Download Nucleotide sequences of normal and rearranged RNA segments 10

Journal o f General Virology (1992), 73, 633-638.
Printed in Great Britain
633
Nucleotide sequences of normal and rearranged RNA segments 10 of
human rotaviruses
Anna Ballard, 1 M. A. McCrae 2 and U. Desselbergerl*f
1Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5 S T and 2Department o f Biological Sciences,
Warwick University, Coventry CV4 7AL, U.K.
Normal and rearranged R N A segments 10 of group A
rotaviruses isolated from a chronically infected
immunodeficient child were amplified by the polymerase chain reaction as full-length cDNA copies, and
were subsequently cloned and sequenced. Compared
with the nucleotide sequence of the normal R N A
segment 10, the rearranged form contains a partial
non-coding duplication at its 3' end and several point
mutations. The normal R N A segment 10 was similar to
that of bovine rotavirus.
Introduction
partial duplications have been found which had been
generated by reiteration of genomic sequences after the
termination codon of the normal open reading frame
(ORF).
We have described numerous subpopulations of
rotaviruses isolated from a chronically infected
immunodeficient child which contain various forms of
rearrangements of RNA segment 8-, 10- and 11-specific
sequences (Hundley et al., 1987). Here we report the
sequences of the normal (standard size) R N A 10 and of
its rearranged form in one of these viruses, and compare
the sequences both with each other and with those of
other RNA segments 10 (Both et al., 1983; Baybutt &
McCrae, 1984; Okada et al., 1984; Ward et al., 1985), as
well as with the structures of other rearranged genome
segments.
Group A rotaviruses, the main cause of viral gastroenteritis in infants and in the young of a variety of
mammals and birds (Flewett & Woode, 1978; Estes et al.,
1984; Kapikian & Chanock, 1990), usually possess a
genome of 11 segments of dsRNA of well conserved size
(Estes, 1990). However, within Group A, rotaviruses
with atypical R N A profiles have been observed in which
segments of standard size are replaced by rearranged
forms of larger size. Such genomes were first found in
rotaviruses isolated from chronically infected immunodeficient children (Pedley et al., 1984; Eiden et al., 1985;
Hundley et al., 1987), but have also been isolated from
both human (Besselaar et al., 1986) and mammalian
(Thouless et al., 1986; Pocock, 1987; Bellinzoni et al.,
1987; Tanaka et al., 1988) immunocompetent hosts. It
has also been possible to generate genome rearrangements in tissue culture-adapted bovine rotaviruses by
serial passage at high m.o.i. (Hundley et al., 1985).
Variants of bluetongue virus, a member of the orbivirus
genus of the Reoviridae family, also exhibit rearranged
genomes (Ramig et al., 1985; Eaton & Gould, 1987).
Thus, genome rearrangements may be an important
mechanism of variation and evolution of dsRNA viruses
(Desselberger, 1989).
Several of these rearranged genes have been
sequenced (Gonzalez et al., 1989; Gorziglia et al., 1989;
Scott et al., 1989; Matsui et al., 1990). In most cases
t Present address: Public Health Laboratory, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QW, U.K.
The nucleotide sequence data reported in this paper will appear in
the DDBJ, EMBL and GenBank nucleotide sequence databases under
the accession numbers D01145 and D01146.
0001-0630 © 1992 SGM
Methods
Viruses and cells. Rotaviruses isolated from a child with severe
combined immunodeficiency carrying various forms of rearrangements of RNA segments 8, 10 and 11 (Hundley et al., 1987) were
investigated. Initially, two viruses, A28 and A64, which carry normal
and rearranged RNA segments 10, respectively, were studied. Both of
these viruses also carry rearranged segments 8 and 11 (genotypes 5 and
7 as shown in Fig. 3 of Hundley et al., 1987). Bovine rotavirus (UK
Compton strain) was propagated as a control. Viruses were grown on
confluent monolayers of MA104 cells infected at low multiplicity
(m.o.i., 0-1) to prepare stock suspensions (Hundley et al., 1987).
Electrophoretic fraetionation of rotavirus dsRNA and of amplified D NA.
Viral genomic dsRNA was extracted from virions and fractionated by
electrophoresis in 10~ polyacrylamide gels as described previously
(Rodger & Holmes, 1979). The dsRNA was visualized by silver
staining (Follett et al., 1984).
Amplified DNA bands were fractionated on 1 ~ agarose gels using
TAE buffer (Sambrook et al., 1989) and visualized by staining with
ethidium bromide.
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634
A. Ballard, M. A. McCrae and U. Desselberger
Preparation ofrotavirus mRNA. MA104 cells were infected at high
multiplicity (1 to 5) and grown overnight in the presence of 0.5 p-g/ml of
trypsin (Sigma Type IX) until c.p.e, was observed. Cells were harvested
and lysed by incubation on ice in Tris-NP40 buffer (100 mM-Tris-HC1
pH 8.0, 50 mM-NaCI, 10 mM-EDTA and 0-5% NP40). Subsequently,
unlysed cells and nuclei were removed by brief centrifugation (11000 g,
15 s), and the cytoplasmic supernatant was digested for 3 h at 37 °C with
250 p-g/ml of proteinase K (Sigma) in the presence of 0-2% SDS,
followed by repeated extractions with phenol (saturated with 100 m i Tris-HCl pH 8.0) and ether, and ethanol precipitation. The precipitate
was centrifuged (11000g, l0 min) and resuspended in a small volume of
distilled water (McCrae, 1985). Starting with one 75 cm x Roux bottle of
MA104 cells, 100 gl containing a mixture of mRNA and dsRNA was
obtained; the RNA content was not quantified.
Synthesis o f full-length cDNA. Full-length cDNA corresponding to
RNA segment 10 was made by reverse transcription of mRNA from
virus-infected cells followed by amplification in a polymerase chain
reaction (PCR). The oligonucleotide primers used are shown in Fig.
1 (a). In the 3' halves of the primers the nucleotide sequences are
complementary to the termini of viral RNA or cDNA segment 10; in
their 5' halves they contain several restriction endonuclease recognition
sites as indicated (Xu et al., 1991). The oligonucleotides were produced
on an ABI DNA synthesizer (model PCR-mate 391).
For synthesis of cDNA from normal-length RNA segment 10, 5 to
10 p-1 of mRNA was mixed with 100 ng of each primer and dimethyl
sulphoxide to a final concentration of 10 % in a final volume of l 2.5 p-l.
Under these conditions, amplification from dsRNA (purified from
virions) was never successful; the products obtained were likely to have
originated from mRNA molecules. The reaction was then heated in a
Techne PHC-2 Thermocycler to 94 °C for 2 min and cooled to 42 °C.
An equal volume (12.5 p.1) of reverse transcriptase/PCR amplification
mixture was added [final concentrations: 10 mM-Tris-HC1 pH 8.3,
50 mM-KCI, 1.5 mi-MgCl2, 0.3 mM-DTT, 0.05% gelatin, 0.2 mMdATP, 0.2 mM-dGTP, 0.2 mM-dCTP, 0.2 mM-TTP, 6 units avian
myeloblastosis virus reverse transcriptase (Pharmacia) and 0-5 units
Taq DNA polymerase (Perkin-Elmer Cetus)] and incubation was
continued at 42 °C for 30 min. Reverse transcription was followed by
PCR amplification of the cDNA using 25 cycles of denaturation at
94 °C for 2 min, annealing at 55 °C for 1 min and synthesis at 70 °C for 4
min (Xu et at., 1991). The product of this PCR reaction was isolated
from an agarose gel, purified using Isogene (Perkin-Elmer Cetus) and
resuspended in 20 pl of distilled water. The product was diluted 1 : 100
and 5 p-I was used in a second PCR amplification reaction, as described
above but omitting the reverse transcription step. In this way large
quantities of full-length cDNA for cloning were generated.
For synthesis ofcDNA from mRNA of rearranged RNA segment 10
the procedure described above was used, but only 10 ng of each primer
was added and the annealing time was increased to 5 min. The decrease
in primer concentration was crucial for obtaining full-length cDNA
from rearranged segment 10 mRNA. When the higher primer
concentration was used, cDNA of the size of normal RNA segment 10
was always obtained (results not shown), suggesting intermolecular
base-pairing of short cDNA products in the duplicated regions.
Cloning o f full-length ds cDNA. Prior to cloning, all PCR products
were end-filled using the Klenow fragment of D N A polymerase I
(Gibco-BRL) and standard procedures (Sambrook et al., 1989).
Cloning of full-length cDNA was achieved with two different
procedures. Cloning of cDNA corresponding to RNA segment 10 of
standard length was carried out by G-C tailing and annealing into
pAT153 (Sambrook et al., 1989). cDNA corresponding to rearranged
RNA segment 10 was digested with SphI, which cuts in the region of
primer AB 101 only; the other end was left uncut, i.e. blunt-ended. This
product was ligated into pBluescribe which had been digested with
SphI and SmaI (Sambrook et at., 1989). The plasmids were transfected
into Escherichia coli strains MC1061 and JMI09, respectively, and
colonies with insert-containing plasmids were detected using standard
procedures (Sambrook et al., 1989).
DNA sequence analysis. Sequencing was carried out by the
dideoxynucleotide chain termination method (Sanger et al., 1977) using
the Sequenase Version 2.0 Kit (United States Biochemical Corporation), after subcloning of suitable DNA fragments into M 13mp 18 and
M13mpl9 sequencing vectors.
Results
The RNA profiles of rotaviruses A28 and A64 with
normal and rearranged RNA segments 10, respectively,
are shown in Fig. 1 (b). Full-length cDNA clones of
segments 10 of both viruses were obtained by combined
reverse transcription-PCR (Fig. 1c) and transfected into
E. coli strains as described in Methods. Cloned rotavirus
cDNA inserts were isolated, and subfragments (obtained
by digestion with HinclI, PstI and SphI) cloned into
M13mpl8 and M13mpl9 for sequencing. All nucleotides
were confirmed by sequencing on both strands, and two
independent clones of the rearranged cDNA (cl 6 and cl
2) were fully sequenced.
The nucleotide sequences of RNA segment 10 of
normal length (A28) and of rearranged RNA segment 10
from A64 virus (cl 6) are shown in Fig. 2 and 3,
respectively. It was found that the gene rearrangement
consists of a partial duplication of the normal ORF
which stretches from nucleotides (nt) 41 to 569. In the
rearranged gene, a reiteration of the ORF had occurred
two nucleotides after the termination codon. This
repeated portion of the ORF start at nucleotide 81, i.e. 40
nucleotides downstream of the initiation codon of the
normal ORF. Thus, nucleotide 81 becomes rearranged
nucleotide 572. The sequence is then completed to the 3'
end, nucleotide 751 of the normal gene, which becomes
nucleotide 1242 in the rearranged gene. An interesting
feature is the occurrence of a seven base direct repeat in
nucleotide positions 75 to 81 and 563 to 569 (Fig. 3). The
overall relationship between normal and rearranged
genes is diagrammatically represented in Fig. 4. A
second clone (cl 2) of the rearranged gene from the same
cloning experiment was sequenced fully and the junction
point 571/572(81) confirmed.
When the sequences were compared, a number of
point mutations were found using the sequence of the
normal gene as a reference (Table 1). Comparison of the
nucleotide sequences between positions 1 and 751 of the
normal and rearranged genes (cl 6) revealed five point
mutations in positions 76, 184, 290, 371 and 519, of
which only one (nucleotide 290) was a silent mutation
(Table 1). The second cDNA clone (cl 2) of the
rearranged gene had different point mutations compared
with the standard sequence (Table 1). Within the ORF
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Sequences of rearranged rotavirus RNAs
(a)
Primer AB 102
5'
EcoRI
bide[
4,
4,
NcoI
'
,~ :
GAATTCATATGGCCAI~GGTCACACTAAGACCATTCC
Ball
3'
, Complementary to 3' end of m R N A
Primer AB101
SphI
~,
5'
Narl
4'
PvulI Ij
¢ ,
GCATGCCGGCGCCAGCT~GCTTTTAAAAGTTCTGTTc
NaeI
3'
I Complementary to 3' end of negative-strand R N A
(c)
(b)
1
2
3
1
2
3
tlO
~10
Fig. 1. (a) Oligonucleotide primers for reverse transeription-PCR of rotavirus RNA segment 10. (b) RNA profiles of human
rotaviruses with normal gene 10 (A28, lane 1) and rearranged gene 10 (A64, lane 2), and of bovine rotavirus (UK Compton strain, lane
3). RNA segments were separated by eleetrophoresis on a 10% polyaerylamide gel and silver-stained (Sambrook et al., 1989). RNA
segments are designated, the prefix R indicating rearranged forms. (c) Product of reverse transeription-PCR showing full-length eDNA
copies of normal and rearranged RNA 10 of human rotaviruses A28 and A64, lanes 2 and 3 respectively. In lane 1, molecular size
markers (1 kb D N A ladder Gibco-BRL) are shown. Eleetrophoretic separation was on a 1% agarose get which was stained with
ethidium bromide (Sambrook et al., 1989).
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635
636
A. Ballard, M. A. McCrae and U. Desselberger
1
51
GGCTTTTAAA
AGTTCTGTTC
CGAGAGAGCG
CGTGCGGAAA
GATGIGAAAAG
!
GGCTTTTAAA AGTTCTGTTC CGAGAGAGCG CG~GCGGAAA GATGGAAAAG
CTTACCGACC
TCAATTACAC
ATTGAGTGTG
ATCACTCTAA
TGAACAGTAC
51
CTTACCGACC TCAATTACAC ATTGAATGTGVATCACTCTAA TGAACAGTAC
I
ATTGCATACA ATACTAGAAG ACCCAGGAAT ~GCGTATTTT CCTTATATTG
10L
ATTGCATACA
ATACT~GAAG
ACCCAGGAAT
GGCGTATTTT
CCTTATATTG
151
CATCTGTTCT AACAGTTTTG TTCACTTTAC ATAGAGCGTC AATTCCAACG
151
CATCTGTTCT
AACAGTTTTG
TTCACTTTAC
ATAAAGCGTC
AATTCCAACG
201
ATGAAGATCG CACTAAAGAC ATCAAAATGC TCGTATAAGG TAGTGAAGTA
201
ATGAAGATCG CACTAAAGAC ATCAAAATGC TCGTATAAGG TAGTGAAGTA
251
CTGCATTGTG ACAATTTTTA ATACACTATT GAAACTGGCA GGTTATAAAG
251
CTGCATTGTG ACAATTTTTA ATACACTATT GAAACTGGCG GGTTATAAAG
301
AACAAATTAC TACTAAAGAT GAAATAGAAA AGCAGATGGA CAGAGTCGTC
301
AACAAATTAC TACTA~AGAT GAAATAGAAA AGCAGATGGA CAGAGTCGTC
351
AAAGAAATGA
CTACACGTGA
351
AAAGAAATGA
GACGTCAGTT
GGAAATGATT
GATAAGTTGA
CTACACGTGA
TTGATGGTGC
401
AATTGAGC~A
GTTGAACTAC
TTAAACGCAT
TTATGATAAA
TTGATGGTGC
CGGAATAGAT
ATGACGAAAG
AAATAAATCA
AAAAAACGTA
I01
GACGTCAGTT
TGAAATGATT
GATAAGTTGA
401
AATTGAGCAA
GTTGAACTAC
TTAAACGCAT
451
GAGCAACTGA
CGGAATAGAT
ATGACGAAAG
AAATAAATCA
AAAAAACGTA
451
GAGCAACTGA
501
AAAACGCTAG
AAGAATGGAA
GAGTGGAAAA
AATCCTTATG
AACCAAAGGA
501
AAAACGCTAG AAGAATGGGA GAGTGG~AAA AATCCTTATG AACCAAAGGA
551
AGTGACTGCA
GCAATGTGAG
AGGTTGAGCT
GCCGT~GACT
GTCTTCGGAA
55$
AGTGACTGCA
GCAATGTGAG
601
GCGGCGGAGT
TCTTTACAGT
AAGCCCCATC
GGACCTGATG
GCTGGCTGAG
bOl
AATACTAGAA
GACCCAGGAA
651
AAGCCACAGT
CAGCCATATC
GCGTGTGGCT
CAAGCCTTAA
TACCGTTTAA
651
TAACAGTTTT
GTTCACTTTA
TTAGTGTGAC
701
GCACTAAAGA
CATCAAAATG
CTCGTATAAG
701
CCAATCCGGT
751
C
CAGCACCGGA
CGTTAATGGA
TTATGATAAA
AGGAATGGTC
Fig. 2. N u c l e o t i d e sequence o f normal R N A segment 10 ( c D N A form,
positive strand) o f h u m a n rotavirus A28. Stop and start c o d o n s are
underlined.
were nine point mutations (at nucleotides 58, 76, 290,
292, 349, 371, 414, 470 and 476), of which only three
(nucleotides 76, 290 and 371) were identical to those ofcl
6. Two changes in cl 6 (nucleotides 184 and 519) were not
found in cl 2. Comparison of nt 81 to 751 of the normal
gene with nt 572 to 1242 of the rearranged genes showed
one further point mutation (nt 1181) in both clones of the
rearranged gene (Table 1).
1. Nucleotide and predicted amino acid changes
between RNA segments 10 of human rotaviruses A28
(normal RNA 10; NIO) and A64 (rearranged RNA 10;
clones 6 and 2).
A~ATCACTCTA
ATGAACAGTA
CATTGCATAC
TGGCGTATTT
TCCTTATATT
GCATCTGTTC
CATAGAGCGT
CAATTCCAAC
GATGAAGATC
GTAGTGAAGT
ACTGCATTGT
751
GACAATTTTT AATACACTAT TGAAACTGGC GGATTATAAA GAACAAATTA
801
CTACTAAAGA
TGAAATAGAA
AAGCAGATGG
ACAGAGTCGT
CAAAGAAATG
851
AGACGTCAGT
TGGA~ATGAT
TGATAAGTTG
ACTACACGTG
AAATTGAGCA
901
AGTTGAACTA
CTTAAACGCA
TTTATGATAA
ATTGATGGTG
CGAGCAACTG
951
ACGGAATAG~ TATG~CGAAA GAAATAAATC AAAAAAACGT ~AAAACGCTA
iOO1
GAAGAATGGA AGAGTGGAAA AAATCCTTAT GAACCAAAGG AAGTGACTGC
1051
AGCAATGTG~
GAGGTTGAGC
TGCCGTCG~C
TGTCTTCGGA
ii01
TTCTTTACAG
TAAGCCCCAT
CGGACCTGAT
GGCTGGCTGA
GAAGCCACAG
~1~I
TCAGCCATA,
CGCGTGTGGC
TCAAGCCTTA
ATCCCGTTTA
~CAATCCGG
1201
TCAGCACCGG
ACGTTAATGG
AA~GAATG6~
CTTAGTGTGA
CC
AGCGGCGGAG
Fig. 3. N u c l e o t i d e sequence o f rearranged R N A segment 10 (cl 6;
c D N A form, positive strand) o f h u m a n rotavirus A64. Start and stop
c o d o n s are underlined. T h e junction point b e t w e e n nucleotides 571 and
572 is indicated by an arrow. T h e arrow b e t w e e n nucleotides 80 and 81
denotes the position o f the duplication starting point. A direct s e v e n
base repeat is delineated by dotted lines.
Table
Base position
N10
58
76
184
290
292
349
371
414
470
476
519
519
692
R10
C1 6
76
184
290
371
519
1181
Base change
R10
Cl 2
58
76
290
292
349
371
414
470
476
~010
1181
* NC, N o n - c o d i n g .
A m i n o acid change
N10
R10
Cl 6
R10
Cl 2
A
G
G
A
G
T
T
G
T
G
A
A
A
A
A
G
G
G
C
T
A
G
A
C
G
A
C
C
G
C
N10
R10
C1 6
R10
C1 2
D
S
R
A
G
V
E
E
D
T
K
K
NC
N
K
G
E
N¢
V
N
D
A
G
K
~c*
NC
Comparison of the normal gene 10 of rotavirus A28
with those of human rotavirus Wa (serotype 1 ; Okada et
al., 1984), simian rotavirus SA11 (serotype 3; Both et al.,
1983) and bovine rotavirus UK strain (serotype 6;
Baybutt & McCrae, 1984; Ward et al., 1985) showed a
high degree of nucleotide and amino acid similarity (Fig.
5), especially with the bovine rotavirus genes. There was
an accumulation of amino acid changes in positions 131
to 141 which has already been noted by others (Estes &
Cohen, 1989).
Discussion
Rearrangements of rotavirus R N A segments have now
been described in a number of human and animal
rotavirus strains (Pedley et al., 1984; Hundley et al.,
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Sequences o f rearranged rotavirus R N A s
Normal gene 10
1 4181
I '
569
'
751
,
I
5'
3'
Met
"'.
Stop
""
141
I ,
569
"
"
572
,
"",. "1(/
" -.
"
"..
1242
"
,.
Met
4 3,
Stop
Rearranged gene 10
Fig. 4. D i a g r a m o f t h e s t r u c t u r e s o f n o r m a l and rearranged genes 10of
h u m a n rotavirus, m , Complete O R F ; [[], duplicated part of O R F of
normal gene (untranslated); solid lines indicate 5' and 3' untranslated
regions.
Normal 10
Wa
U K Bovine
Bovine
SAI 1
N o r m a l 10
Wa
U K Bovine
Bovine
SAIl
N o r m a l 10
Wa
U K Bovine
Bovine
SAIl
Normal 10
Wa
U K Bovine
Bovine
SA11
Wa
U K Bovine
Bovine
SAIl
l
MEKLIDLNYTLSV
ITLMNSTLHTILEDPGMAYFPYIASVLTVLFTLHRA5
-D--A ..........
S--D---S-IQ
.......
L .............
K--
..................
K--
N ............................
30
....................................
V ..........
K-..................
N ......................
G--A-NK--
51
IPTMKIALKTSKCSYKVVKYCIVTIFNTLLKLAGYKEQITTKDEIEKQMD
.................
[ . . . . . . . z. . . . . . . . . . . .
..................................................
i00
v . . . . . . . Q---
..................................................
..................................................
I01
RVVKEMRRQFEM
I DKLTTR E I EQVELLKR
-I .......
L ....................
150
IYDKLMVRATDG
I DblTKE l NQ
,,-,-,,-,,-,---,--,--
.........
.........
L ...........................
L ....................
H ....
.........
L ........................
151
KNVKTLEEWKSGKNPYEPKEVTAAM
--I---D--E
---R .....
S-GE .........
I-TV-E .........
T-QT~GE
.........
175
........
S .... SE ...............
---R ...... N-R ............
---R ..............
R ......
Nucleic acid
similarity (~)
83
91
90
89
A m i n o acid
similarity (~o)
83
95
94
92
Fig. 5. Comparison of the predicted amino acid sequence of the R N A
10 product of h u m a n rotavirus serotype 10 with those of other group A
rotaviruses: Wa, serotype 1 (Okada et al., 1984); U K bovine, serotype 6
(Baybutt & McCrae, 1984); bovine, serotype 6 (Ward et al., 1985); SA
11, serotype 3 (Both et al., 1983). The percentage nucleotide and a m i n o
acid similarity is indicated at the bottom.
1985; Thouless et al., 1986; Besselaar et al., 1986;
Bellinzoni et al., 1987; Pocock, 1987; Tanaka et al., 1988)
and are not, as initially observed, confined to viruses
isolated from chronically infected immunodeficient
hosts (Pedley et al., 1984; Eiden et al., 1985; Hundley et
al., 1987). A number of these rearrangements have been
sequenced (Gonzalez et al., 1989; Gorziglia et al., 1989;
Scott et al., 1989; Matsui et al., 1990) and, with a few
exceptions, the normal ORF was found to be retained.
637
The rearrangement involved partial duplication of the
gene in the non-coding Y-terminal sequences and thus
increased its length. This overall strategy has been
confirmed by sequencing gene 10 of human rotaviruses
(Hundley et al., 1987). Compared to the ORF region of
the normal gene, the ORFs of the rearranged gene
contained five and nine nucleotide changes in clones 6
and 2, respectively.
Of the rearranged genes sequenced previously (all
from R N A 11), one (Scott et al., 1989) had duplications
almost identical to the original sequences, whereas the
others (Gorziglia et al., 1989; Gonzalez et al., 1989;
Matsui et al., 1990) had numerous point mutations and
one (Gorziglia et al., 1989) had a partial deletion. The
rearranged sequence reported here would fall in between.
Whereas some of the 'super-short' electropherotype
viruses (VMRI; Matsui et al., 1990) contain a clear
partial duplication in the 3' end untranslated region, the
'short' electropherotype virus DS-1 and the 'super-short'
electropherotype virus M69 contain sequences in their 3'
end untranslated regions which are similar to each other,
but not to any other available rotavirus gene sequence
(Matsui et al., 1990).
The differences in point mutations between clones 2
and 6 of cDNA of rearranged R N A segment 10 are likely
to be due to errors produced by the retrovirus reverse
transcriptase (Leider et al., 1987) and the double PCR
procedures used before sequencing. To what extent these
changes reflect true heterogeneity of m R N A remains to
be elucidated.
With regard to the mechanisms which produce R N A
rearrangements, it has been suggested that the rotavirusspecific RNA-dependent R N A polymerase (Cohen,
1977) falls back on its template at various stages of
primary transcription and retranscribes from that template (Hundley et al., 1987; Tanaka et al., 1988;
Gorziglia et al., 1989; Matsui et al., 1990). It is
remarkable that in all the cases sequenced (this sequence
and those described in references below) the reinitiation
has occurred close to the termination codon: after zero
(Scott et al., 1989), two (this sequence), four (Gorziglia et
al., 1989) and six nucleotides (Gonzalez et al., 1989). This
could be due to selection against any other intramolecular recombination events, or to a predilection for
rearrangement at this site on structural grounds. The first
possibility may be true in most cases, but Hundley et al.
(1985) described a rearrangement of the bovine rotavirus
genome resulting in a novel R N A segment 5 product of
larger than normal size, which was generated through an
in-frame reiteration within the ORF (Tian Ye et al.,
unpublished results).
With regard to the possibility of the site near the
termination codon being a preferred site for recombination, it should be noted that it has been implicated in
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638
A. Ballard, M. A. McCrae and U. Desselberger
intramolecular base pairing (Okada et al., 1984, for R N A
segment 11; Gorziglia et aL, 1989). Such base pairing
may be an important signal for polymerase binding and
replication, as suggested for other viruses (Hsu et al.,
1987). On the other hand, it is of interest that a direct
seven base repeat is found to the left of the junction and
reinitiation sites (AATGTGA, positions 75 to 81 and 563
to 569, Fig. 3). It seems possible that the polymerase
becomes less processive near the termination codon and
preferentially jumps to a site identical to the one it has
just passed.
In any case, rearrangements, whether they are
produced by this or a similar mechanism, do require an
extended single-stranded region of transcript template,
whether it is positive- or negative-strand RNA. Thus, the
phenomenon of rearrangement may help to unravel the
mechanism of R N A replication in rotaviruses and other
dsRNA viruses.
U.D. acknowledges fruitful discussions with P. Palese. This work
was funded by a grant from the Medical Research Council to U.D.
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