Comparative sequence analysis of rotavirus genomic segment 6--the gene specifying viral subgroups 1 and 2.

(1)

Comparative Sequence Analysis

of

Rotavirus

Genomic Segment

6-the Gene

Specifying Viral Subgroups

1

and

2

GERALD W. BOTH,1 LINDA J. SIEGMAN,' A. RICHARD BELLAMY,2 3* NOBUKO IKEGAMI,4 AARON

J. SHATKIN2 AND YASUHIRO FURUICHI2

Division of Molecular Biology, Commonwealth ScientificandIndustrial Research Organization, North Ryde, NewSouth

Wales, 2113Australia1; Roche Instituteof Molecular Biology, Roche Research Center, Nutley, New Jersey 071102; Department ofCell Biology, Universityof Auckland, Auckland, 1, NewZealand3;and Clinical ResearchInstitute, Osaka

NationalHospital, Osaka, 540, Japan4

Received2February 1984/Accepted 22 March 1984

Cloned DNA copies of rotavirus genomic segment 6 from simian 11 (subgroup 1) and human strain Wa (subgroup 2) rotaviruses have been usedtodetermine thenucleotidesequencesof thegenethatdetermines viral subgroup specificity. Both genomic segments are 1,356 nucleotides in length andpossess5'- and 3'-terminal untranslatedregions of 23 and142nucleotides, respectively. The inferred amino acidsequencerevealsVP6 to beapolypeptide of 397 amino acids in whichmorethan 90% of the amino acidsequenceisconserved between the two viruses. Thereare34 amino acid changes between the subgroup 1 and 2 polypeptides,mostclustered in threeregions of themoleculeatresidues 39through 62, 80 through 122, and 281 through 315.

Rotaviruses,

members of the family Reoviridae, are im-portant causative agents of severe infantile

diarrhea

in children andanimals (10, 12). Therotavirusgenome consists

of eleven segments of double-stranded RNA(dsRNA) that areenclosedin adouble-layeredprotein shell typical ofmost

membersof the family Reoviridae(14).However, unlikethe

closely related reoviruses, rotavirus

preparations

derived from infected cells or from diarrheal stool samples also

includeparticles with asingle-layeredproteincoat(10, 12). Thesesingle-layered particles have also beentermed"rough particles," "dense particles," and "cores" (1). They are

readily prepared from whole virions by removal of the

proteins of the outer shell, and like reovirus cores, they contain an active virion-associated transcriptase (9). The

single-layered particles, therefore, are most conveniently referred to as cores. The modified single-shelled rotavirus

particles prepared by treatment with calcium chloride and

termed cores (1) are better described as spikeless cores

since, like alkali-treated reovirus cores (30), they lackboth

themajor peripheral protein anddetectable RNA

transcrip-taseactivity (1).

Thepresenceof bothcoresandwhole virionsinrotavirus preparations used for initial serological studies created

un-certainty concerningtheidentity of the antigenically

impor-tant rotavirus surface proteins. The two sets of

surfTace

proteins on cores and virions are distinct (10, 12) and, perhaps predictably, induce different antibodies when a

mixture ofparticles is used to prime the immune system.

Consequently, the serological classification of rotaviruses

recently has required modification (16, 27): separate sub-group and serotypic antigens havebeendefined (16, 27, 28, 32, 33) which are located on the core and whole virion, respectively.

There are at least four serotypes of human rotaviruses (33), theserotypicdeterminantbeingVP7, a

35,500-molecu-lar-weight glycoprotein that is present among the outer

proteins oftheintact virion(10, 12). VP7 has been identified asthe translation product ofgenomicsegment 9 (4, 15), and theprimaryamino acid sequence of simian 11 VP7 has been

*Correspondingauthor.

97

deduced fromthe nucleic acidsequence ofa clonedcDNA copy ofthe dsRNA segment(4).

Lessbiochemicalinformationisavailablefor thesubgroup determinant, whichhasbeenidentified bygenetic studiesas VP6(15), a42,000-molecular-weight non-glycosylated poly-peptide that is coded for by genomic segment 6. Two

rotavirus subgroups havebeenidentified, principallyonthe

basis of the immune adsorption hemagglutination test (16)

butalso in a morerefined fashion byuseoftwomonoclonal antibodies (11) capable ofdistinguishing subgroup 1 and 2

strains. Most rotavirus isolates so far examined with these

monoclonal reagents fall into one of these two subgroups.

However, since somemonoclonal antibodiesreactedwithall

viruses studied, the VP6proteins must also share common

epitopes. This conclusion is supported by the ability of polyclonal antiserum that has been raised against a single

rotavirus type to detect most other rotavirus strains. The

exception is therecently defined pararotaviruses (2), which have different nucleic acid sequences and are

serologically

distinct (22).

The availability of cloned DNA copies of rotavirusgenes nowenables therotavirusgroup andsubgroup determinants

to be further characterized. Here we report the complete nucleotidesequenceofthegenomicsegmentcoding theVP6

of simian 11 rotavirus (subgroup 1) and human strain Wa

rotavirus (subgroup 2). The aim ofthis work has been to

identify thoseregions ofthe VP6molecule whicharesimilar inprimary aminoacid sequence(presumablyresponsible for cross-reactivity) and those regions of the polypeptide that

contain amino acid substitutions which could form the

subgroup epitopic determinants.

MATERIALS ANDMETHODS

Virus and viral RNA. Simian 11

(SAl1)

rotavirus was

obtained from I. H. Holmes, Department of

Microbiology,

University of Melbourne, Australia. Human strain Wa

rota-virus was obtained from H. B.

Greenberg,

Laboratory of InfectiousDiseases, NationalInstitutesofHealth,Bethesda,

Md. Both viruseswere

propagated

in MA104 cells

(26),

and

genomic dsRNAs were extracted from

purified

virions as

previously described(13, 26).

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(a)

--.

4

0-b

(b)

=:-4

FIG. 1. Strategy ford cloned DNA copy of dsR Wa rotavirus. The numb

102.

The sequences w4

mRNA

(OI)

(4,24),by the the dideoxy method afte

BamHI

site of phage

ml:

Molecular cloning. S were applied to the m derived from each viru N. Ikegami, A. Nom4

publication). A library 13) was screened forrn

copy of genomic segm

from segment 6dsRNY amide gel electrophore in this fashion were cc

by using a modified N Nucleic acidsequenci the two cDNA clones three methods

descrit

strategy used is outline

the SAl clone was strands of the

moleculc

dent methods. The st

determined, principall region between residi Maxam-Gilbert

sequer

residues commencing

sequence found fort

region of the sequence on the messenger RN from Wa cores

(4,

19).

Nucleotide sequence

sents the completeseqi of SAl rotavirus

tol

found for human strain

the correct number of'

._.,.__ _ found in the Wa clone must have resultedfrom an in vitro

mutation which presumably occurred during reverse tran-+-u *-E 4-~-U

scription

of the cDNA. Point mutations of thistypehavealso been found to occur occasionally duringthe in vitro synthe-sis of influenza virus cDNA (7). The two nucleic acid

_

812

8 sequences are 78% homologous with base changes located

throughout the molecule. However, 91%of the amino acid sequence is conserved because many of the base substitu-tions in the nucleic acid sequence are located in the third position of the altered codons.

Both genomic segments possess a 5'-terminaluntranslated

region of 23 nucleotides that precedes an initiator codon at residues 24 through 26, resulting in a single open reading frame that codes for a polypeptide of397 amino acids. The other two reading frames contain multiple termination co-dons, the longest product (28 aminoacids) being initiated at

_

, . residue 28,the site ofan

ATG,

which would be

only

aweak initiator (17). The single termination codon at nucleotides

1215

through

1217 is followed by a

3'-terminal

untranslated

8 12 region of 142 nucleotides. The termini of both genomic segments exhibit conserved nucleotide sequences at the 5'-etermining the nucleotide sequence of the (5' GGCTTT 3') and 3'-(5' ATGTGACC 3') ends of the NA genome segment 6 of (a)SAl and (b) molecules. These sequences have also been found for other ers refer to the distance in nucleotides x

ere determined by copying cDNA from

rotavirus

genomic segments (3, 4, 6, 14), indicating thatfor Maxam and Gilbert method (U) (20), or by genomic segment 6, the cloningprotocols used have yielded r subcloning

Saou3Al

fragments into the

full-length

copies

ofboth viral RNAs.

3mp8

(0) (21, 24). The information presented in Fig. 2 also enables the dsRNA segments to be searched for the consensus sequence that surrounds the translational initiation site on the

messen-tandard molecularcloning techniques ger RNAs of eucaryotes (17). For the ATG thatprecedes the ixture of eleven segments of dsRNA open reading frame, a purine at position -3 has been

Is(3, 5, 13; Y. Okada, M. Richardson, conserved for both rotavirus mRNAswith an A-- G change

oto, and Y. Furuichi, submitted for found between the simian and human sequences. Both of rotavirus genomiccDNAclones (3, sequences contain aG residue at position +4 downstream ecombinant plasmids thatcontained a from the AUG. Neither sequence contains the 3'-terminal

lent

6 by using a cDNA probe copied AAUAAA polyadenylation signal (23) as anticipated from that had been purified by

polyacryl-

the absence of polyadenylate on the messengerRNA of the sis (13, 26). Selected clones obtained Reoviridae (14).

nfirmed as being copies of segment 6 Comparison of the amino acid sequences of the subgroup

orthern hybridization procedure (26). specific genes. Since SAl and Wa rotaviruses are

repre-ing.

Complete nucleotide sequences of sentative members of the two major rotavirus subgroups

were determined by a combination of (11), it is of interest to compare the amino acid sequences

)ed previously (20, 21, 24, 25). The inferred from the

two

nucleic acid sequences. The 34 amino d in Fig. 1. The complete sequence of acid substitutions found for the Wa strain are presented in established first, either from both Fig. 3 in conjunction with the hydrophobicity profile for the or by a combination of two indepen-

SAll

protein that has beencomputed by themethodofKyte equence of the Wa clone was then and Doolittle (18). The amino acidsubstitutionsfound for the y on one strand except for a small inferred Wa protein sequence do not generate substantial ues 750 through 850 in which the changes in the overallhydrophobicity of the molecule, since ice on both strands indicated four T the hydrophobicity plot for the Wa strain is similar to that at position 810 rather than the

(T)5

shown in Fig. 3 for SAl (data not shown). Many of the -he SAl sequence. Therefore, this amino acid changes found between the two subgroup pro-was also checked by primer extension teins are conservative: forexample,the Asp - Gluchanges

A prepared by in vitro transcription (at positions 2, 45, 86, 114 and369) and Ile= Valchanges (at positions 39, 56, 89, 92, 109, 281, 385). Thebasic amino acids Arg, Lys, and His are completely conserved between the RESULTS two sequences. With regard to the location of the amino acid for genomic segment 6. Figure 2 pre-

changes,

ca. 70% of the changes are clustered in three uence of the cloned copy of segment 6

regions

ofthemolecule at residues 39through62, 80 through

gether with the nucleotide changes

122,

and 281

through

315. The significance of the location

Wa rotavirus. Ambiguity concerning and nature of the amino acid changes is of interest as

T residues in the Wa clone at residues discussed below.

810 through 815 was resolved by results obtained whenthis region was also sequenced by primer extension on the

messenger RNA. The sequence determined in this manner yielded the sequence (T)5, indicating that the (T)4 sequence

DISCUSSION

Definitive information on the structural location of the subgroup protein VP6 is not available, but its removal by

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100

A G G T G A C A T G T T A A T T C

GGCTTTTAAA CGAAGTCTTC AAC ATG GAT GTC CTA TAC TCT TTG TCA AAG ACT CTT AAA GAC GCT AGA GAC AAA ATT GTC GAA GGC ACT TTG TAT TCT MC GTG AGT GAT

M D V L Y S L S K T L K D A R D K I V E G T L Y S N V S D

E

200

T G C G A T G T A T G T C C G T T

CTA ATT CM CAA TTT MT CAA ATG ATA ATT ACT ATG AAT GGA AAT GAA TTT CM ACT GGA GGA ATC GGT AAT TTG CCA ATT AGA MC TGG MT TTT MT TTC GGG

L I Q Q F N Q M I I T M N G N E F Q T G G I G N L P I R N W N F N F G

V D V T D

300

C TA T A C TT G T G AT C T A T A T C A T T T A

TTA CTT GGA ACA ACT TTG CTGMC TTA GAC GCT AAT TAT GTT GAA ACG GCA AGA MT ACA ATT GAT TAT TTC GTG GAT TTT GTA GAC MT GTA TGC ATG GAT GAG

L L G T T L L N L D A N Y V E T A R N T I D Y F V D F V D N V C M D E

N T I E I I

400

CA T A T G A G A T A G T G T A G G GA T G C A A

ATG GTT AGA GM TCA CAA AGGMC GGA ATT GCA CCT CM TCA GAC TCG CTA AGA MG CTG TCA GCC ATT AAA TTC AAA AGA ATA AAT TTT GAT MT TCG TCG GAA

M V R E SQ R N G I A P Q S D S L R K L S A I K F K R I N F D N S S E

A V E A A G

500

T C A C C C A T GT T A A C T T T T

TAC ATA GM MC TGG MT TTG CM MT AGA AGA CAG AGG ACA GGT TTC ACT TTT CAT AAA CCAMC ATT TTT CCT TAT TCA GCA TCA TAT ACA CTA MAT AGA TCA

Y I E N W N L Q N R R Q R T G F T F H K P N I F P Y S A S Y T L N R S

V F

600

A ATG A A C C T T T A G C C A T G C

CM CCC GCT CAT GAT AAT TTG ATG GGC ACA ATG TGG TTAMC GCA GGA TCG GM ATT CM GTC GCT GGA TTT GAC TAC TCA TGT GCT ATT MC GCA CCA GCC AAT

Q P A H D N L M G T M W L N A G S E I Q V A G F D Y S C A I N A P A N

M

700

G A C C AG T A G C C CA C G T T A T T T A G A A T

ATA CAA CM TTT GAG CAT ATT GTG CCA CTC CGA AGA GTG TTA ACT ACA GCT ACG ATA ACT CTT CTA CCA GAC GCG GAA AGG TTT AGT TTT CCA AGA GTG ATC AAT

I Q Q F E H I V P L R R V L T T A T I T L L P D A E R F S F P R V I N

Q A

800

T C G C T T T A A A C T A G A A T G A T T T A r,

TCA GCT GAC GGG GCA ACT ACA TGG TTT TTC AAC CCA GTG ATT CTC AGG CCG AATAAC GTT GAA GTG GAG TTT CTA TTG MT GGA CAG ATA ATA AAC ACT TAT CAA

S A D G A T T W F F N P V I L R P N N V E V E F L L N G Q I I N T Y Q

900

T T A C A G A C T T T T G C T T T T T AAT C G T A

GCA AGA TTT GGA ACT ATC GTA GCT AGAAAT TTT GAT ACT ATT AGA CTA TCA TTC CAG TTA ATG AGA CCA CCA AAC ATG ACA CCA GCA GTA GCA GTA CTA TTC CCG

A R F G T! V A R N F D T I R L S F Q L M R P P N M T P A V A V L F P

A L N A

1000

C A A T T C G C T C T TAC T A G A A G T G C A G A

AAT GCA CAG CCA TTC GAA CAT CAT GCA ACA GTG GGA TTG ACA CTT AGA ATT GAG TCT GCA GTT TGT GAG TCT GTA CTC GCC GAT GCA AGT GAA ACT CTA TTG GCA

N A Q P F E H H A T V G L T L R I E S A V C E S V L A D A S E T L L A

Q Q N

1100

G C GG G C T A T C G A C T A T T C G A A A

AAT GTA ACA TCC GTT AGG CAA GAG TAC GCA ATA CCA GTT GGA CCA GTC TTT CCA CCA GGT ATG AAC TGG ACT GAT TTA ATC ACC AAT TAT TCA CCG TCT AGG GAG

N V T S V R Q E Y A I P V G P V F P P G M N W T D L I T N Y S P S R E

A E

1200

T C G T C G A T G G GA G C TC TC A T C C

GAC AAT TTG CAA CGC ATA TTT ACA GTG GCT TCC ATT AGA AGC ATG CTC ATT AM TGAGGA CCAAGCTAAC AACTTGGTAT CCAACTTTGG TGAGTATGTA GCTATATCAA

D N L Q R I F T V A S I R S M L I K

V

1300

TCA CAG TC CAT A T T ATG GT CTGTCT GA A G

GCTGTTTGM CTCTGTMGT AAGGATGCGT ATACGCATTC GCTACACAGA GTAATCACTC AGATGGTATA GTGAGAGGAT GTGACC

FIG. 2. Nucleotide sequence and inferred amino acid sequences ofSAl and Wa rotavirus genomic segments6. Thecomplete nucleotide

and amino acid sequences of the coding strand of theclonedSAl segment arepresented togetherwithnucleotide andamino acid changes

found in the corresponding Wa sequence.

high concentrations of calcium chloride (1), accessibility to Only 34 amino acid changes are found when the inferred iodination (P. Gunn and A. R. Bellamy, unpublished data), amino acid sequences of VP6 proteins of SAl and Wa and marked immunogenicity (10, 12) suggest that this protein rotaviruses are compared. Major amino acid sequence con-probably constitutes the peripheral spikes of the icosahedral servation between VP6 proteins of rotaviruses of the two

rotavirus core. Although immunogenic, VP6 must be almost different subgroups is consistent with the known ability of completely masked in the virion because little or no VP6- antibody primed by one rotavirus to detect most other specific antibodies were formed when rabbits were chal-

rotaviruses

(12), with the exception of the newly defined lenged with intact human, simian, or bovine rotaviruses

(5a).

pararotaviruses (2), which are distinct (22). The

group-The minimal changes in protein sequence found here for specific determinants that enable rotavirus VP6 proteins to the two VP6 proteins implies that variation in this major cross-react probably are located on the regions of the structural protein may be limited by the constraints imposed molecule in which the amino acid sequence is extensively

by the conformational requirements of the rotavirus core. conserved. Incontrast, thoseepitopes that enablesubgroups

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._

2

X:

.2

Z220 240 20O 3U30 34396J 3O0

Amino

Acid Number

FIG. 3. Amino acid substitutions found between subgroup 1 (SAil) and subgroup 2 (Wa) rotaviruses. The amino acid changes are

indicatedby solidsquaresandarelocatedon ahydrophobicity plot of the SAil polypeptide. The SAil amino acidpresentateach position is shownabove thesquareand theamino acidpresentintheWasequenceis shown belowthesquare.Therelativehydrophobicity indexwas

generated by computer, using the method of KyteandDoolittle(18). Analysis of the Wasequence by the samemethodyieldedasimilar

hydrophobicity profile. Horizontal bars indicate the three putative antigenic regions which wouldencompass morethan 70% of theamino acid changes found between the twopolypeptides.

1 and 2 rotaviruses to be distinguished probably reside in threeregions (Fig. 3,horizontal bars) which contain mostof the amino acid changes.

In theabsenceofanyinformationonthetertiarystructure ofVP6, itobviously isnotpossibletoidentifyprecisely the regions of the molecule that constitute the subgroup

epi-topes: widely separated regions of the molecule could be brought into close proximity by folding of thepolypeptides,

as has been shown elegantly for the influenzavirus

hemag-glutinin (31). However, by analogy with the amino acid changes found to occur among H3 influenza viruses (8, 29, 31), the best studied system involving variation in a viral

antigen, the amino acid differencesatresidues 62(Asn-Asp), 213 (Pro-Gln), and 315 (Glu-Gln) could contribute to the antigenic differences between the rotavirus subgroups. Oth-er changes, for example, those located at residues 84 and 172, which arealso likelytohave surface locations in VP6 (Fig. 3) could also contribute totheantigenic determinants. Since most of the amino acid changes found between subgroup 1 and 2 proteins are clustered in a fewrelatively restrictedregions of the molecule, peptide synthesis should provideafeasiblemethod by which thenatureof the epitopic determinantscould be furtherinvestigated. The synthesis of peptides corresponding to putative epitopic regions should enable thegeneration of site-specific antibodiesthatcouldbe

tested for subgroup specificity. Alternatively, blocking

ex-periments could be carried out with selected synthetic peptides and the subgroup-specific monoclonal reagents

already developed (11). Either of these two experimental approaches should in the future enable the major subgroup-specific epitopes to be unambiguously located on the VP6

molecule.

ACKNOWLEDGMENTS

Wethank Jeanette StreetandMark Lin forassistanceinscreening theSA-11androtavirus(Wa)cDNA libraries forgenomic6 clones.

Thisworkwassupportedinpartbygrantsfrom the NewZealand MedicalResearchCouncil and theWorld HealthOrganization.

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D I E I NN TNTD V V v DS SA T

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140

l

16

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-3L 7A A° T AVN E s s D Vl

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