Download Nucleotide Sequence Analysis of the Small (S) RNA Segment of

J. gen. Virol. (1989), 70, 1281-1285. Printedin Great Britain
1281
Key words: nucleotide sequence/Bunyamweravirus
Nucleotide Sequence Analysis of the Small (S) RNA Segment of
Bunyamwera Virus, the Prototype of the Family Bunyaviridae
By R I C H A R D
M. E L L I O T T
Institute o f Virology, University o f Glasgow, Church Street,
Glasgow G l l 5JR, U.K.
(Accepted 18 January 1989)
SUMMARY
The nucleotide sequence of the small (S) RNA segment of the Bunyamwera virus
genome has been determined. The S RNA is 961 bases in length and, in common with
other bunyaviruses, encodes two proteins, N and NSs, in overlapping reading frames.
A six-way alignment of the amino acid sequences of the N and NSs proteins of viruses
representing three serogroups within the Bunyavirus genus indicates regions which are
strongly conserved, and provides targets for future analysis of protein function.
The family Bunyaviridae is classified into five genera, Bunyavirus, Hantavirus, Nairovirus,
Phlebovirus and Uukuvirus, on the basis of serological and biological criteria. Bunyamwera virus
is the prototype of both the Bunyavirus genus and the family Bunyaviridae and was isolated from
infected mosquitoes in Uganda (Smithburn et al., 1946). The bunyavirus genome comprises
three segments of negative-sense RNA designated L (large), M (middle) and S (small). The S
RNA encodes the nucleoprotein, N, and a non-structural protein, NSs; the M RNA encodes the
two virion glycoproteins G1 and G2, and a second non-structural protein, NSm (reviewed by
Bishop, 1985); the L RNA encodes the large protein L (R. M. Elliott, unpublished data) which is
presumed to be a component of the virion-associated RNA polymerase.
Nucleotide sequence studies of cloned cDNA copies of bunyavirus RNAs have shown that
the N and NSs proteins are encoded in overlapping reading frames (Bishop et al., 1982;
Cabradilla et al., 1983; Akashi & Bishop, 1983; Akashi et al., 1984; Gerbaud et al., 1987; R. M.
Elliott & A. McGregor, unpublished data), and the M segment encodes a polyprotein precursor
containing G1, G2 and NSm (Eshita & Bishop, 1984; Lees et al., 1986; Grady et al., 1987;
Pardigon et al., 1988). This laboratory has cloned cDNA copies of the Bunyamwera virus
genome segments (Lees et al., 1984; Pringle et al., 1984), and has reported the sequence of the M
RNA segment (Lees et al., 1986). Here the sequence of the Bunyamwera virus S RNA segment is
presented, together with a detailed comparison of six bunyavirus S RNA sequences which
highlights regions of conservation in their N proteins.
Complementary DNA clones containing Bunyamwera virus S segment-specific sequences
were generated and identified as described by Pringle et al. (1984). DNA sequence
determination was by both the chemical degradation method (Maxam & Gilbert, 1980) as
modified in Lees et al. (1986) and the dideoxynucleotide chain termination method using DNA
fragments subcloned into bacteriophage M13 (Sanger et al., 1980).
The nucleotide sequences of five independent but overlapping cDNA clones were determined
and yielded a contiguous sequence corresponding to bases 15 to 961 of the Bunyamwera virus S
RNA (complementary, positive RNA sense). None of the clones examined contained the 5'terminal consensus sequence of bunyavirus RNAs (Clerx-van Haaster et al., 1982); hence the
terminal sequences were determined by primer extension of a synthetic oligonucleotide
(complementary to bases 108 to 127) in the presence of dideoxynucleoside triphosphates
(Geliebter et al., 1986) using infected cell positive strand RNA as template (Fig. 1a).
0000-8831 © 1989 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 13:57:15
Short communication
1282
(a)
1-108
228-400
Primer extension
5'
3' + RNA
11
894
pBUNS14
,I
!
298
952
I
pBUN3/59
I
311
952
I
pBUN308
!
310
943
t
795
pBUN309
961 pBUN93
I
(b)
~ i
1
AGTAGTGTACTCCACACTACAAACTTGCTATTGTTGAAAATCGCTGTGCT~TTAAATCC~CAG~GGTCATTAAAGGCTCTTT~TGAT
90
91
M M S L L T P A V L L T Q R S H T L T L S V S ~ P L
E L E F H D V A A N T S S T F D P E V A Y A N F K R V H T T
TGAGTTGG~TTTCATGATGTCGCTGCT~CACCAGcAGTACTTTTGACCCAGAGGTCGCATACGCT~CTTT~GCGTGTCCACACCAC
180
181
G L V M T T Y E S S T L K D A R L K L V S Q K E V N G K L H
G L S Y D H I R I F y I K G R E I K T S L A K R S E W E V T
TGGGCTTAGTTATGACCACATACG~TCTTCTACATTAAAGGACGCGAGATTAAAACTAGTCTCGCAAAAAG~GTG~TGGG~GTTAC
270
LTLGAGRLLYIIRNIFLATGTTQFLTMVLPS
271
L N L G G W K I T V Y N T
F P G N R N N P V P D D G L T L
ACTT~CCTTGGGGGCTGG~GATTACTGTATAT~TACG~TTTTCCTGGC~CcGG~C~CCCAGTTCcTGACGATGGTCTTACCCT
360
T A S V D S L P G T Y L R R C *
H R L S G F L A R Y L L E K M L K V S E P E K L I I K S K I
361
CCACcGCCTCAGTGGATT~CTTGCCAGGTAC~TACTTGAG~GATGCTG~GTCAGTG~CCAGAG~TTGATTATTAAATCAAAAAT
450
451
I N P L A E K N G I T W N D G E E V Y L S F F P G S E M F L
~TC~CCCTTTGGCTGA~G~TGGGATCACTTGG~TGATGGAGAGG~GTTTATCTCTCTTTCTTCCCAGGATCAGAGATGTTCTT
540
541
G T F R F Y P L A I G I Y K V Q R K E M E P K Y L E K T M R
AGG~CTTTCAGATTCTACCCCTTAGC~TCGGGATCTAC~GTTCAGCGC~GG~TGG~CCAAAATACCTTGAGAA~C~TGCG
630
631
Q R Y M G L E A A T W T V S K L T E V Q S A L T V V S S L G
GCAGAGGTACATGGGACTAG~GCAGC~CTTGGACTGTTAGTAAATTGAcAG~GTTCAGTCTGCACTGACAGTTGTCTCTAGCTTAGG
720
721
W K K T N V S A A A R D F L A K F G I N M *
TTG~GAAAACC~TGTTAGTGCAGCTGCCAGGGACTTCCTTGCT~TTCGG~TC~CATGT~GCAGGGATGCATTTTT~TCGGG
810
811
CTAAAGTCATCTGTTTT~TTTGGCTAAAAGGGTTGTTTC~CCCACA~T~CAGCTGCTTGGGTGGGTGGTTGGGGACAG~GACA
900
901
GCGGGCTAAATC~CATTATATTGTT~TGGTATTTT~GTTTTAGGTGGAGCACACTACT
961
Fig. 1. Sequencing strategy and nucleotide sequence of the Bunyamwera v i e s S RNA segment. (a)
Relationship of the five overlapping cDNA clones and the bases they contain relative to the
complementa~ positive sense RNA. Also shown are the two regions that were sequenced by primer
extension on mRNA (.-) or vRNA (~). (b) Nucleotide sequence of the S RNA and deduced amino acid"
sequences of the N and NSs proteins. The sequence presented is of the complementary positive RNA
strand, written as DNA.
The Bunyamwera virus S R N A segment is 961 nucleotides in length (Fig. 1 b) and has a base
composition of 2 7 - 2 ~ A, 2 2 . 4 ~ C, 19-5~ G and 3 1 - 0 ~ U. Fourteen out of the 15 terminal
nucleotides at the 5' and 3' ends of the R N A are complementary, the exception being A-C at
positions 9 and 953. This mismatch was also noted in the complementary sequence at the termini
of the M R N A segment (Lees et al., 1986).
Two AUG-initiated open reading frames were found in the sequence: the first, of 233 codons,
from bases 86 to 787 presumably encodes the N protein, and the second of 101 codons from base
105 (a tandem A U G ) to base 410, presumably encodes the N S s protein. The N protein is
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 13:57:15
1283
Short communication
V
S
D
G
S
F
L
L
P
A
(371) AGT GGA TTC CTT GCC (385)
AGT GGA TGT CTT GCC
NSs
N
G
C
L
A
N
V
D V
L
P
NSs
Fig. 2. Discrepanciesbetweenthe sequencesofcDNA clonesat positions378 and 379.The upper lines
show the nucleotide sequence and deduced amino acid sequencesobtained from clones pBUN3/59,
pBUN308 and pBUN309; the lower lines show the sequenceobtained from cDNA clone pBUNS14.
Sequencedeterminationby primer extensionon vRNA as template showedthe consensus sequencein
the population to be that shown in the upper part of the figure.
S
deficient in Cys residues, in agreement with data on the radiolabelling of infected cell proteins
reported previously (Elliott, 1985); the NSs protein contains a single Cys residue at its carboxy
terminus, which, is sufficient to radiolabel this protein, a feature used in the mapping of NSs
(Elliott, 1985).
Discrepancies between the sequences of cDNA clones pBUNS14 and clones pBUN308,
pBUN309 and pBUN3/59 were observed at positions 378 and 379 (Fig. 2). These were resolved
by primer extension of an oligonucleotide complementary to virion RNA (bases 269 to 288 of
positive sense RNA) to obtain the consensus sequence of the RNA population; the sequence
obtained was the same as that from pBUN308, pBUN309 and pBUN3/59. Therefore,
pBUNS14 may have been derived from a minor variant in the RNA population, and is
interesting since it predicts a Cys residue at position 98 in the N protein. The possibility that the
difference in pBUNS14 arose by a reserve transcriptase-induced artefact, however, cannot be
discounted.
Nucleotide sequences are now available for the S R N A segments of six bunyaviruses which
represent three serogroups in the Bunyavirus genus: Bunyamwera, Maguari (R. M. Elliott & A.
McGregor, unpublished data) and Germiston (Gerbaud et al., 1987) viruses of the Bunyamwera
serogroup, snowshoe hare (Bishop et al., 1982) and La Crosse (Akashi et al., 1983; Cabradilla et
al., 1983) viruses of the California serogroup and Aino virus of Simbu serogroup (Akashi et al.,
1984). The N proteins of these viruses are very similar in size, ranging from 233 to 235 amino
acids; the NSs proteins are more variable in length, from 91 to 109 amino acids. The N and NSs
proteins have been compared by pairwise comparison using the GAP program, followed by
alignment of the 'gapped' sequences using the program PRETTY (both programs from the
University of Wisconsin Genetics Computer Group; Devereux et al., 1984). The six-way
alignment of the proteins is shown in Fig. 3. Overall the N proteins show about 40% similarity
and the NSs proteins about 25 % similarity. Viruses within a serogroup show considerably more
relatedness, 80% or greater between N proteins and 70% or greater between NSs proteins.
Examination of the alignment of the N protein sequences in Fig. 3a shows that certain regions
of the N proteins are well conserved, particularly between residues 62 and 102, residues 123 and
169 and the carboxy-terminal 15 residues. Presumably these conserved regions are of functional
significance to the protein. Furthermore, these regions may give rise to the complement-fixing
antibodies that cross-react throughout the Bunyavirus genus (Bishop, 1985). Noteworthy also is
the strict conservation between all six proteins of the positions of some basic amino acid residues
(at positions 51, 61, 66, 70, 94, 95, 102, 168, 181,184, 186, 199, 217 and 225). These residues may
be involved in the interaction of the nucleocapsid protein with viral RNA.
In contrast to the relatedness of the N proteins, the NSs proteins show greater diversity (Fig.
3 b). However, these proteins can be readily aligned with the minimum of inserted gaps, and the
difference in length of the~NSs proteins can be accounted for by the variability at the carboxy
termini of the individual proteins. The region showing greatest similarity is between residues 71
and 91, which represents the carboxy terminus of the shortest NSs proteins. Outside this region
there are only single or pairs of amino acid residues that are conserved. The significance of these
residues in the, as yet unknown, function of the NSs protein awaits investigation.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 13:57:15
1284
Short communication
(a)
60
1
BUN N
.MiELeFhDV
MAGN
.MiELeFnDV
GERN
.MIELeFeDV
LACN
.MsDLvFyDV
SSHN
.MsDLvFyDV
AINO.N
manqFiFqDV
Consensus - M - E L - F - D V
AantssTFDP
AantssTFDP
pnnigsTFDP
AstgangFDP
AstgangFDP
pqrnlaTFnP
A ..... TFDP
E v a Y a n F k r v httgLsYDhI
EiaYvnFkri httgLsYDhI
E s G Y t n F q r n ylpgvtLDqI
DaGYmdFcvk naesLnLaaV
DaGYmaFcvk yaesvnLaaV
EvGYvaFiak hgaqLnFDtV
E-GY--F . . . . . . . L-LD--
RIFYikgrei
RVLYikgrei
RIFYikgrei
RIFFlnaaka
RIFFlnaaka
RfFFlnqkka
RIF .......
61
eVtLnLGGWK
eVtLnLGGWK
eVtLnLGGWK
kanpkFGeWq
kanpkFGeWq
sVdLtFGGiK
-V-L--GGWK
ItVyNtnFPG
VaVfNtnFPG
VpVINtnFPG
VeViNnhFPG
VeVvNnhFPG
ftlvNnhFPq
V-V-N--FPG
NRNNPVPDdg
NRNsPVPDdg
NRNNaVPDyg
NRNNPIgnnd
NRNNPInsdd
ytaNPVPDta
NRNNPVPD--
LTLHRLSGFL
LTLHRLSGFL
LTFHRiSGYL
LTiHRLSGYL
LTiHRLSGYL
LTLHRLSGYL
LTLHRLSGYL
120
A E Y I L E k . m l kvsEpekliI
A E Y i L E k i l k vsd.pekliI
A E Y I L g k Y I a .etEpeklim
A E W v L D q Y n e nddEsqhelI
A E W v L E q Y k e nedEsrrelI
AkWvaDqC.. ktnqiklaea
A E - - L E - Y . . . . . E ..... I
121
k s K I I N P L A E kNGITWndGe
k s K I I N P L A E kNGITWadGe
rtKIVNPLAE kNGITWesGp
r t t I I N P i A E sNGVgWdsGp
k t t I I N P i A E sNGVrWdsGa
meKIVmPLAE vkGcTWteGl
- - K I I N P L A E -NGITW--G-
EVYLSFFPGs
EVYLSFFPGs
EVYLSFFPGa
EIYLSFFPGt
EIYLSFFPGt
tmYLgFaPGa
EVYLSFFPG-
EMFLgTFrFY
EMFLgTFkFY
EMFLgTFrFY
EMFLeTFkFY
EMFLeTFkFY
EMFLeTFeFY
EMFL-TF-FY
P L a I G I Y k V q rkeMEPkYLe
P L a I G I Y k V q kkeMEPkYLe
P L a I G I Y k V q rkeMDPkFLe
P L t I G I h r V k qgmMDPqYLk
P L t I G I Y r V k qgmMDPqYLk
P L v I d m h r V l kdgMDvnFMr
P L - I G I Y - V . . . . MDP-YL-
181
KtMRQRYmgL
KtM-RQRYmgL
KtMRQRYIgi
KaLRQRYgtL
KaLRQRYgsL
KvLRQRYgtL
K--RQRY--L
eAatWtvsK1
eAatWtvsKV
dAqtWtttKl
tAdkWmsqKV
tAdkWmsqKV
tAeqWmtqKI
-A--W---KV
teVqsALtvV
neVqaALtvV
geVeaALkvV
aaIaksLkdV
taIaksLkeV
daVraAFnaV
--V--AL--V
ssLgWkKtnv
sgLgWkKtnv
sgLgWkKtnv
eqLkWgKggl
eqLkWgrggl
gqLsWaKsgf
--L-W-K---
SaAARdFLaK
SaAAReFLaK
SsAAReFLsK
SdtAktFLqK
SdtARsFLqK
SpAARaFLaq
S-AAR-FL-K
LTqrshtlTL
LTqrlhtlTL
LTqsqdtlTF
LilmqgiwTs
Lilmqgiwhs
LTrrsgmwhL
LT ...... TL
svstplglVM
svstplglVM
svttcqglrL
vlkmqnhstL
vlnmqnqsIL
llnmgpnsIs
......... L
ttYeSStlkd
ttFeSStlkd
tkFaSStlkd
lqLgSSssml
lqLgSSssmp
ipLdSSssir
--L-SS ....
FLaTGTtQFI
FLaTGTvQFq
sLeTGTmQcl
FLeTGTtQLv
FLeTGTiQLt
F..pnTqQil
FL-TGT-Q--
TmVLPSTasV
TmVLPSTdsV
TtVLPSTvsV
TtILPSTdyl
atILPSTdcq
cqtLPSlsiV
T-VLPST--V
iii
Dslpgtylrr c* . . . . . . . . .
Dslpgtylrk f* . . . . . . . . .
Dtlpgtyles tlqrqnqkss *
gi* . . . . . . . . . . . . . . . . . .
Di* . . . . . . . . . . . . . . . . . .
sqdi* . . . . . . . . . . . . . . . .
D ....................
BUN.N
MAGN
GERN
LACN
SSHN
AINO.N
Consensus
BUN.N
MAG.N
GERN
LAC.N
SSH.N
AINO.N
Consensus
BUN.N
MAG.N
GERN
LAC.N
SSHN
A]NON
Consensus
KtsLaKrsEW
KtsLtKrsEW
KnsLSKrsEW
KaaLSrkpER
KaaLSrkpER
KmvLSKtaqp
K--LSK--EW
180
237
FGInM*.
FGInM*.
FGIrM*.
FGIrLp*
FGIrLp*
FGIni*.
FGI-M--
(b)
BUN.NSS
MAG.NSS
OERNSS
LACNSS
SSHNSS
AINONSS
Consensus
1
MMSLltpavl
MMSLltpavl
.MSLitsgvl
MMShqqvqmd
MMShqqvqmd
.MfLngislr
MMSL ......
61
BUN.NSS
MAG.NSS
GERNSS
LACNSS
SSH.NSS
AINONSS
Consensus
AGRIIYiIrI
AGRIIYIIqI
AGRylYsIrI
sGRwrlsIiI
sGRwrlsIiI
AsslhWiItI
AGR--Y-I-I
60
aRlkLvSqke v n G k L h L t L g
aRlkLvSqke v s G r L r L t L g
aRlkivSqke vnGkLrLtLg
qRprLiSrvs qrGrLtLnLe
qRprLl Srvs qrGrqiLnLe
rRprwySvrr h n q v L i L h L v
-R--L-S . . . . . G-L-L-L-
Fig. 3. Six-way alignments of (a) the nucleocapsid (N) proteins and (b) the non-structural (NSs)
proteins of Bunyamwera (BUN), Maguari (MAG), Germiston (GER), La Crosse (LAC), snowshoe
hare (SSH) and Aino viruses. The alignments were prepared using the GAP and PRETTY programs of
Devereux et al. (1984). The 'consensus' is based on a plurality of 4, and is a convenient way of
highlighting regions of strong conservation.
The comparisons described above highlight the conserved regions of the S RNA gene
products. Once systems expressing these gene products have been established, the conserved
areas of the proteins will provide targets for specific mutagenesis which should help both in
designating function and delineating functional domains within the proteins.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 13:57:15
Short communication
1285
I thank Mairi Smith for technical assistance and Martina Scallan for performing direct R N A sequence analysis
to resolve bases 378 and 379. The author is a Medical Research Council Senior Fellow, and this work was
supported by a MRC project grant.
REFERENCES
AKASHI,H. & BISHOP, D. H. L. (1983). Comparison of the sequences and coding of La Crosse and snowshoe hare
bunyavirus S R N A species. Journal of Virology 45, 1155-1158.
AKASHI,H., GAY,M., IHARA,T. & BISHOP,D. H. L. (1984). Localized conserved regions of the S R N A gene products of
bunyaviruses are revealed by sequence analyses of the Simbu serogroup Aino virus. Virus Research I, 51-63.
BISHOP, D. H. L. (1985). Replication of arenaviruses and bunyaviruses. In Virology, pp. 1083-1110. Edited by B. N.
Fields. New York: Raven Press.
BISHOP, D. H. L., GOULD, K. G., AKASHI,H. & CLERX-VANHAASTER,C. M. (1982). The complete sequence and coding
content of snowshoe hare bunyavirus small (S) viral R N A species. Nucleic Acids Research 10, 3703-3713.
CABRADILLA,C. D., HOLLOWAY,B. P. & OBIJESKI, J. P. (1983). Molecular cloning and sequencing of the La Crosse
virus S RNA. Virology 128, 463-468.
CLERX-VANHAKSTER,C. M., CLERX, J. P. M., USHIJIMA,H., AKASHI,H., FULLER, F. & BISHOP, D. H. L. (1982). The 3'
terminal R N A sequences of bunyaviruses and nairoviruses (Bunyaviridae): evidence of end sequence generic
differences within the virus family. Journal of General Virology 61, 289-292.
DEVEREUX,J., HAEBERLI,p. & SMITHIES,O. (1984). A comprehensive set of sequence analysis programs for the VAX.
Nucleic Acids Research 12, 387-395.
ELLIOTT, R. M. (1985). Identification of non-structural proteins encoded by viruses of the Bunyamwera serogroup
(family Bunyaviridae). Virology 143, 119-126.
ESHITA, Y. & BISHOP, D. H. L. (1984). The complete sequences of the M R N A of snowshoe hare bunyavirus reveals
the presence o f internal hydrophobic domains in the viral glycoprotein. Virology 137, 227-240.
GELIEBTER, J., ZEFF, R. A., MELVOLD, R. w. & NATHENSON, S. G. (1986). Mitotic recombination in germ cells
generated two major histocompatibility complex mutant genes shown to be identical by R N A sequence
analysis: K bin9 and K bin6. Proceedings of the National Academy of Sciences, U.S.A. 83, 3371-3375.
~,RBAUD, S., VIALAT, P., PARDIGON, N., WYCHOWSKI, C., GIRARD, M. & BOULOY, M. (1987). The S segment of
Germiston virus R N A genome can code for three proteins. Virus Research 8, 1 13.
GRADY, L. J., SANDERS,M. L. & CAMPBELL,W. P. (1987). The sequence of the M R N A of an isolate of La Crosse virus.
Journal of General Virology 68, 3057-3071.
LEES, J. F., PRINGLE, C. R. & ELLIOTT, R. M. (1984). Molecular cloning of the Bunyamwera virus genome. In
Segmented Genome Viruses, pp. 13-16. Edited by D. H. L. Bishop & R. W. Compans. New York & London:
Academic Press.
LEES,J. F., PRINGLE,C. R. & ELLIO'I'f,R. M. (1986). Nucleotide sequence of the Bunyamwera virus M R N A segment:
conservation of structural features in the Bunyavirus glycoprotein gene product. Virology 148, 1-14.
MAXAM,A. i . & GILBERT, W. (1980). Sequencing endqabeled D N A with base-specific chemical cleavages. Methods
in Enzymology 65, 499-560.
PARDIGON,N., VIALAT,P., GERBAUD,S., GIRARD, M. & BOULOY,M. (1988). N ucleotide sequence of the M segment of
Germiston virus: comparison of the M gene product of several bunyaviruses. Virus Research 11, 73-85.
PRINGLE, C. R., LEES,I. F., CLARK, W. & ELLIOTT, R. M. (1984). Genome sub-unit reassortment among bunyaviruses
analysed by dot hybridization using molecularly cloned complementary D N A probes. Virology135, 244-256.
SANGER, F., COULSON, A. R., BARRELL, B. G., SMITH, A. J. H. & ROE, S. A. (1980). Cloning in single-stranded
bacteriophage as an aid to rapid D N A sequencing. Journal of Molecular Biology 143, 161-178.
SMITHBURN, K. C., HADDOW, A. J. & MAHAFFY,A. F. (1946). A neurotropic virus isolated from Aedes mosquitoes
caught in the Semliki Forest. American Journal of Tropical Medicine 26, 18~208.
(Received 9 December 1988)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 12 May 2017 13:57:15