Download Comparative Sequence Analysis of the Genomic Segment 6 of Four

J.
gen. Virol. (1988), 69, 1659-1669. Printed in Great Britain
1659
Key words: rotavirus VP6/sequence homology~antigenic epitopes
Comparative Sequence Analysis of the Genomic Segment 6 of Four
Rotaviruses Each with a Different Subgroup Specificity
By M A R I O G O R Z I G L I A , * Y A S U T A K A H O S H I N O , K A Z U O N I S H I K A W A ,
W. L E E M A L O Y , 1 R O N A L D W. J O N E S , A L B E R T Z. K A P I K I A N AND
R O B E R T M. C H A N O C K
Laboratory o f Infectious Diseases and 1Biological Resources Branch, National Institute o f Allergy
and Infectious Diseases, National Institutes o f Health, Bethesda, Maryland 20892, U.S.A.
(Accepted 25 March 1988)
SUMMARY
The nucleotide sequences of the genes that code for the major inner capsid protein,
VP6, of the human rotavirus strain 1076 (subgroup I), porcine rotavirus Gottfried
(subgroup II), equine rotavirus strain H-2 (non-I/II) and equine rotavirus strain FI-14
(both subgroups I and II) have been determined. The sixth segment positive-stranded
RNA encodes a protein of" 397 amino acids in all strains with the exception of strain
H-2 in which it encodes a protein of 399 amino acids. Alignment of amino acid sequences
of the VP6 protein of strain FI-14 and subgroup II rotaviruses (Wa and Gottfried)
indicates a high degree of homology (94~), while homology between strain FI-14 and
subgroup I rotaviruses (SA-11, RF and 1076) was somewhat less (90 to 92~). On the
other hand a high degree of conservation of amino acid sequence (95 to 97~) was
observed between the H-2 strain and subgroup I rotaviruses. Five regions that may
contribute to subgroup epitopes were identified. Region A (amino acids 45, 56) and
region C (amino acids 114, 120) may contribute to subgroup I epitopes and regions B
(amino acids 83, 86, 89, 92), D (amino acids 312 or 314, 317 or 319) and E (amino acids
341 or 343, 350 or 352) may contribute to subgroup II epitopes. When analysed using
the Western blot technique monoclonal antibodies specific for VP6 epitopes shared by
all rotaviruses were observed to react with both monomeric and trimeric forms of VP6,
while monoclonal antibodies specific for a subgroup I or II epitope reacted only with
the trimeric form of VP6. This observation and the sequence analyses suggest that
subgroup antigenic specificity is determined by conformational epitopes produced by
the folding of VP6 or the interaction between VP6 monomers.
INTRODUCTION
Rotaviruses possess two outer capsid proteins, designated VP7 and VP3, which are
considered to be independent neutralization antigens (Hoshino et al., 1985; Offit & Blavat,
1986). The major inner capsid protein of rotaviruses, p45K (Novo & Esparza, 1981), also
referred to as VP6 (Espejo et al., 1981; Ericson et al., 1982) is present in the virion in an
oligomeric, possibly trimeric form (Gorziglia et al., 1985; Sabara et al., 1987). VP6 is an
important component of the virion not only because of its abundance, accounting for
approximately 80 ~ of viral protein, but also because of its constituent antigenic sites. Moreover,
VP6 may be a component of the viral polymerase or other enzyme complexes which are active in
single capsid particles (Holmes, 1983). The analysis of reassortant rotaviruses initially led to the
identification of the sixth RNA segment as the gene which encodes VP6 (Kalica et al., 1981).
This protein can be detected readily with a variety of antigen assay systems including
complement fixation, immune adherence haemagglutination assay, radioimmunoassay and
ELISA. VP6 contains a group antigen common to all group A rotaviruses (Greenberg et al.,
1983). Also, in initial studies two distinct non-overlapping antigenic specificities, designated
subgroup I and subgroup II, were identified and localized to the VP6 protein (Kapikian et al.,
0000-8033 © 1988 SGM
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1660
M. GORZIGLIA AND OTHERS
1981 ; Greenberg et al., 1983; Taniguchi et al., 1984). When tested with specific hyperimmune
rotavirus antisera or subgroup-specific monoclonal antibodies group A rotaviruses usually
exhibited either subgroup I or subgroup II specificity. In subsequent studies a more complex
pattern emerged when it was observed that some animal and avian rotavirus failed to react with
either subgroup I- or II-specific antibodies and were designated n o n I/II (Hoshino et al., 1983,
1984). In addition, Hoshino et at. (1987a) recently identified a fourth category of subgroup
reactivity in an equine rotavirus strain, FI-14, which reacted with both subgroup I and subgroup
II monoclonal antibodies.
The complete nucleotide sequence of gene 6, the gene that encodes VP6, of two subgroup I
animal rotaviruses, vervet monkey rotavirus SA-11 (Estes et al., 1984) and bovine rotavirus R F
(Cohen et al., 1984) and one subgroup II h u m a n rotavirus Wa (Both et al., 1984) have been
previously determined. Comparison of the deduced amino acid sequences indicated a high
degree of homology; more than 90 ~ of the amino acids were conserved between subgroup I and
subgroup II rotaviruses.
We describe the deduced amino acid sequence of the VP6 protein of four additional rotavirus
strains, each with a different subgroup specificity: h u m a n rotavirus strain 1076 (subgroup I),
porcine rotavirus strain Gottfried (subgroup II), equine rotavirus strain H-2 (non-I/II) and
equine rotavirus strain FI-14 (both subgroup I and II). This report presents a comparison of the
VP6 amino acid sequences of rotaviruses with dual or no known subgroup reactivity with those
of rotaviruses belonging to subgroup I or II. This approach was taken in an attempt to identify
epitopes responsible for subgroup specificity. In addition, the importance of the trimeric
conformation of the VP6 protein to subgroup epitopes was investigated.
METHODS
Viruses and cells. The following rotavirus strains were studied: human rotavirus 1076 (subgroup I, serotype 2),
porcine rotavirus Gotffried (subgroup II, serotype 4); equine rotavirus H-2 (subgroup non-I/II, serotype 3) and
equine rotavirus FI-14 (both subgroup I and II, serotype 3). These viruses were propagated in a continuous line of
African green monkey kidney cells, MA104 (Hoshino et al., 1984). Trypsin (Type IX, Sigma) was used at a final
concentration of 10 Bg/ml for 60 rain at 37 °C to activate virus infectivity. After adsorption of virus, cell
monolayers were fed with Eagle's MEM supplemented with 1 ~g of trypsin per ml.
In vitro transcription. Viruses were purified by Freon 113 extraction of rotavirus-infected cells followed by
pelleting through a cushion of 30~ (w/v) sucrose. The virus pellet was resuspended in buffer containing 20 mMTris-HC1 pH 7.4 and 15 mM-EDTAand incubated at 37 °C for 1 h. Single capsid viral particles produced by this
treatment were purified by isopycnic centrifugation in a CsCI gradient. Single-stranded RNAs were prepared by
in vitro transcription of rotavirus single capsid particles. The ssRNAs were purified by lithium chloride
precipitation (Flores et al., 1982). The ssRNA pellet was washed twice with 100~ ethanol, vacuum-dried and
resuspended in 20 to 40 BIof sterile distilled water. The integrity and concentration of ssRNA were determined by
electrophoresis in 1~ agarose followed by staining with ethidium bromide.
Dideoxynueleotide sequencing. The procedure used followed the method described by Gorziglia et al. (1986).
Briefly, 40 to 70 ng of a DNA primer 18 nucleotides long, which was synthesized on an Applied Biosystems
synthesizer, was mixed with 4 to 7 ~g of rotavirus ssRNA transcripts, heated at 95 °C for 1 min, and then allowed
to anneal at 42 °C for 5 min. Primer extension was performed as a set of four reverse transcription reactions that
contained 3 pM-dATP, 50 ~Meach of dCTP, dGTP and dTTP, 600 units of reverse transcriptase per ml, and I mCi
of [~x-32p]dATPper mL In addition, 1 gM-ddATPor 14 ~M each of ddCTP, ddGTP or ddTTP was included to
terminate extension. Reaction mixtures were incubated at 42 °C for 30 min, combined with 7 ~tlof sequencing gel
sample buffer (95~ formamide, 10 mM-EDTApH 8.0, 2-5 mM-Tris-HC1pH 8.3, 0.1 ~ xylene cyanol and 0.1
bromophenol blue), heated at 95 °C for 3 min and analysed on 6~ sequencing gels. An oligonucleotide primer
corresponding to the last 17 nucleotides of the 3' end of gene 6 (Estes et al., 1984; Both et al., 1984) was used
empirically to initiate nucleotide sequence extension of all rotavirus strains. Subsequentlyoligonucleotideprimers
18 nucleotides long were used at intervals of about 200 to 300 nucleotides to sequence the plus strand of strains
1076, Gottfried, H-2 and FI-14.
S D S - P A G E and eleetroblotting of virus polypeptides. Polypeptides were analysed by polyacrylamide slab gel
electrophoresis using the method of Laemmli (1970), with 10~ running and 4 ~ stacking gels as previously
described (Gorziglia et al., 1985). Before electrophoresis, single capsid particles were dissociated by incubation at
100 °C for 7 rain in Laemmli sample buffer, with or without a final concentration of 5% 2-mercaptoethanol or by
incubation at 37 °C for 30 min in the same buffer, with or without 2-mercaptoethanol. Samples were loaded in
duplicate and followingelectrophoresis half of the slab gel was fixed in 7 ~ acetic acid-30 ~ methanol. Bands in
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Sequence analysis o f rotavirus gene 6
1661
the slab weredetected by staining with CoomassieBrilliant Blue R-250.The other half of the gel was soakedfor 60
min in transfer buffer, containing 20 mM-Tris-HC1, 150 mM-glycineand 20~ methanol. The polypeptides from
the gels then were transferred electrophoreticallyonto nitrocellulosemembranes (Schleicher& Schuell)for 14 h at
50 V in a Bio-Rad Trans-Blot unit.
Immunological detection ofpolypeptides on nitrocellulose. After electrophoretic blotting, the polypeptides were
detected using a commercial enzyme immunoassay kit (Bio-Rad Immunoblot; goat anti-mouse, horseradish
peroxidase). Monoclonalantibodyraised against FI-14VP6 whichrecognizesa rotavirus groupA-common(4B5),
FI-14-specific(4F12), subgroup I (6C10)or subgroup II (5F5) epitope and monoclonalantibodyraised against Wa
subgroup II (631/9) were used as first antibody in this assay (Hoshinoet al., 1987a; Greenberg et al., 1983). As a
second antibody we used goat anti-mouse IgG-horseradish peroxidase conjugate.
RESULTS
Gene 6 nucleotide sequence
Of gene 6 of rotavirus strains 1076, Gottfried, H-2 and FI-14 98-75 ~o was sequenced directly
by the dideoxynucleotide method using synthetic oligonucleotides to prime cDNA synthesis
from m R N A (Fig. 1). The remaining 1.25~ represents the 17 nucleotide long primer used to
initiate the primer extension. The sequence of this primer corresponds to the 3' end of SA-11
gene 6 (Estes et al., 1984; Both et al., 1984). We chose this sequencing method because it is rapid,
reproducible and specific. Also, this method provides the advantage of sequencing a
representative population of the specific m R N A rather than dealing with a single m R N A
sequence through the cloned cDNA. One artefact observed is premature termination (bands in
all sequencing lanes); however, carrying out the reverse transcriptase reaction at 50 °C or using
different primers helps to eliminate this problem. The gene 6 of three strains, 1076, Gottfried
and FI-14, contains 1356 nucleotides, while the fourth strain, H-2, has six additional
nucleotides, CCACCA, inserted at position 911. The gene 6 of the rotaviruses previously
sequenced, i.e. simian strain SA-11, bovine strain RF and human strain Wa, possesses a 5'
terminal non-coding region of 23 nucleotides which precedes the first A U G codon. This codon
initiates the only long open reading frame which in these instances codes for a polypeptide of 397
amino acids. The sixth gene of each of the four rotaviruses analysed in this study also has a 5'
untranslated region of 23 nucleotides preceding the A U G which initiates the long open reading
frame. This long open reading frame also codes for a protein of 397 amino acids in the case of
strains 1076, Gottfried and FI-14, while the corresponding protein of strain H-2 is 399 amino
acids in length. Moreover, H-2 possesses a second potential initiation codon at residues 132 to
134, within a sequence favourable for translation, i.e. C A A A U G G . This second in-phase A U G
could initiate a polypeptide of 363 amino acids. A single termination codon is present at
nucleotides 1215 to 1217 in the VP6 of strains 1076, Gottfried and FI-14 or at nucleotides 1221 to
1223 in the case of strain H-2. The termination codon is followed by a 3' terminal untranslated
region of at least 125 nucleotides in each of the four strains sequenced. The actual length cannot
be determined by this sequencing method since the 3' 17 nucleotide long primer region was not
actually sequenced.
Amino acid homology among subgroup I, subgroup II, subgroup I and II, and subgroup non-I/H
rotaviruses
Comparison of the deduced amino acid sequence of VP6 of the four rotavirus strains
determined in this study as well as that of three rotaviruses analysed in previous studies indicates
that seven regions are completely (residues 8 to 29, 63 to 74, 176 to 204, 226 to 238, and 316 or 318
to 326 or 328) or highly conserved (residues 122 to 171, 371 or 373 to 397 or 399) (Fig. 2).
We observed a high degree of homology (95 ~ or more) among the subgroup I and subgroup
non-I/II strains (1076, SA-11, RF and H-2) and a somewhat lower homology (90 to 93~o) of these
strains with the subgroup I and II and subgroup II strains (FI-14, Wa and Gottfried) (Table 1).
The VP6 of the subgroup I and II rotavirus strain FI-14 was most homologous (94~) to the two
subgroup II strains, Wa and Gottfried.
There are 18 positions in VP6 among the subgroup I and subgroup non-I/II viruses at which a
specific amino acid is conserved, while a different amino acid is conserved at each of these 18
positions in the protein sequence of the subgroup I and II and/or subgroup II rotavirus strains.
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]
1076
F[-14
I
1076
Fl-14
C .....
C.
C ........
T ........
-T .................
...........
G ......
G ......
A .....
A .......
A- -C ....
G .....
T .....
. . . . . . . .
C-AC .....
ATC ....
C .....
G .....
AT- -G ........
. . . . . . . . . . .
C-G ........
T-G
C-AC
C- -C .....
A .......
A .......
A- *C----AT-
A .......
T ..............
C ........
G-*G-
C ........
-C--TT-G
....
C--C
C--C
........
.............
-AT ....
-C--AT
A- -C-
C- -A-
C--A-
-C .....
G" "C
AT ...........
AT ...........
- -A-H-C
C ........
AT .......
- -GT .......
AT .......
. . . . . . . . . . .
G" "C . . . . . . . . . . .
AT ...........
T . . . . . . . . . . . . . .
G-
--A
TG----A
GTTC .......
AT .....
AC ......
........
G--G
........
G- - -GTT-
-G ....................
GTT ..........................
G- - -GTT ..............
C ......
T ............
A .......
A .......
T ......
C ....
T ............
G ...........
C .............
G-TC ....
G- -C ........
...........
....
C--G
.....
.....
TC-AC-A---T
T- --C----CT
TC-A--A--C
C ...........
.............
C- -A--G
A--A
.....
.....
........
A- -CT ....
T ....
CA-A .....
TATA--GT
A- -CT-G---A-AA--
T .......
....
.....
-C .....
A--C
G--A-
....
.....
A ........
.....
-A-
GA-A-
-A-
A-A--G--T--
GA-G-
-T
-T-
-C--
..........................
C- -G- -G .....
C--T
C .....
. . . . . . . . . . . . . . . . .
-T ....................
-CACA .....
-ACA--T
-CACT-
-ACA--T
A .....
-G- -A .....
A .....
G-A-
. . . . . . . . . . . . . .
T .....
.....
T .....
T" -A*
T- -A-
A- -A-
A .....
A- -A-
A .....
-GT-
- -T-
-A .....
-G-A
-A .....
........
AC .....
C ....
..............
T ...........
.................
-G-G-
- -AC ....
A--C--G--G
-A-A-
A--C--AC
-GT-GA-A-
- -T-CA-A-
G--T
A .....
G ......
A .....
.....
.....
G .......
.....
"T-
-G .....
A" "T--G"
A- -T-
.....
G--C"
C- -C- -C ........
"T ..................................
T .....
T--C
........
l"-T
A-T--T
T--A
........
........
A- -A .....
C- -C . . . . . . . . . . . . . . . .
C .....
"C ........
G. . . . . . . . . . . .
T . . . . . . . .
T--T--G
-T . . . . . . . .
A--T
C ........
..............
C .......
C--A
G- -A-
.....
. . . . . . .
T--T
T .....
T ......
G .....
A .....
.....
l .....
T--T
.....
.....
A .....
G ...........
T . . . . . . . . . . . .
G--G--G
C--A
T .......................
T .....
C ....
T .....
A ........
. . . . . . . . . . . . . .
G .....
G .....
G ........
A ......
A .....
T .....
T" -A-
T- -A-
- -CC-
-A-
....
......
A--T
.....
.....
.............................
A--T
A .................
A .................
C ......
........
G ...........
G ....
G--C
....
T ...........
T ........
T .....
C--C-
T .......
C--C
.....
.....
A ............
G .....
G- - -C-Y-
.....
-G ........
...........
~---C-C--G--G
G- - -C-T
G .....
..............
--G ..............
T ......
G--G--A--T
G--C-T-
G ....
AC- -C ........
---A---C
.....
T ........
-T--A
T ...........
-C--G-
-A-
G- -T .......
G .....
T--T---A---
C--C
..........................
G- -T ........
A--T
C--T
C- -T-
C--T
.....
T .....
.....
C--GT--
........
G- -T-
.....
.....
A- -C-
---T
---T
[ ]
1|
........
........
GC'A-
"C ...........
G- -A .............................
G .....
C ........
T- -T-
T .....
C ...........
-G- -T ........
T .....
-C-
C ........
C ...........
T---C
T" --C
..........
.............
C ........
C ........
G--G"
G ........
-GT-G
TT-GT-G
"T"
........
........
A .....
G--A
A--T
T ....
T-
-T-
T ....
A .....
A- -T-
A .....
-
T-
840
T ....
T-G--
C-T-
C-T-
C-TC-
A--T-AT-
.....
A ........
.....
-G ........
..............
G--C
......
T" "G ......
.........
700
C
A
T- -A
T---C-TC-G ......
.....
-T- -T ......
-C--A--T--T
G--T--A-
-GT-G
- -A- - - -G- -T .....
.....
.....
G--C
G--G--T---T-G
........
T--GA-T
C*-C--G
T--C--G
ACCAGATGCAGAAAGGTTTAGTTTTCCAAGAGTAATTAATTCAGCAGACGGAGCAACAACATGGTTTTTTAATCCAGTAATTTTAAGACCAAATAATGTAGAAGTAGAATTCCTACTTAATGGACAGATAATTAACACAT
C ........
......
]
1076
F1-14
GottfrJed
G--A
T ........
C .......
-A--T
T .....
-T-
-T-
A--T
C--A
T--G
. . . . . . . . . . .
G-"A .............
G--A-C
T- -A-
. . . . . . . . .
A .....
GA .....
- -TGT-G-
-A-A-TGT
T ......
T .....
-G- "CC'GA'A'T
-G- - -C-
T- -G-C-
A--G--CC--A-A-T
C ....
A. . . . . . . . . . .
560
C--A
C .......
-- -G .....
---G
G-
420
T--
T--
T--
T- -
--
28O
C . . . . . . .
C ..........
GCT- - -G- -A ..........
GGCT---G--A-C--G
CGCT- - -G- -A-
GCT---G--A-
G ...........
G ........
.....
T-T---T--
C .............
. . . . . . . . . . . . . . . . . . . .
A ....................
A
A- -C .......................
G ......................
G-A .........
.....
.....
AT .....
C-T-AT
G--CC-C-AT
G ........
G ......
.....
G- - -A-T
G- --A-T
G- --A-T
G
140
GG-'A-T
CC-T-AT--G--T
T" "C" " G - - G
........
T ........
T ........
........
G .......
G .....
"T .................
C ............
A .....
-G--T
A .....
T- -A-
T- -A .....
C- -C--C
A ...........
CT-G .................
-G- -G- -A-CC
C-T .....
A---T-T--CA-A---T
C--GC
G--T
T- -A .....
T .....
. . . . . . .
C- "TC'T-
"TC
TC ....
TC ....
TC ....
TC ....
C .................
A''C-
A .....
A .....
.....
A .....
..........
....
........
. . . . . . . . . . . . .
AT ................
AT
A--T--TC-AC-A--CT
A .....
A
A .....
A .....
A .....
T-A .........
.....
A ........
C--A--A
............
T .......................
T--C
T . . . . . . . . . . . .
G- -T
G. . . . . . . . . .
GA-A-AG-
T . . . . . . . . . . . .
G ...........
-T . . . . . . . .
T- -T ........
-T-
.......................
T .....
C--G
...............
......
I
]
............
........
RF
Wa
....
......................
........
T ..............
G .............
T ................
..............
and
T ........
C ........
C--A--C--CT
ATGTGGCTAAATG•AGGATCAGAGATACAAGTCGCAGGATTTGAT•ATTCATGTGcTA•TAACGcTCCAGCAAATACACAACAATTTGAACATATAGTTCAACTTAGACGGGCAcTAACAACTGCAAcAATAACTATATT
......
I I
non(I/If)
G--T
T
T .....
T .....
C .....
T ....
SA-11
H-2
A . . . . . . . .
C ..............
C ................................
T .......
II
| |
I
RF
|I
......
........
......
]
and
-T-
. . . . . . . . . . . . . . . .
.......
non(]/||)
Gottfried
Ua
T-G ........
T .................
G- -T ........
C ..............
•AGAATATATAGAAAACTGGAATTTGCAAAATAGAAGACAA•GTACTGGATT•GTGTT•CATAAA•cAAACATTTTTCCATATTCTGCTTC••TTACTTTAAA•AGATCACRA•CAcTGCATAATGATTTAATGGGAACT
H-2
SA-11
| I
11
C .....
G .....
....
I
and
Wa
G-C-
. . . . . . . . . . .
Fl-14
-T
1076
fried
......
-G .....
Gott
.....
G .....
G .....
. . . . . . . .
A ........
A
C- -A ........
A .....
-TG-G-
-T--TG
G-A--T--TG
CG-G-
. . . . . . . . . . . . . . .
.......
II
T ......
]
I)
......
CT-G ........
T ...........
ATATTTTATTGACTTCATAGACAATGTATGCATGGATGAAATGACAAGAGAATCACAAAGAAATGGAATTGCTCCTCAATCCGATGCACTACGTAAACTATCTGGTATTAAGTTTAAAAGAATAAATTTTGATAATTCAT
|
|
T .....
T ......
RF
]
] [
1
G .......
A .....
- - -A
G .....
T--C---G----T--TG-G--T
SA-11
H-2
Wa
non(]/|
I
and
1076
] ]
[
Gottfried
l
| )
.................
RF
non(!/I
................
[ I
SA-11
H-2
T--C--G
........
TT-G .....
TT-A
ACTATGAATGGAAATGAGTTTCA•ACTGGA••CATTG•TACATTACCAATTAGGAATTGGACATTTGATTTTGGTCTACTTGGTACAACATTGTTGAATCTAGATGCTAATTATGTTGAAACTGCAAGAACAACAATAGA
..............
....................
.................
...........
XI
and
]
RF
non(|/|!)
......
A .............
............................
!!
............................
.....
. . . . . . . .
........
T--C--G
T--C
T--C
GGCTTTT~C~GTCTT~ATGG~GTTCTATACTC~TTTCA~CGCTTAAAGATGCTAGAGAT~TTGTTGAGGGTACGCTATATTCT~TGTTAGTGACAT~TACAGC~TTC~TC~TCATAGTA
| |
|
Fl-14
Gottfr~ed
Wa
11
............................
............................
............................
| !
SA'11
H-2
Gottfri~
•a
!
1076
FIoI~
and
l
I
SA-11
~n(|/II)
RF
H-2
o
Z
0
0
o
p0
N
b~
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........
-T ......
l
1
I andll
RF
1076
FI-14
1
1076
.....
1076
I
l
and
RF
1076
FI-14
T .....
T" -A .....
...........
.......
G--T .....
G...........
.....
G'-G---A-A
"G ......
G........
A'A
.....
A--C
G- -G ......
............
A' ....
AC-T-
.......
T- - "G-G'GC
.....
-C- -CG- --GC .....
....
T- -C- --T ....
T'-A'''T-C''T
A'-GT
A .....
A .....
T- -T - -A- -GT ....
.....
T .....
T ........
- .............
- ....
A ...........
A ........
A,CA"
-AGC""
.......
.........
"AC ........
G-'A--
C ......
A--TG-A
.....
......
"G-A .....
TG'A .....
"G" "G" -A-'TG'A
A'T ........
A-T-
.....
TA-T ....
TAC" - - "C- "GA'T
GCA'-AC
G- "AGCG-'G
A" - "A"""
A .....
A ........
T .........
T .....
G .....
............................
C- - -A ................
C--GA ................
G'-C--'A
G--T ..............
AC'G .................
"AC"" - "C" "G ...........
........
T- -A ........
........
T''A-'G
T ........
'''-G''"
.....
1120
C" -T
A ...........
T
T
T
T
980
G--Q--
T ...........
A .....
T--T
A" "' ........
CCACCAAATATGACGCCAGCAGTTAATGCCTTATTTCCACAAGCACAACCGTTCCAGCATCAC
" .............
C .....
"C .....
- .............
....
C'AT'G
. .....
. .....
......
......
......
- -A""
* .......
CCCACCA-A-A ......
AC-T''T'--G-C'G'--G--G'''T
'--'--,,-,--'---,---,
G .....
-A-TA
AC-T .....
TG" -- -TC-T-
A--,'-G,
A .....
A .....
TGTCGT TTCAATTAATGCGT
T" "T" -T- -A .....
A- -T .................
G--T
T .....
T,-A-
A .....
C- -A .....
C- -T- -C .....
-C- -T .....
T ...........
G ........
T- -G- *G .....
T .....
A" -G" "T- "G" "G" -0-
A .....
A" "T- "G ..............
A" -T .................
A- -C .....
.....
T- "A" -C ..........
T- -A .....
,- - -T ..........
G- -A- - "T-G--C
C .....
AT .......
C" "C " - C"" "T ..........
T--T
.......
T .......
T .......
..............
G .....
A--T--C
G...........
.....
CA'C""
CA-C-
-T'AT
T. . . . . . . .
"CAG ..............
" "TCAT "CAG .....
A .............................
.....
TAC .....
.........
OTA'A--A
........
GG" -T'A-TT-A
G" " "T'A'TT
......
-A-G ....
G .....
T ......
A .....
, ..................
TG ....................
C" "T ..................
UTA" -T .......
CTA" "T .......
.........
- -A-G-
NNNNNNNNNNNNNNNNN
C G " A G T G A G A G G A T GT GACC
AGTGAGAGGATGTGACC
NNNNNNNNNNNNNflNNN
1362
T-G- "T- -G .........
T'G--T-'G
CTG'CTGA-
CTG-CTCA-
- "A'G'AGTGAGAGGATGTGACC
- - A - G" NNNNNNWNNNNflNPOINN
T C T GA T GGT A T N N N N f l N N N N N N N N N N N N
C-TCA-
C" - A .....
C ...........
CTG-A ..........
-A ..................
G--A ..................
-C" "a-
CTA - A ...........
CTA'A-T
AOTA ..........
C" "'"
A" - -C-T .....
C" "G- "GC'T ...........
GA . . . .
.......
C- - --T .......
CC---T
CA-- -T .......
A- -C ........
1260
G-AC-~T
.....
-°
G-G- -
"AT-T--
G-C----G-,
A-C ....
G'T""
G- -A ...........
G .....
CA-TCT .......
CA . . . . . . . . . . . . .
TA ..........
6A ....
.......
GC-TCT .......
GA----AC-TCT
A'TC"
A-''-T-"
"AG-T"
T--
T T A C A G T G G C T T , C A T T A G A A G C A T GC T A A T C A A A T G = A G G A C C A A G C T A A T T A C T C G G T A T C C T A G T T T G A T A A G
T .....
T CACGTATTCGTACCGCAGAGTAATCAT
G-TGTA-'T-
A .......................
T .....
"G" -T ........
Fig. 1. Comparison of the nucleotide sequence of the VP6 gene of equine FI-14 with homologous genes from equine H-2, simian SA-11 (Estes et al., 1984), bovine
RF (Cohen et al., 1984), human 1076, porcine Gottfried and human Wa (Both et al., 1984) rotaviruses. The sequence shows the DNA strand that corresponds to the
viral plus RNA strand. Underlined bases indicate the positions of the initiation and the single termination codons. N indicates the conserved region used to initiate
primer extension.
C .........
C .........
l l
! I
-T-TA
........
G .....
- "AT- - "C .....
G" TG"" "CT .........
G'TG"
G-T-TA-
T*-T'GC- -T .......
CA-A .............................
A ...........
CG . . . . . . . . . . .
T- -G- -G ......................................
G-'G
-T" -G .....
T ........
GC-T .....
AC'T ......
G--C--TC-G
T ..............
.....
T A T G T A G C T A T G T T A A G C T G CT T G A A C T C T G C A A G T A A G A A C A C G T
C ............
..........
.....
T- -C .....
T-'C
T ..........
- -T-ATACAG
A'CA-C ......
...........
A .....
C .......
C ..........
C .................
C ...............
Got t fried
II
l)
-T- "AC ....
T" "T--T--AC-A--A--C"
T--G-
AATGAAT TGGACTGAAT T GAT TACCAATTAT TCTCCATCAAGAGAAGATAACTTACAACGTGTGT
C ..............
•a
I
I
SA-11
non(I/I
II
Wa
H-2
II
I and I I
T ..............
I
l
RF
C ........
T .....
C ..............
I
Fl-14
A-A .....
.....
TA-A .....
A--C--A--T--GA
..............
TG'C ....................
C--C
G--TA-A
.....
.....
T .....
T .....
"C" -A ......
A ........
G--'T''
A'''T-A''T
- "A ...........
- -A--T
non(l/II)
Gottfried
A- "T ...........
...........
"T ...........
"C- "A--T
,G-A-
-T- -A ..............
A" "A .....
A--G"
h .....
A--A-
G CCA CA G T T GGA C T C A C A C T A C G T A T T ' A T T C ' G C A ' T T T G T GA A T C A ' T C T T A GCG G A T T C A A A T ' A A A C T A T G ' T A G C A A A C ' T GA CA G ' T G T A A GGCAAGAA T A T GCA G TA C CA G T T G GACCAG T C T T 7 C CA C CA G "
- -T'-G--A-
""A .....
--A .....
• "T .....
.....
TA-A .....
SA-11
II
A-A .....
A-A .....
"T- "'--TA'A
-T .....
H-2
Wa
| a M 11
II
]
RF
F|-14
Gottfried
I
SA-11
non(fill)
11
H-2
lI
Gottfried
Wa
.....
A-A .....
--A-A
ACCAAGCACGGTT T GGCACTATAATTGCAAGAAATTTTGACACAATCAGGT
-' ......
SA-11
-T--G-
non(I/11)
!
H-2
0',
1664
M. G O R Z I G L I A A N D O T H E R S
A
H-2
non(Ill,)
SA-11
I
RF
1076
Fl-14
l
I and
I1
"0 ....
L ......................
L ......
- o ....
, ......................
-0 ....
L......................
-o . . . .
c ......................
'
N
.....................
100
~
L ......
.-, ....
1 ........
L......
M-I . . . . I . . . . . . . .
N'''m'''N. . . . . . . . . . . . . . . . . . . . . .
~N'-O--V--V]- .......
~ ......
.,
"
~--o--v.-~
....
'
B
MV| . . . .
" I " " "
I ........
~
~
....................
.......
r"'°v"v)"
" ......................
NEVLYS I SKT CI(DARDK | VE GT LYSNVSO I | QQFV~ [ [VnqGI~EFQTGGI GTLP I~NWTFOFGLLfiTTLLNLOANYVETARITT
Gottfried
l[
......
L......................
~......
. ......
~0 . . . . . . .
.--~- .........................
Wa
II
......
c ......................
c ......
. ......
-~ .......
.--v~ ......................
.......
.......
1EYF IDFI~NVCMDEIq
~ .........
,-+
t .....
~ .......
:--~
.......
C
H-2
non(I/H)
2o0
V ...........
~
...........................
I(--T ....................
SA'11
l
V...........
1-S .....
~ .............................
RF
,
V...........
~-S-l---
I. . . . . . . . . . . . . . . . . . . . .
1076
FI-16
,
V ...........
~-S .....
I. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I
Gottfried
~a
and II
T............
p ........
A-ON . . . . . . . . . . . . . . . . . . . . . . . . .
Y.......
A-DN . . . . . . . . . . . . . . . . . . . . . . . . .
T....................
A-ON . . . . . . . . . . . . . . . . . . . . . . . . .
T ....................
A-DN . . . . . . . . . . . . . . . . . . . . . . . . .
TRE.~DR~G~APQ~iDALRKLS~G~KFKR~FDN~EY'ENWNLQNRRQRTGF~FHKPN~FPYSA~FTLNRSQPLHN~L~GTHWLNAGSE~QVAGF0YSCA|N
II
A ...........
'l
A.......
-IE-F---~ - .............................
V'--~E .....
l ....................
.........................
M-ON
A~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
R'DN . . . . . . . . . . . . . . . . . . . . . . . . .
300
H-2
non(I/[
I)
................
V.......
L ..................
SA-11
I
....
RF
I
................
V .......
L .............
1076
I
................
V.......
L ......................
Fl-14
I and
II
I .......
P---V
.......
T---Y .....
F. . . . . .
[ ..........
14
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
PP-Q
.
V ...............
L .......................................................
T ........
.
Y ............
o.--
I ...................................
..'"
Y................................................
. .--
A~A~T~F~H~VQLRRAL~AT~T~LPDAERF~FPR~AD~LT~WFFNPV~LRPNNVEVEFLLNGQ~N~Y~ARFG~ARNFD~RL$FQLMR~PP
Gottfried
II
....
I ...................
L .......................................................
Wa
II
....
1...................
L .............................................................
V ...............
..--
A--'L .....
..-298
D
H-2
SA-11
non( I/I l)
......
A" ""-~-
,
......
Av--?,
....
. . . . . .
' 1 "
....
....
RF
1076
,
FI-14
' and '1
....
T-A---ls
E
E--I .......
~S--------S~
e r. . . . . . . . . . .
E ..........
,~--L
El
E . . . . . . . . . .
EI. . . . . . . . . . .
E..........
P--El ...........
. . . . . . . . . . .
z99
...................
.....
~ ......
. . . . . . . .
. . . . . .
~ ........
~ .....
,
D ............................
.............
0 ...............
. . . . . . . . . . . . .
D . . . . . . . . . . . . . . . . . . . . . . . . . .
, .............
, ............
v-
0 ............................
N~AVNALF~AQ~FQ~HHATVGLTLR~SAVCE~LA~s~NETMLA~VTA~/RQE~AV~VGPVFPPGMNWTEL%T~YSP$RE~NLQRVFTVAs'R~L|K
Gottfried
I[
......
Wa
1'
..........
0-'"
1 ......
I. . . . . . . . . . .
E ..........
~---L-
"; . . . . . .
]...........
E..........
A~'--L . . . . . .
.....
I. . . . . .
l ..........................................
t. . . . . .
I ..........................................
397
Fig. 2. Comparison of the amino acid sequences of VP6 from seven different rotaviruses representing
four different subgroups (in roman numerals). Simian SAq 1 (Estes et al., 1984), bovine RF (Cohen et
al., 1984) and human Wa (Both et al., 1984) are presented for the purpose of comparison. Boxes A to E
are regions within a frame of six to 12 amino acids, where rotaviruses with subgroup non-I/H specificity
and both subgroup I and II specificity share two or four amino acids with the strain of subgroup I and II
specificities.
T a b l e 1. Percentage amino acid (or nucleotide) homology in VP6 among three subgroup L two
subgroup 11, one subgroup non-I/H and one subgroup I and H animal rotavirus strains
Rotavirus
H-2 (subgroup non-I/II)
1076 (subgroup I)
SA-11 (subgroup I)
RF (subgroup I)
FI-14 (subgroup I and 1I)
Wa (subgroup II)
No. unique amino acids*
H-2
1076
SA-I 1
-
97(85)
-
95(82)
97(87)
-
10
1
6
RF
96(84)
98(88)
96(87)
-
4
FI-14
Wa
90(79)
92(81)
91(80)
91(81)
-
90(80)
92(79)
91(78)
91(80)
94(82)
11
5
Gottfried
(subgroup II)
90(81)
93(82)
93(80)
92(80)
94(83)
98(88)
3
* An amino acid at a given position in the sequence of VP6 which is not shared by any of the other six rotavirus
strains.
A m o n g these positions n i n e a m i n o acids o f the subgroup I or n o n - I / I I are shared w i t h subgroup
II and are therefore unlikely to d e t e r m i n e subgroup specificity (Table 2).
T h e r e are four positions in the V P 6 of rotaviruses b e l o n g i n g to subgroup I, subgroup n o n - I / I I
and subgroup I and II at w h i c h a specific a m i n o acid is c o n s e r v e d (residues 45, 56, 114 and 120).
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Sequence analysis of rotavirus gene 6
1665
Table 2. Conservation of VP6 amino acid sequence among rotaviruses with four different
subgroup specificities : L II, I and H or non-I/H
Amino acid present in rotavirus strains with
indicated subgroup specificity
~k
I or non-I/II
Amino acid
(n = 4)
I and II
(n = 1)
2
7
30
37
39
45
53
56
60
83
86
89
92
101
114
115
120
151
172
174
175
217
225
305 or 307
310 or 312
315 or 317
327 or 329
338 or 340
339 or 341
348 or 350
369 or 371
Asp
Leu
Leu
Met
Ile
Glu
Asn
Ile
Asn
Asn
Asp
Val
Val
Val
Asp
Ser
Ser
Thr
Ala
Asp
Asn
Val
Leu
Ala
Asn
Glu
Glu
Ala
Ser
Ser
Asp
Glu
Ile
Ile
Ile
Val
Glu
Thr
Ile
Thr
Tlar
Glu
Ile
Ile
Thr
Asp
Ala
Ser
Val
Leu
Asn
Asp
Ala
Ile
Asn
Gin
Gin
Asp
Ser
Asn
Ala
Glu
II
(n = 2)
Glu
Leu
Leu
Met
Val
Asp
Asn
Val
Thr
Thr
Glu
Ile
Ile
Ala
Glu
Ala
Ala
Ile or Val
Met
Asp
Asn
Ala
Leu
Asp or Asn
Gin
Gin
Glu
Ala
Asn
Ala
Glu
In contrast, a different amino acid is conserved at each of the four positions in the protein of the
subgroup II strains (Fig. 2 and Table 2). Three of these conserved differences represent
conservative changes and the other is a change from a polar to a non-polar amino acid in the
subgroup II strains. There are six positions in the VP6 protein among the subgroup II
rotaviruses at which a specific amino acid is conserved (Fig. 2, Table 2). There are 14 positions
in the VP6 protein among the subgroup II and subgroup I and II rotaviruses at which a specific
amino acid is conserved while a different amino acid is conserved at each of these 14 positions in
the protein sequence of the subgroup I and subgroup non-I/II rotaviruses. Ten of these
conserved differences represent conservative changes.
The VP6 protein of the subgroup I and II strain, FI-14, contains 11 positions at which unique
amino acids are present. The VP6 of the subgroup non-I/II strain contains ten unique amino
acids, most of which are found between residues 244 and 314. In addition, in this region two
extra prolines are present at residues 297 to 298. The other five rotavirus strains, 1076, Gottfried,
R F , W a and SA-11, contain one and three to six amino acids respectively at a given position
in the sequence of VP6 which are not shared by any of the other six rotavirus strains (Fig. 2,
Table 1).
Immunological detection of native polypeptides
A typical electrophoretic profile of single capsid particles incubated at 100 °C (Fig. 3) shows
four polypeptides with approx. Mr of 120K (VP1), 99K (VP2), 88K (VP4) and 45K (VP6). In
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1666
M. GORZIGLIA AND OTHERS
(a)
(b)
1
2
3
4
1
2
3
4
VP1
VP2
VP6
Fig. 3. Effect of heat and reduction on VP6 present in single capsid particles of subgroup I SA-I l
strain. (a) PAGE analysis of rotavirus SA-11 single capsid proteins stained with Coomassie Brilliant
Blue. Single capsid particles were incubated at 37°C without (lane 1) or with (lane 2) 2mercaptoethanol, or at 100 °C without (lane 3) or with (lane 4) 2-mercaptoethanol. (b) SA-11 viral
proteins identified in (a) were electroblotted to nitrocellulose paper and reacted with monoclonal
antibody 6C10 which recognizes subgroup I specificity. Mr of the proteins is indicated on the left.
1
2
3
4
5
6
7
8
140K
45K
Fig. 4. Immunoblot analysis of different forms of V P6 (lanes 1 and 2, H-2; lanes 3 and 4, FI-14; lanes 5
and 6, Wa; lanes 7 and 8, SA-11). Single capsid particles were incubated at 37 °C (lanes 1, 3, 5 and 7) or
100 °C (lanes 2, 4, 6 and 8) in sample buffer containing 2-mercaptoethanol. Polypeptides were separated
by 10~ PAGE, electroblotted to nitrocellulose paper and reacted with monoclonal antibody raised
against VP6 of strain FI-14, which recognizes the common epitope (Hoshino et al., 1987). Mr of proteins
is shown on the left.
contrast, i n c u b a t i o n of single capsid particles of strain H-2, W a or S A d 1 at 37 °C, with 2m e r c a p t o e t h a n o l , resulted in a decrease in i n t e n s i t y of the 4 5 K b a n d ( m o n o m e r i c VP6) a n d the
a p p e a r a n c e of a high Mr b a n d of approx. 140K (Fig. 3, 4) w h i c h corresponds to the trimeric form
o f VP6 (Gorziglia et al., 1985; S a b a r a et al., 1987).
Fig. 3 shows the reactivity of a m o n o c l o n a l a n t i b o d y specific for a subgroup I epitope with the
blotted SA-11 VP6 polypeptides derived from single capsid particles of SA-11 strain rotavirus
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Sequence analysis o f rotavirus gene 6
1667
incubated at 37 °C (lanes 1 and 2) or 100 °C (lanes 3 and 4). The VP6 oligomer was antigenic in
either its unreduced or reduced conformation, indicating that disulphide bonds were not
required for the integrity of the subgroup I epitope recognized in this assay. Smear bands
detected in Fig. 3(b), lanes 1 and 2, would be explained by the high sensitivity of the
immunoblotting assay; such bands could correspond to different configurations of the trimer.
Monomers of VP6 expressed in baculovirus were detected with subgroup I monoclonal antibody
(Estes et al., 1987). Our data differ from those of Estes et al. (1987) in that we do not detect
binding of subgroup I monoclonal antibodies or subgroup II (data not shown) antibodies to
virion VP6 monomers. Thus when expressed in insect cells infected with baculovirus
recombinants, VP6 monomers possessed an immunoreactivity different from virion VP6
monomers.
Monoclonal antibodies that react with an epitope common to all mammalian rotaviruses
recognized both the reduced and unreduced forms of p45K and the pl40K in all the rotaviruses
studied (Fig. 4). Immunodot blot assay of the viral proteins treated in the same way as the
electrophoresis samples yielded the same results (data not shown).
DISCUSSION
A high degree of VP6 amino acid homology among simian rotavirus SA-11 (subgroup I),
bovine rotavirus RF (subgroup I) and human rotavirus Wa (subgroup II) has been observed in
previous studies (Both et al., 1984; Cohen et al., 1984). In the present study these relationships
were confirmed and extended by sequence analysis of the VP6 genes of four additional rotavirus
strains, human rotavirus strain 1076 (subgroup I), porcine rotavirus strain Gottfried (subgroup
II), horse rotavirus strain H-2 (subgroup non-I/II) and horse rotavirus strain FI-14 (subgroup I
and II).
A comparison of the deduced amino acid sequence of the sixth gene product among
rotaviruses with four different subgroup specificities indicated that the subgroup II strains were
most closely related to each other and the subgroup I strains also formed a closely related family.
The subgroup non-I/II strain was more closely related to the latter strains, while the subgroup I
and II strain was more closely related to the former strains. The observed conservation of amino
acid sequences among the different subgroups of rotaviruses occurred independent of serotype
and host species. This suggests that the diverse rotaviruses which have been studied may have
acquired their sixth gene by reassortment (Hoshino et al., 1987b; Midthun et al., 1987).
The VP6s of the two equine strains (H-2 and FI-14) contain the highest number of unique
amino acids (10 to 11) suggesting that from an evolutionary point of view the VP6 of these
strains is more distant from that of the other rotavirus strains. Hoshino et al. (1987a) have
suggested that FI-14 strain has a "proto' VP6 gene. The nucleotide sequence suggests that the
VP6 proteins of different subgroups and strains within the subgroups represent divergent
evolution from a common progenitor: thus (i) the length of VP6,397 amino acids, is similar in all
subgroups, except subgroup non-I/II rotavirus, which contains 399 amino acids, (ii) several
regions of the VP6 are highly conserved and (iii) a cysteine residue is present at the same three
locations in each of the VP6s.
It is now well known that the major inner capsid protein contains domains which specify the
common and subgroup antigens (Hoshino et al., 1987a). Recently, Pothier et al. (1987) used a
competitive binding assay to identify three non-overlaping antigenic sites common to all
rotaviruses. Monoclonal antibody 4B5, against the FI-14 VP6 epitope, which recognized all
other strains tested (i.e. common epitope or conserved regions of VP6) reacted in
immunoblotting with both the monomeric and trimeric forms of VP6. Moreover, preliminary
results, arresting mRNA gene 6 translation at predetermined sites by oligodeoxynucleotides,
indicated that monoclonal antibody 4B5 immunoprecipitated a polypeptide that corresponds to
the first 80 amino acids of VP6 (data not shown). These results as well as the immunoblot ELISA
reactions observed with partial digests of VP6 by Sahara et al. (1987) suggests that common
epitopes of VP6 are continuous determinants. By contrast, the immunoreactivity of subgroup
specificity appears to be complex. VP6 monoclonal antibodies with subgroup I or subgroup II
specificity reacted with single capsid particles and with the trimeric, but not the monomeric,
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M. G O R Z I G L I A
AND OTHERS
form of VP6. This suggests that the oligomer is the native conformation of VP6 in the virion and
that these subgroup-specific antigenic sites are dependent upon interaction between VP6
monomers for their mature configuration.
In an attempt to predict the region most likely responsible for subgroup II specificity we noted
that eight amino acids clustered in regions B (Thr 83, Glu 86, Ile 89 and Ile 92), D (Glu 310 and
Glu 315) and E (Asn 339 and Ala 348) are conserved in FI-14 and subgroup II rotaviruses.
However, six additional amino acids shared among these rotaviruses are scattered in other
regions of VP6, and these may also be involved in subgroup II antigenic specificity (or
specificities). In contrast, there are two clusters, A (Glu 45 and Ile 56) and C (Asp 114 and Set
120), among the subgroup I viruses, in which four specific amino acids, two in each region are
shared with the FI-14 strain.
Whether or not the different conserved regions contribute to the various subgroup-specific
antigenic sites cannot be decided at present; however, since most of the amino acid changes
found among subgroup I, subgroup II and subgroup I and II strains are clustered in five different
regions, expression and site-directed mutagenesis of these regions of gene 6 may allow us to
elucidate the currently recognized antigenic determinants on VP6.
We express our thanks to A. Buckler-White for preparation of oligonucleotide primers. W e also t h a n k Ms
Sandra C h a n g and Ms Linda Jordan for editorial assistance in the preparation of this manuscript.
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