
ccTATATGCTcACcTATcACTGccGcAGCAAGATTCcAcTcCTGCAAATGTGGGAATTGcccCGcAAAGTGCGATcAGTG
P
I
L
C
Y
S
A
P
H
I
L
T
S
A
L
A
P
A
Q
R
Q
F
D
H
S
S
T
C
P
K
A
C
N
G
V
N
G
C
I
P
A
A
P
K
Q
C
S
D
A
Q
C
I
S
740
760
780
800
CGAACGAGATTATGGACCTCCTTAGGGGGATGGATGCTCGCCTGCAACATCTTGAACAAAAGGTGGACAAGGTGCTTGCA
E
R
D
Y
G
P
P
*
A
N
E
I M
D
L
L
R
G
M
D
A
R
L
Q
H
L
E
Q
K
V
D
K
V
L
~
•
•
•
NSI
P
•
820
840
860
cAGGGcAGCATGGTGAccCAAATAAAGAATGAATTATCAACAGTAAAGACAAcATTAGCAACAATTGAAGGGATGATGGc
Q
G
S M V T Q I
K
N
E
L
S T V
K
T
T
L
A
TI
E
G'M
900
920
.
940
AACAGTAAAGATCATGGATCCTGGAAACCCGACAGGGGTCCCAGTTGATGAACTTAGAAGAAGTTTTAGTGATCATGTGA
T
V
K
I M
D
P
G
N
P
T
G
V
P V
D
E
L
R
R
S
F
S
D
H
NSI
P
880
M
AP
960
V
980
I000
1020
1040
CAATTGTTAGTGGACCAGGAGATGTGCCGTTCAGCTCCAGTGAAGAACCCACACTGTATTTGGATGAGCTGGCGAGGCCC
T
I V
S
G
P
G
D
V
P
F
S
S
S
E
E
P
T
L
Y
L
D
E
L
A
R
P
1060
1080
1100
1120
GTcTcCAAGccTCGTCcTGcAAAGcAGACAAAAcccCAAcCAGTAAAGGATTTAGcAGGAcGAAAAGTGATGATTACcAA
V S K P R P A K Q T K P Q P V K D L A G R K V M
I T K
1140
1160
.
1180
1200
AATGATCACTGATTGTGTGGCTAACCCTCAAATGAAGCAGGCGTTCGAGCAAcGATTGGCAAAGGCCAGCACCGAGGATG
M
I
T
D
C
V
A
N
P
Q
M
K
Q
A
F
E
Q
R
L
A
K
A
S
T
E
D
1220
.
1240
.
1260
CTCTGAACGACATCAAGAGAGACATCATACGAAGCGCcATATGAATTcACCAGAAGcACcAGAcTCAAGGAAAAATcCAT
A
L
N
D
I K R
D
I I
R
S
A
I
*
1280
1300
GAACTGGAAGCCACAATGATTCCCTATTAAATAAAAAA
Fig. 2. NucleotidesequenceofthePgeneofmumpsvirus. Thesequencerep resentedisthep°sitiveantigen°me'P°ssibletranslati°n
productsareindicated.
1558
G. D. Elliot and others
MIY
END1
END2
SBLI
SBLI
SBL2
BF
500
510
520
530
540
550
GAAAAACCAAGAACCTCAACGCCGGTAACAGAATTTAAGAGGGGGGGGCCGGGAGCGGCTGCT
.'.G . . . . . T . . . . T . . . . . . . . . . . .
G ....................................
..G ..... T .... T ............
G ...................
--. ........
CG ....
..-G . . . . . T . . . . . . . . . . . . . . . . .
G ...................
--. ........
CG ....
..G ..... T .................
G ...................
--. .......
C.- ....
..G ..... T .................
G .....................
--. ..............
........
C .....................................
--. .......
C.G ....
E
K
P
R
T
S
T
P
V
T
E
F
K
R
G
G
P
G
A
A
A
(I)
(R)
cell
VERO
VERO
VERO
VERO
VERO
EGG
VERO
RNA
M/G?
M/G?
M
M/G
G
G
M/G
(G)
Fig. 3. Sequence variation in the published sequences around the insertion site in the m u m p s virus P gene. MIY and E N D 1 , Miyahara
and Enders strain sequences determined by Takeuchi et al. (1988). These probably represent edited transcripts and not antigenomic
sequences; SBL1, sequence of the Veto cell-grown SBLI strain determined by Elango et al. (1989); SBL2, sequence of the egg-grown
SBL1 strain (this work); END2, Enders strain sequence (this work); BF, sequence of the BF strain of m u m p s virus (this work). The
sources of R N A used are indicated as either M ( m R N A ) or G (genomic R N A ) or both. The amino acid sequence of the Miyahara strain
and expressed mutations is indicated.
1
2
3
4
5
6
7
8
9
10
ll
12
13
14
ooGG
GbGG~
~G
A
C
G
T
Fig. 4. Sequence variability at the insertion site. Cloned amplified product from the m R N A extracted from Enders virus-infected Vero
cells was sequenced. The G reaction products of 14 subclones are shown. Clones with insertions containing one, two, three or four extra
G residues are presented. Clones with five or more inserted G residues have not been observed.
Table I. Strain-dependent sequence variation at the editing
site o f mumps virus
N u m b e r of G
residues inserted
Protein
encoded
Enders
strain
SBL1
strain
BF
strain
0
1
2
3
4
NS1
NS2
P
NSl-like
NS2-1ike
12"
2
12
1
3
35
0
14
1
0
59
0
1
7
6
* N u m b e r of c D N A clones with the indicated number of G residues.
without exception all had six G residues after position
530. However, analysis of c D N A clones obtained from
po!y(A)+ RNA, reverse-transcribed so that only positivestrand templates were converted and amplified, showed
variation in the number of G residues at this site (Fig. 4).
Clones of the P m R N A of the Enders, SBL1 and BF
strains were analysed and the distribution of the number
of clones with various numbers of inserted G residues is
shown in Table 1. c D N A clones with more than four G
residues inserted were not observed among the 153
clones analysed.
It was apparent that the SBL1 strain was more
accurate in inserting only the correct two G residues than
the other two strains. This insertion process has been
referred to as editing of the transcripts (Thomas et al.,
1988; Cattaneo et al., 1989; Vidal et al., 1990). It was
clear that the SBL strain was accurate in this process and
very few misedited transcripts were observed. The
Enders strain appeared to be much more prone to
misediting. These results were observed in a number of
experiments with the different strains and with R N A
samples extracted at different times post-infection.
These, therefore, appeared to represent real strainspecific differences in the editing phenomenon. They
also implied that the editing function is probably virusand not host-encoded. Very few edited P-encoding
transcripts were observed when the BF strain was
analysed. These data are discussed below.
M u m p s virus P gene
Discussion
The sequence of the P gene and its transcripts have
allowed us to suggest how the generation of the NS1
protein takes place in mumps virus. We have found that
at position 531 in the sequence given in Fig. 2 there are a
variable number of extra G residues incorporated in the
mRNA transcripts. These will give rise to mRN. A
molecules that encode the P and NS2 proteins, whilst
direct unedited transcripts from the P/NS1 gene encode
the NS1 (V) protein. It has previously been shown that
the NS proteins contain a nested set of tryptic peptides
also present in the P protein (Herrler & Compans, 1982;
Simpson et al., 1984). These NS proteins were shown to
have no precursor-product relationship and were primary translation products from mRNA molecules
similar in size to that of the P protein (Simpson et at.,
1984), The identification of a cysteine (C)-rich domain in
the NS 1 protein, the absence of C in the NS2 protein and
the very low C content of the P protein (Simpson et al.,
1984) are inconsistent with the suggestion of Takeuchi
et al. (1988) and Elango et al. (1989) that the NS proteins
are generated from the P mRNA by internal starts of
translation. We conclude therefore that the mumps virus
NS proteins and P proteins are generated from the
mRNA by the same process as has been shown to be
responsible for the synthesis of the V and P proteins of
SV5, but with the difference that the inaccuracy of the
process or misediting in some strains also appears to be
able to lead to the generation of another protein, NS2. In
about 40~ of the transcripts two extra G residues have
been inserted to generate transcripts that contain an
ORF of sufficient size to encode the P protein.
The derived amino acid sequence of the mumps virus
P protein indicates very high levels of homology (37%)
between mumps virus and SV5, 19% homology between
mumps virus and NDV and insignificant levels of
homology with the other paramyxoviruses. This is
further evidence for the proposed close relationship
between SV5, NDV and mumps virus (Rima, 1989).
Hightower et al. (1984) reported that the non-structural
proteins of NDV shared amino-terminal peptides with
the NDV P protein. Recently McGinnes et al. (1988)
suggested that the non-structural proteins of NDV were
generated from the same ORF as the P protein. The
mechanism responsible for the generation of NS proteins
in NDV, the third member of this subgroup of the
paramyxoviruses (Rima, 1989) is not yet clear. It will be
interesting to establish the precise sequence of the P gene
and its transcripts for this and other paramyxoviruses.
The alignment of protein sequences of the paramyxoviruses reveals no conservation of amino-acid residues in
the P proteins (Rima, 1989) or in the C proteins, whereas
in the V/NSl-like proteins there are a substantial
1559
number of conserved residues (Thomas et al., 1988).
Thus, it may well be that the shorter V-like proteins
expressed from this gene are functionally important, as
indicated by the level of transcripts encoding them.
The incorporation of non-templated residues appears
to be due to the presence of sequences, related to the
polyadenylation signal, containing all the bases normally
found in front of a set of U residues at which the
polymerase, by slippage, generates the poly(A) tails at
the end of mRNA, except that these are replaced by C
residues in the insertion site. Sequences similar to
polyadenylation sites have also been identified in the
other paramyxoviruses in which the phenomenon of the
incorporation of non-templated residues has been documented (Thomas et al., 1988; Cattaneo et al., 1989) or
postulated. Thomas and coworkers (1988) suggested that
the secondary structure of the RNA template around the
insertion site was important. However, we were not able
to fold the template around the switch site into a
thermostable structure similar to the one proposed for
SV5.
The data presented here indicate strain variability in
the generation of the NS1 and NS2 proteins. In the SBL
strain only transcripts encoding the NS1 and P proteins
were detectable in roughly a 2 : 1 ratio and this correlated
with the synthesis of these proteins in vivo. In the Enders
strain it was noted that the process of insertion of nontemplate residues was less accurate and transcripts with
between one and four inserted G residues were observed.
The ones with one or four residues were supposed to give
rise to the NS2 protein and this was demonstrated in
Enders virus-infected cells. Earlier we reported that the
Belfast (BF) strain of mumps virus induced synthesis of
greater amounts of NS proteins in infected cells than the
Enders strain. Scanning of l~C-labelled protein profiles
in SDS-PAGE showed that in the BF strain the relative
molar proportions of P, NS1 and NS2 protein were 1.0,
1.8 and 0.5 respectively and in the Enders strain the
proportions were 1.0, 1-0 and 0-7 (Simpson et al., 1984).
The values for the Enders strain correlate well with the
relative numbers of transcripts for each of the proteins
and indicate strain variation in the ability to generate
various non-structural proteins. However, the data for
the BF strain were surprising in the sense that the
number of transcripts that would putatively encode the P
protein was rather smaller than expected. The data given
are the results of repeated experiments and extractions of
RNA. We have shown that the mRNA fraction that was
analysed is capable of generating the P protein in in vitro
translation and thus we cannot yet conclude that
insertion of G residues into transcripts is the 0nly
mechanism for the generation of the P and NS2 proteins.
Further experiments with specific antisera are under way
to elucidate this further. We do not consider that PCR is
1560
G. D. Elliot and others
the cause of the aberrant results (low levels of P
transcripts) seen for the BF strain, because we have not
observed any problems in the other strains and the
genomic R N A sequence of the BF strain. Direct cloning
of the region without PCR has also demonstrated that
insertion occurs at the editing site. In many ways the
difficulty with the results for the BF strain are opposite to
those experienced with Sendai virus where 31% of the
transcripts seem to encode a V-like protein instead of the
P protein, but no such protein has been found in vivo
(Vidal et al., 1990). In the measles virus editing system
we have recently been able to identify the X protein
observed earlier (Rima et al., 1981) as the V-like cysteinerich translation product in infected Veto ceils.
In conclusion, by establishing the sequence of the
genome of the SBL strain of mumps virus we have
obtained indications that the organization of the ORFs
and expression of the P/V gene of mumps virus is similar
to that of SV5, except that additional proteins are
generated in mumps virus-infected cells by the reduced
accuracy of insertion of non-templated residues in
different strains. The proposed mechanism not only
explains the data in our earlier paper (Simpson et al.,
1984), which we found difficult to explain using the
models of gene expression then available, but also the
distribution of cysteine over the P and NS proteins,
whereas the suggestion in earlier reports that the latter
were generated from internal starts of translation does
not. The observed variability in the transcripts from a
single genomic sequence showing different numbers of G
residues inserted after position 531 supports the interpretation that mumps virus uses a similar mechanism to SV5
and measles virus for the expression of non-structural Vlike proteins.
We thank the Medical Research Council (U.K.) for grant support
and the Government of Pakistan for studentship support to M.A.A.
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sequence of the entire coding region of canine distemper virus
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(Received 22 December 1989; Accepted 6 March 1990)