0022-538X/91/073738-08$02.00/0
CopyrightC) 1991, AmericanSocietyfor Microbiology
Rotavirus YM Gene 4: Analysis of Its Deduced Amino Acid
Sequence and Prediction of the Secondary Structure of
the
VP4 Protein
SUSANA
L6PEZ,*
IMELDAL6PEZ,
PEDROROMERO, ERNESTO MENDEZ,XAVIERSOBERON, ANDCARLOS F. ARIAS
Departamento de Biologia Molecular, Centrode Investigaci6n sobreIngenieriaGeneticayBiotecnologia, Universidad Nacional Aut6noma de Mexico, ApartadoPostal510-3, Colonia Miraval, Cuernavaca, Morelos62271, Mexico
Received 28 January 1991/Accepted 15 April 1991
We havedetermined thecomplete nucleotidesequenceof the VP4geneofporcinerotavirus YM. It is2,362 nucleotides long,withasingleopenreadingframe coding foraproteinof 776 amino acids. Aphylogenetictree wasderived fromthededucedYM VP4amino acidsequenceand 18otheravailableVP4sequencesofrotavirus strainsbelongingtodifferentserotypesand isolated from different animal species.In thistree, VP4proteins
weregroupedby thehosts that the corresponding virusesinfect rather thanbytheserotypesthey belongto, suggesting that this protein is involved in the host specificity of the viruses. In an attempt to predict the secondarystructureof the VP4protein,weselected themoredivergentVP4sequencesandmadeasecondary structureanalysisof each protein. Inspiteof variations within the individual structurespredicted,therewas
ageneralstructural pattern whichsuggested the existenceof at least twodifferent domains.One,comprising
the amino-terminal 63% of the protein, is predicted to be a possible globular domain rich in ,-strands alternated withturns andcoils. The seconddomain, represented bytheremaining, carboxy-terminal partof VP4,is rich inlongstretchesofa-helix,oneofwhich,63 amino acidslong,hasheptadrepeatsresemblingthose found inproteinsknowntoform a-helical coiled-coils. Thepredicted secondarystructurecorrelatesweil with the availabledataontheprotein accessibility delineatedby immunologicalandbiochemicalfindings and with
thespike structureoftheprotein, which has beendeterminedbycryoelectron microscopy.
GroupArotaviruses are amajorcauseofacute gastroen-teritis in infants and youngchildren. They are also associ-ated with diarrhea in the young of many mammalian and avian species (28). These viruses contain a genome com-posed of 11 segments of double-stranded (ds) RNA sur-rounded by two layersof proteins. Theouterlayer contains atleast twoproteins,VP4and VP7, whichplayacrucial role in the first interactions of the virus with its host cell (11). Both VP4 and VP7 are able to elicit antibodies capable of
neutralizing virusinfectivity (44, 45). VP7,aglycoprotein of
37 kDa (2, 9), has beenimplicated in the attachment of the virus to the cell (15, 37, 50), and studies with reassortant viruses have shown that the serotype-specific neutralizing phenotype primarily segregates with the gene that encodes thisprotein(21, 24, 53).
VP4, the minor component of the outer capsid, has a molecular mass of 84 to 88 kDa and is encoded by RNA segment 4 (32, 36). Genetic and biochemical data have
shownthatthisprotein is the viralhemagglutinin,and it has been suggested to be responsible for the host specificity of rotaviruses (23, 27, 34). VP4 is also known to influence virus growth in cell culture (21, 22).
Inthe presenceof trypsin, VP4 in the virus is cleaved into VP5and VP8, which results in the enhancement of rotavirus
infectivity(10, 12),probably by promoting virus penetration into cells (5, 15, 25).
Recently, cryoelectron micrographs of the complete un-cleaved virus have shown that the virus contains 60 spikes protruding from the virion (48, 55), and staining of the virus with Fab fragments of monoclonal antibodies (MAbs)
di-*Corresponding author.
rected to VP5 has shown that these spikes mightbe formed by dimersofVP4(47).
Recently, we reported the isolation ofporcine rotavirus YM, which was shown, through its immunological charac-terization and the sequence ofits VP7 gene, torepresenta newrotavirus serotype(3, 49). In this work, wereport the sequenceofthe VP4 geneof this strain,andonthe basis of the deduced amino acid sequence of the encoded VP4
polypeptideand otheravailable VP4amino acidsequences, we present a prediction ofthe secondary structure ofthis
protein.
MATERIALSANDMETHODS
Virus.Rotavirus YM(3, 49)wasgrownin MA104cellsand
purified essentiallyasdescribed by Espejoetal. (10). Molecularcloningand nucleic acidsequencing.The unfrac-tionated 11 dsRNA segments derived from rotavirus YM were cloned essentially as described by Arias et al. (1). Briefly, the total viral dsRNA was polyadenylated with
poly(A) polymerase, and oligo(dT) was used as primer for
the reverse transcriptase reaction. EcoRI linkers were
li-gatedtothe resultantdscDNA,whichwaspreviously meth-ylated with EcoRI methylase. After EcoRI digestion, the dscDNA was ligatedinto the EcoRI site ofplasmidpMT21 (1). Transformant, ampicillin-resistant colonies, containing
hybrid plasmids with cDNA inserts corresponding to YM RNA segment4, were selected by hybridization of random recombinant plasmids labeled by nick translation to YM RNA segment 4 isolated by gel electrophoresis (2). The inserts in thehybridclonesweresequenced by the method of MaxamandGilbert(38). To obtain the sequences of the gene 4 regions that were not present in the selected clones, 3738
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ROTAVIRUS YM GENE 4 3739
FIG. 1. Complete nucleotidesequence ofgene4ofporcine rotavirus YM. The DNAsequenceof the mRNAsensestrandisshown; the
initiation and termination codonsareunderlined.
including the 3' end of thegene,theisolated dsRNAsegment
4 was hybridized with selected synthetic oligonucleotides,
and the sequence was determined by the dideoxy-chain
termination method with avian myeloblastosis virusreverse
transcriptase, asdescribed previously (33).
Construction of the phylogenetic tree. The amino acid sequencesof the VP4proteins publishedtodate(17, 20, 26, 27, 32, 35, 41, 42, 46, 52) were pairwise aligned by the
method of Myers and Miller (39), and the degreeofsimilarity betweenthemwascalculated. Toattain the best alignment, agapofoneaminoacidatposition 136wasintroduced in all
humanrotavirusstrains and the porcine rotavirus Gottfried, since all these strains have 775 residues, one less than the rest of the strains analyzed. In addition, the mutation dis-tances werecomputed by the method of Fitch and
Margo-liash (14). Mutation distances are defined as the minimal
numbersofnucleotides thatareneededtobe altered inorder forthegeneofone VP4proteintocodefor the VP4protein ofadifferent rotavirus strain.
A dendrogram was constructed from the mutation dis-tancesby using the Fitchprogramof the Phylip 3.0package (J. Felsenstein, University of Washington, Seattle, Wash.). This program is based on the algorithm described by Fitch
andMargoliash (14).
Hydropathic index and secondary structure predictions. Thehydropathicindex ofeachindividual proteinwas
calcu-lated by the method of Kyte and Doolittle (29) using a
window of 6 amino acids, and the valuesateachequivalent positionwere averaged. To make an aligned average of all the strains atposition 136, where the Gottfried, Wa, ST3, and RV5 strains contain one less amino acid, the average value ofthe restof the strains was arbitrarily used.
The secondary structure prediction of the selected
se-quenceswasmade with thealgorithms of Gamieretal. (16) andChou and Fasman (4) by usingthe Wisconsin UWGCG package (8).
RESULTS
Nucleotide sequenceofrotavirus YM gene4. The general characteristics of theYM VP4proteingenearethe same as
those reported for homologous genes from other animal rotavirus strains. It is 2,362 nucleotides long, and its 5' (GGCTATAAAA)and3' (TTGTGACC)ends have the
con-served terminal sequences present in other rotavirusgenes
(11) (Fig. 1). It has a single long open reading frame
beginningwithanATG codonatresidue 10andending with
a single stop codon (TAA) 22 bases from the 3' end. The open reading framecodes for aprotein of 776 amino acids,
with apredicted molecularmass of 86,772 Da.
Comparisonbetween the YM VP4proteinandhomologous proteins from animal and human rotavirus strains. The de-duced amino acid sequence of the YM VP4 protein was
comparedwith 18 otherreportedVP4amino acidsequences ofrotavirus strains isolated from differentanimalspecies and belongingtodifferentserotypes(17, 20, 26, 27, 32, 35, 41, 42, 46, 52) (Table 1, upper right portion). The YM VP4 amino acidsequencewas morecloselyrelatedtothatoftheporcine rotavirusOSUstrain, having 97%overall identity, while the lowest homology was with the human strains, ranging from
68.5 to70.7%.
In general, three groups that share >80% homology
among its members were observed. One group is repre-sentedbyallanimalstrains, with theexception ofthebovine UK and the porcine Gottfried strains; a second group is formed by all the symptomatic human strains; and a third
groupcontainsall thenewbornasymptomatichumanstrains plustheporcineGottfriedstrain. ThebovinestrainUKdoes not share more than 75% identity with any of the strains analyzed, although, ingeneral,it seemedtobemoreclosely
related tothe animal strains.
In order to study the genetic relatedness of the VP4 proteins derived from different hosts and/or belonging to different serotypes, we calculated the mutation distances between eachpairof viruses(seeMaterialsand Methods and lower left portion of Table 1), and from these data a
predictedphylogenetictree wasconstructed(Fig. 2). Inthis phylogenetic tree, VP4 proteins weregrouped bythe hosts from which the corresponding viruses were isolated rather than by the serotypes to whichthey belong. There were a
fewexceptions tothisgrouping; theporcine Gottfriedstrain ismore relatedtothe newbornasymptomatic strains thanto
theotherporcine (OSUandYM) strains,and the bovine UK strain is classifiedinabranchseparatefromthe other bovine
sequencesanalyzed. TheVP4proteinofthe SA114fM strain was grouped with the NCDV and C486 bovine strains, in
accordancewithpreviousreportswhich havesuggestedthat
this strainmightrepresentareassortant between bovineand
simian strains(27, 31).
GGCTATAAAATGGCTTCGCT CAATTATAGA CAACTACTTA CTAATTCATA TACAGTCAAT CTTTCTGACG AAATTCAAGA AATTGGATCA GCTAAGGCAC 100 AGAATGTTAC CATAAATCCT GGTCCATTCG CGCAAACGGG TTATGCACCA GTTAATTGGG GGGCAGGTGA GACTAACGAC TCAACAACTG TCGAGCCATT 200 ACTAGATGGT CCATATCAAC CAACCACTTT CAATCCACCT ACGAGCTATT GGGTATTACT TGCGCCAACT GTAGAGGGTG TGATTATCCA AGGAACAAAC 300 AATACCGACA GATGGTTAGC TACTATATTG ATTGAACCAA ACGTGCAAAC GACTAACAGAACATACAATC TTTTTGGTCA ACAAGTAACT CTGTCGGTAG 400
ATAACACATC ACAAACACAA TGGAAGTTCA TTGATGTGAG TAAAACTACG CTGACAGGAAACTACACGCA ACACGGACCA TTATTCTCTA CACCAAAATT 500 ATACGCTGTA ATGAAATTCA GCGGTAGAAT ATATACATAT AACGGAACCA CGCCAAATGC AACAACGGGA TACTATTCAA CTACTAACTA TGACACAGTA 600 AATATGACGT CGTTTTGCGA TTTTTATATT ATACCAAGAA ATCAAGAAGA AAAATGTACT GAGTATATCA ATCATGGATT ACCCCCTATA CAAAATACGA 700 GGAATGTAGT GCCAGTATCT TTATCGGCTA GAGAAATAGT GCATACAAGA GCCCAAGTTA ATGAAGATAT TGTTGTTTCA AAAACTTCAC TTTGGAAAGA 800 AATGCAATAT AACAGAGATA TAACGATAAG ATTTAAGTTT GATAGGACGA TTATTAAGGC TGGAGGATTA GGATACAAAT GGTCAGAAAT ATCTTTTAAA 900 CCAATTACTT ATCAGTATAC ATACACTAGA GATGGAGAAC AGATTACAGC GCACACTACA TGCTCAGTTA ATGGAGTTAA CAATTTTAGT TATAATGGCG 1000 GTTCATTACC GACGGACTTT GCTATATCAA GATATGAAGT GATTAAAGAA AATTCATTTG TTTATATTGA CTACTGGGAT GATTCGCAAG CATTCAGAAA 1100 CATGGTGTAT GTTCGGTCAC TTGCTGCTAA TTTGAATACA GTAACGTGCA CTGGTGGTAG TTATAGCTTC GCCTTGCCTT TAGGTAACTA TCCAGTTATG 1200 ACTGGTGGTA CAGTTACACT ACACCCAGCT GGAGTTACGT TATCTACCCA ATTTACTGAT TTTGTATCTC TTAATTCATT GCGCTTTAGG TTCAGATTAA 1300 CTGTGGGAGA ACCTTCATTT TCTATAACGA GAACTAGGGT AAGTAGATTA TACGGACTTC CAGCAGCTAA TCCAAACAAT CAGAGGGAAC ATTATGAAAT 1400 AAGTGGCAGA CTTTCGTTAA TATCATTAGT GCCATCAAAT GATGATTATC AAACGCCAAT TATGAACTCA GTTACCGTGA GACAAGATCT AGAGAGACAA 1500 TTGGGAGAGC TACGGGATGA GTTCAATTCA TTGTCACAGC AAATAGCGAT GTCACAACTG ATTGATTTGG CACTATTGCC ATTAGATATG TTTTCAATGT 1600 TCTCAGGAAT TAAAAGTACAATAGATGCTG CGAAATCTAT GGCAACAAAT GTAATGAAAAGATTTAAACG GTCAAACCTA GCAAGTTCAG TTTCTACGTT 1700
AACTGATGCA ATGTCTGACG CAGCATCGTC TATTTCAAGA AGTTCATCAA TACGATCAAT AGGATCATCA GCATCCGCTT GGACGGAAGT GTCAACTTCA 1800 ATCACAGACA TTTCTACTAC AGTTGATACA GTATCAACGCAAACTGCTAC TATTGCTAAA CGGTTACGAT TGAAAGAAAT AGCGACTCAA ACCGATGGTA 1900 TGAATTTTGA TGACATATCT GCAGCGGTAT TAAAAACGAA AATTGATAAA TCAGCGCAAA TAACACCAAG TACGTTACCG GAGATAGTTA CTGAAGCTTC 2000
AGAGAAATTT ATTCCAAATAGGACATACAG AGTTATTAAC AATGATGAAG TCTTTGAGGC TGGAATGGAT GGGAAATTTT TTGCATACCG TGTAGATACA 2100 TTCGATGAAA TACCATTTGA TGTACAGAAA TTCGCAGATT TAGTTACGGA TTCACCGGTT ATTTCTGCAA TAATTGATCT CAAGACGTTA AAAAATCTAA 2200
AAGATAACTA CGGAATAAGT AAGCAGCAAG CTTTTGACCT ATTACGATCA GATCCAAAAG TATTACGTGA ATTTATCAAT CAAAATAATC CAATAATACG 2300 AAATAGAATT GAAAACTTAA TAATGCAATG TAGACTGTAA GTAGTGTCTC GAGGTTGTGA CC 2362 VOL. 65, 1991
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TABLE 1---Continued
ST3 1076 MCN13 M37 WA VA70 PR1 KU RV5 DS1
69.9 69.8 70.3 70.7 68.8 68.5 68.6 68.6 69.5 70.1
70.8 70.2 70.7 71.5 69.3 68.9 69.0 69.0 70.1 70.3
72.9 72.8 73.2 73.8 71.1 70.8 71.6 71.2 71.3 72.0
73.4 73.0 73.5 74.7 71.4 71.0 70.5 71.2 71.3 71.7
72.0 71.4 72.1 72.9 71.0 69.9 70.3 71.3 70.8 71.4
72.1 71.7 72.3 73.0 71.4 70.2 70.8 71.5 71.2 71.6
70.5 70.5 70.7 71.6 70.2 68.9 69.6 70.4 69.6 70.2
69.6 71.3 68.7 70.1 68.2 67.6 68.6 68.6 69.0 69.3
87.2 88.1 87.5 88.2 78.1 77.0 77.4 78.8 76.6 76.9
95.7 97.0 96.2 76.7 76.2 76.3 76.5 75.3 75.7
35 96.3 95.0 77.0 76.3 76.3 76.7 75.3 75.7
25 32 96.2 76.6 76.2 76.1 76.3 75.0 75.3
32 42 34 77.8 77.2 76.9 78.1 76.3 76.7
226 226 232 222 96.7 94.0 93.8 89.1 89.6
229 229 218 225 29 93.1 92.3 89.1 89.4
233 236 241 232 48 59 94.1 90.1 90.4
227 227 233 219 49 62 47 90.8 91.2
234 236 241 228 89 92 83 76 98.7
234 236 241 228 88 93 84 76 13
acids that are almost completely conserved between the strainscompared(residues 1 to 14, 223 to 236, 256 to 271, 312 to 331, 339to 376, 407 to 430, 477 to 498, 516 to 539, 626 to 646, 715 to 736, and 751 to 776). All but two of these
conserved regions fall intheVP5 partofVP4. Intotal, there are361positionsamong the strainscompared(46.5%) which haveidentical residues.
Secondary structure prediction. Recent data from
cryo-electron micrographsoftherotavirus particle with a
mono-clonal antibody bound have shown that the VP4 protein is forming the viral spikes (47, 48, 55), indicating that this proteinhas adistinctivefold. Tolearnabout thestructure of
this protein, thesecondarystructure of each oftheselected aminoacid sequencesshowninFig.3waspredictedbyusing
two different algorithms (4, 16). The prediction of each
individual sequence was carried out, since comparison of
homologous proteins from different origins canimprove the
accuracyofthe prediction ofsecondary structures (7, 51). The diagrammatic representation ofthe resulting predic-tions, in which thecontentsof a-helixand
3-strands
foreachindividual protein are depicted, is shown in Fig. 4A. Even
though there are variations in the predicted secondary
structures of each protein, there is an overall common pattern in which the VP4 protein can be divided into two
distinct parts. The first comprises the NH-terminal 63% portion, inwhich mostly
1-strands
were predicted, andthesecond is represented by the remaining carboxy-terminal region ofthe protein, in which ahigh content of
a-helices
werepredicted. It wasinterestingthatstarting atamino acid 494, there is aregion of about 63 amino acids which has a
high potential for a-helix formation andwhich is conserved betweenthe proteins studied.
We analyzed this region looking for the characteristic motif found in sequences with a propensity to form an
a-helical
coiled-coil: a sustained progression of heptads (abcdefg)n inwhich residues a and d are hydrophobic and theothersare mainly polar(6). Weobservedthat theregion between amino acids 494 and 548canbe written inanheptadpatterninwhichthemajority of the amino acidsatthe aand dpositions (with theexception of serineatposition515and
threonine at position 537) are hydrophobic and conserved among the strains compared (Fig. 5). To attain the best heptad repeat
pattern,
an irregularity which consists oftheaddition ofoneamino acid(position519)inthefourth heptad
was introduced; this will formally result in a shift in the
hydrophobic face ofthe helixfrom the a anddpositionsto theb and e positions.
The analysis of the predicted secondary structure ofthe
regions around the trypsin cleavage sites shows that in all
VP4sequences, the siteatamino acid 241 is precededby a
region of ,B-strand. Onthe otherhand, thestructures ofthe
regionsbetweenthecleavage sitesandafter amino acid247 are more variable, being predicted either as a-helix, ,B-strand, turns, orcoils.
Figure 4B shows a schematic representation ofthe VP4
proteinin which thehypervariable and the most conserved
regions are marked. Also indicated are the positions ofthe
trypsin-susceptible sites,theresiduestowhicheither homo-typic or heterotypic neutralizing MAbs have been mapped (35, 52), andtheregion(amino acids47 to247) whichseems to contain the hemagglutination domain ofthe protein (13, 31).
Thehydropathic indexeswerecalculatedforeach
individ-ualprotein bythe methodof KyteandDoolittle (29), and the
values ateachequivalentpositionwereaveraged. Figure4C
shows the resultantaveragedhydropathic profile. Thiscurve
is verysimilarto equivalentcurves derived from individual
sequences,because whenthe individual curves were
super-imposed on this averaged profile, there were no obvious opposite predictions (data not shown). This hydropathic profile shows that the amino-terminal60% of VP4 is more
variable, alternating regions ofmarked hydrophobicity and
hydrophilicity, while the remaining,
carboxy-terminal
40% of the protein is in general less variable.DISCUSSION
Previous work on antigenic characterization of porcine rotavirus YM showed that this strain represents a new rotavirus serotypedistinct fromserotypes 1 to 10(3,49). In
rotaviruses, both surface proteins VP4and VP7 have been shown to induce
neutralizing,
serotype-specific antibodies.Comparison
ofthe YM VP7amino acid sequence with thehomologous proteinsequences ofotherrotavirus serotypes
showed thatYMVP7hasno morethan90%
homology
withany of the other sequences compared (3, 49).
Similarly,
inon November 10, 2019 by guest
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ROTAVIRUS YM GENE 4 3741 TABLE 1-Continued
ST3 1076 MCN13 M37 WA VA70 PR1 KU RV5 DS1
69.9 69.8 70.3 70.7 68.8 68.5 68.6 68.6 69.5 70.1
70.8 70.2 70.7 71.5 69.3 68.9 69.0 69.0 70.1 70.3
72.9 72.8 73.2 73.8 71.1 70.8 71.6 71.2 71.3 72.0
73.4 73.0 73.5 74.7 71.4 71.0 70.5 71.2 71.3 71.7
72.0 71.4 72.1 72.9 71.0 69.9 70.3 71.3 70.8 71.4
72.1 71.7 72.3 73.0 71.4 70.2 70.8 71.5 71.2 71.6
70.5 70.5 70.7 71.6 70.2 68.9 69.6 70.4 69.6 70.2
69.6 71.3 68.7 70.1 68.2 67.6 68.6 68.6 69.0 69.3
87.2 88.1 87.5 88.2 78.1 77.0 77.4 78.8 76.6 76.9
95.7 97.0 96.2 76.7 76.2 76.3 76.5 75.3 75.7
35 96.3 95.0 77.0 76.3 76.3 76.7 75.3 75.7
25 32 96.2 76.6 76.2 76.1 76.3 75.0 75.3
32 42 34 77.8 77.2 76.9 78.1 76.3 76.7
226 226 232 222 96.7 94.0 93.8 89.1 89.6
229 229 218 225 29 93.1 92.3 89.1 89.4
233 236 241 232 48 59 94.1 90.1 90.4
227 227 233 219 49 62 47 90.8 91.2
234 236 241 228 89 92 83 76 98.7
234 236 241 228 88 93 84 76 13
acids that are almost completely conserved between the
strainscompared (residues 1 to 14, 223 to 236, 256 to 271, 312 to331, 339 to 376, 407 to 430, 477 to 498, 516 to 539, 626 to 646, 715 to 736, and 751 to 776). All but two of these conservedregions fall in the VP5 part of VP4. In total, there are361 positions among the strains compared (46.5%) which
haveidentical residues.
Secondary structure prediction. Recent data from
cryo-electron micrographsof the rotavirus particle with a mono-clonal antibody bound have shown that the VP4 protein is
forming the viral spikes (47, 48, 55), indicating that this
proteinhas adistinctivefold. To learn about the structure of
thisprotein, the secondary structureof each of the selected
amino acidsequencesshown in Fig. 3 was predicted by using two different algorithms (4, 16). The prediction of each
individual sequence was carried out, since comparison of
homologous proteinsfrom differentorigins can improve the accuracyof the prediction of secondary structures (7, 51).
The diagrammatic representation of the resulting
predic-tions,in which the contentsofa-helix and
,3-strands
for eachindividual protein are depicted, is shown in Fig. 4A. Even
though there are variations in the predicted secondary structures of each protein, there is an overall common pattern in which the VP4 protein can be divided into two
distinct parts. The first comprises the NH-terminal 63% portion, inwhich mostly 1-strands were predicted, and the second is represented by the remaining carboxy-terminal region of the protein, in which a high content ofa-helices werepredicted. Itwasinteresting that startingatamino acid
494, there is aregion ofabout 63 aminoacids which has a high potential fora-helixformation andwhich is conserved between theproteins studied.
We analyzed this
region
looking
for the characteristicmotif found in sequences with a propensity to form an a-helical coiled-coil: a sustained progression of heptads
(abcdefg)n
inwhich residues a and darehydrophobic
and the othersaremainly polar (6).Weobserved that theregion
between aminoacids494 and 548canbe written inan
heptad
patterninwhichthemajority oftheamino acidsattheaand dpositions (withthe
exception
ofserineatposition
515andthreonine at position 537) are
hydrophobic
and conserved among the strains compared (Fig. 5). To attain the best heptad repeat pattern, anirregularity which consists of theaddition ofoneamino acid(position 519) in the fourth heptad was introduced; this will formally result in a shift in the
hydrophobic face of the helix fromthe a and dpositions to the b and epositions.
Theanalysis of the predicted secondary structure ofthe
regions around the trypsin cleavage sites shows that in all VP4 sequences, the siteat amino acid 241 is preceded by a regionof13-strand. Onthe other hand, the structuresofthe
regions between thecleavage sitesand afteramino acid247 are more variable, being predicted either as a-helix,
1-strand,
turns,or coils.Figure 4B shows a schematic representation ofthe VP4
protein in which thehypervariable and the most conserved regions aremarked. Also indicated are thepositions ofthe
trypsin-susceptible sites, theresiduestowhich either
homo-typic or heterotypic neutralizing MAbs have been mapped
(35,52), and theregion (aminoacids 47 to 247) which seems to contain the hemagglutination domain of the protein (13, 31).
Thehydropathicindexeswerecalculated for each
individ-ualprotein bythemethodofKyte andDoolittle(29), and the valuesateachequivalentposition were averaged. Figure 4C shows the resultantaveragedhydropathic profile.Thiscurve is very similartoequivalentcurves derived from individual sequences, because when the individualcurves were
super-imposed on this averaged profile, there were no obvious opposite predictions (data not shown). This
hydropathic
profile shows that the amino-terminal 60% of VP4 is more variable, alternating regions of markedhydrophobicity and
hydrophilicity, while the remaining,
carboxy-terminal
40% of the proteinis ingeneral lessvariable.DISCUSSION
Previous work on
antigenic
characterization ofporcine
rotavirus YM showed that this strain represents a new rotavirusserotypedistinctfrom serotypes1 to10 (3,
49).
In rotaviruses, both surface proteins VP4 and VP7 have been shown to induceneutralizing,
serotype-specific
antibodies. Comparison ofthe YM VP7amino acid sequence with the homologous protein sequencesof other rotavirus serotypes showed that YM VP7 hasno morethan90%homology
with any ofthe other sequencescompared (3, 49).
Similarly,
in VOL.65, 1991on November 10, 2019 by guest
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YM
UK
SA114fM SAll
RRV
ST3 GOTT
WA
RV5 YM
UK
SA114FM
SAll RRV ST3
GOTT WA
RV5
YM UK
SA114fM
SAll
RRV
ST3
GOTT WA RV5
YM UK
SA1i4fM
SA1l
RRV
ST3
GOTT WA
RV5
YM
UK
SA114fM
SAll
RRV
ST3 GOTT
WA
RV5
YM UK
SA114fM
SAll
RRV
ST3 GOTT
WA
RV5
YM UK
SA114fM
SA1l
RRV
ST3
GOTT WA
RV5
YM UK
SA114fM
SAll
RRV ST3 GOTT WA
RV5
MASLNYRQLLTNSYTVNLSDEIQEIGSAKAQNVTINPGPFAQTGYAPVNWGAGETNDSTTVEPLLDGPYQPTTFNPPTSYWVLLAPTVEGVIIQGTNNTD 10 0 ----I---A---A---SV--G-N-R--V---P---P--V----V-Q-V---AP-DL-VGN-M---RP--VVE--D-SG --A-I---E--- T-T----V---N- P -V--- V---M---NA--VVE---N
---D---T-S---p-V---P-- ---VD--M---TP ---VE
---D---T-T---L---p---p---V---S----VD--M---AA--VVE--- E--- NT ---E-S--I--- N----VLESW-V--- I--V
---S-K--SD--I--N--NQQ-VLE---K--- E---KT---E-S--- T----T-RH--V---V---
S-K--ND--I--N-INK--VFK---RS---S-D-H---EQ---E-T---S---R---H--I
---I---T--ND--I-INSNTN--VYES---S---S-D-H---EQ---E-T-S--V---R---NH--I ---V---K--ND--F-ISSNTD--VYES---N-RWLATILIEPNVQTTNRTYNLFGQQVTLSVDNTSQTQWKF IDVSKTTLTGNYTQHGPLFSTPKLYAVMKFSGRIYTYNGTTPNATTGYYSTTNYDTVNMT 2200
---SV---G-ASET---MM--SSKQW-S-V-D-K---VEMV--AVD-D-AEW-T-L-DT---GM--YGR-LFI-E-E---KG-FI---ASAEVR
---Q--VE---T---QVT-S-D---K---V-L--Q-QD--- S---S-L---G---
HG-K---E----N---F---SE----TI--I-EQ-T-S----D---V---AN-SIG-Y---L-S---N--HNEKL---E-Q----R-AH
---S---V----TSET-S-T---T-EQITIAYA---V---QN-S-S-Y---0- ---HN-K---E---V--K---S
----300
I-I -LL-V----TNQS-Q-T----ETKQIT-E-NTN*K----FEMFRSSVSSEFQHKRT-T-DT--AGFL-HYNSVWSFH-E--H---D-S --S-LSE-ET-V-V-IL---OR-PSQD-Q-T----EVKQIT-E-S-D*K----FEMFRNNANIDFQLQR--T-DT--AGFLTHG- -VW-F --E--H---D-ST-S-LPD-EVV
F-T-VVA---H-NPVD-Q-LI--ESKQFN-S-D-N*KK---LEMFRSSSQNEFYNRRT-T-DTRFVGI L-YG--VW-FH-E--R---DSS--A-LNNI
SI-F-T-V-AV--H-SQ----Q-I---ENKQFN -E-N-D*K----F1;MF-GSSQ-DF SNRRT -T -NNR-VGML-YG--VW-FH-E--R---DSSN-ADLNNISIM SFCDFYI IPRN. EEKCTEY INHGLPPIQNTRNVVPVSLSAREIVHTRAQVNEDIVVSKTSLWKCEMQYNRDITIRFKFDRTIIKAGGLGYKWSEISFKPI T -I---AN----S---AN
---ASS-V-S---AN
II---I---NNS---L---AAN
II---GNS-V-L---V---AAN
.---I---GNSV--L
400
TVTLHPAGVTLSTQFTDFVSLNSLRFRFRLTVGEPSF SITRTRVSRLYGLPAANPNNQREHYEISGRLSLISLVPSNDDYQTPINsvTvRDLERLGE 500
a--m---_ ___ _ ___---____---___-_---___ o_ ___ ---_--___--__ t__ V2
---R-N-DA]
--Ei---A---E---
Y---S---K--K---E---E--IT---K---K----4
--E---E---T---V---K--K---E---E---A---LT ----S---K-RK---E- -E---LT----S---K-RK-.
SIRSIGSSASAWTEVSTSITD 600
-EQ-A--NDVSN '-K-VSN VDTFDE 700
-K-A--DSPVI.' 'KVLREFINI
----G--D
NLIMQCRL 776
V-- --D-V---VN-A-A---- - -- --F --- - --NV---TRS--bL--I--- -D-- - -- - - --K-- --Q- -L---
-V----D--VN-A-A---F----N----TRS--L--I----R---D---x-- -L--V----N---E---F---N---- TRTE-LN-IK-N--M--N ---
-H---Q--L---INF---E---F---N---TRIE--N-IK-N-N---N
---Q--L--K-FIG. 3. Comparison of the deduced amino acidsequences of the VP4 protein of nine rotavirus strains. All strains are compared with porcinerotavirus YM. The arrows indicate the trypsin cleavage sites described for rotavirus SA114fM (32). Dashes indicate amino acid identity.Toattain the bestalignment, an asterisk was introduced at position 136 in the Gottfried (Gott) and human strains.
this work we found that the sequence of the YM VP4 protein addition, a set ofMAbs directed to the VP8 region of the
shares no more than 83% identity with the VP4 sequences OSU VP4 protein showed a very similar reactivity pattern
compared,with the exception of that of the porcine rotavirus with these two rotavirus strains. Only one of eight MAbs
OSU,withwhich it has 97% identity. Accordingly, a hyper- was able todiscriminate between the two strains (30).
immuneserum directed to a recombinant OSU VP4 protein Since serotype classification is based primarily on
anti-neutralized OSU and YM rotaviruses to the same titer. In genic epitopes located on the VP7 glycoprotein, it was
I
I I
LAKENSFVYI
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ROTAVIRUS YM GENE 4 3743
A
SAII-^|-*kwv.*L--s
i- - JwSTA
100 200
i .s s T ;
r- -s _,,
300 400
LW
L ] r
FIG. 4. (A)Diagrammatic representationof the secondarystructurepredictions for theVP4proteins of nine rotavirusstrains. Eachline
representsoneVP4protein; the Gamier predictionwasplotted intheupperpartofeachline,and the ChouandFasmanpredictionwasplotted
below each line. a-Helix is shownas shaded bars, p-strands areshown as solid bars, and coil orturn isshown as asimple line. SA11*,
SA114fM; Gott,Gottfried. (B) Schematic representation of relevant features of the VP4 protein. Thearrowsindicate thetrypsin cleavage sites
determined forSA114fM (32). The relative position of the residuestowhich either homotypic(T)orheterotypic(V) neutralizing MAbsreact,
as determined for the RRV (35) andKU(52)strains,areindicated. The solid barbelowtheline shows therelative position oftheputative fusionpeptide (35). Theopenandhatched bars depict themostconserved and variable amino acid regions, respectively,amongtheselected
VP4sequencescompared in this work. The stippledbarrepresentsthetentative hemagglutination domain (13, 31). (C) Averagedhydropathic profile (see Materials and Methods) of the nine VP4 proteins selected for analysis in this work. The degree of hydrophobicity increases above thehorizontal line; that of hydrophilicity increases withdistancebelow thehorizontal line.
interesting to compare the serotype groups to the groups
generated by comparison of the otheroutercapsid protein, VP4. Theanalysis of the constructedVP4phylogenetictree
indicatesthat thisprotein is bettergrouped by the hosts that therotaviruses infect than by the serotypesthey belongto,
suggesting that VP4 is involved in the host restriction of these viruses. Similar conclusions were drawn in a more
limited studyinwhichonly the amino acid region around the trypsin cleavage sites of VP4wasanalyzed (27). In addition,
supporting these observations is the fact that the dose of virus necessary to cause diarrhea in mice infected with reassortant viruses of bovine (NCDV) and simian (SAil) originsegregatedwith the genethatcodes for VP4, suggest-ing that this protein determines the virulence in vivo (43), probably by restrictingthe hostspecificity of the virus.
Recently, Gorzigliaetal. (18),on thebasis of
neutraliza-tionassayswithseratorecombinantVP4proteins,proposed
a b c d e f g
L g e
F N s
I A m
L A 1
M F S I k S
a K S V M k
L E R q 494-4 97 L R e E 498-504
L S Q e 505-511
s Q L I d 512-519
L P L D 520-526
M F S G 527-533
T i d a 534-540
M A T n 541-547 r F K k 548-554
FIG. 5. Possible heptad repeat pattern in the region between amino acids494to554. Thesequenceshownrepresents the consen-sus sequenceamongtheninestrains used in this work. Uppercase
letters represent residues whichareabsolutelyconservedamongall
strains;inpositionswhere therewasamino aciddiversity,the most
frequentresidueis shown in lowercase. Boldface letters indicate the
hydrophobicresidues in theaand dpositionsof theheptads.
a VP4 typing system forhuman rotaviruses. This antigenic classification fits well the grouping generated witha
phylo-genetictreeinthisstudy.Sinceadirectcorrelationbetween
VP4 amino acid homology and antigenic relatedness was
observed,asmeasuredby neutralization,it will be of interest to seehowmanymoreantigenicgroups aregeneratedwhen
the VP4s of animal rotaviruses are analyzed, since they representa separatebranch in thephylogenetic tree.
Since it has recently been demonstrated that VP4 is forming the spikes of the virus (47), it was of interest to predict the secondary structure of this protein. Given that theaccuracy in theprediction ofa secondarystructure can
be improved by the comparative analysis of homologous amino acid sequences from different origins (7, 51), we
included in our analysis the nine most divergent VP4
se-quences. Twodifferentalgorithms (4,16)wereused in order to diminish theoverpredictionofagiven region. Theuseof both algorithms has beendescribed extensively in the liter-ature, and there is an estimated 50 to 60% accuracy of prediction by any of these methods (51). In our case, comparison of the predicted structure for any given VP4
sequence showed variability between the two methods; however, the general observationof the relative abundance of p-strands in the amino-terminal 63% ofthe protein and a-helix in theremainingcarboxy-terminal portion remained, irrespectiveof thepredictive algorithmused. We also found variability among the predicted structures for the different proteins analyzed, but the observation ofthe pattern
men-tioned above holdstrue in every case.
Theanalysisof thepredicted secondarystructuresuggests that the VP4proteincanbe dividedinto at leasttwodifferent structural domains. One comprises theamino-terminal 63%
region of theproteinandisformedby an arrayofp-strands alternated with turns, coils, and small stretches ofa-helix, which is consistent with the folding of this region as a R
c
I
500 610600 700
E'L_ ;; ,I.';:.,:. ;_.,
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globularconformation. The other domain, whichcomprises theremaining, carboxy-terminal part of VP4, is predicted to be rich ina-helical stretches, the longest of which averages about 63 amino acids long, from amino acid 494 to amino acid -557. Ofinterest, although the heptad repeats present in this region do not fit perfectly the canonical pattern,they resemblethose found in proteins which areknown to form
coiled-coils (6).Previously,Estes and Cohen havesuggested
thattheaminoterminusof VP4,prior to the trypsin cleavage site, might have a globular structure (11).
It is alsointeresting that the regions in which the hemag-glutination activity, trypsin susceptibility, and interaction
with MAbsdescribed sofar havebeenmapped allfall in the amino-terminal 56% of VP4, while the carboxy-terminal regionof the protein, rich ina-helices,does not seem to be seen either antigenically or biochemically. Recently,
Gorziglia et al. (19) isolated rotavirus variants thatescaped
neutralization by a hyperimmune serum. Sequenceanalysis of the gene 4 of these variants showed the presence of mutations in the carboxy-terminal part of VP4 intwoof the sevenvariants analyzed. However, since thesetwovariants have more than one mutation in gene 4, thechanges in the carboxy-terminal region of VP4mightnot be related to the immune selection.
Prasaad et al. (47), using Fab fragments of MAb 2G4,
which is known to reactin theregion aroundamino acid393 of VP4 (35), have suggested that the viral spikes might be formed by dimersof thisproteinand that theantigenic sites that reacted with these Fab fragments were located at the distal end of the spikes.
Taking together thepredicted secondarystructureandthe antigenic, biochemical, and electron microscopy data, it is tempting to suggest that about the amino-terminal 60%part
of VP4 forms a globular domain, which is exposed to the
surfaceof thevirion mostprobablyformingthehead of the
spike, whereas thelong a-helixstretch(494to557) located in thecarboxy-terminalportion oftheproteinmightbeforming
the stemof thespike. Since it has been suggested that each spike is formed by dimers of VP4, this portion might be a region where the two molecules of VP4 interact, possibly formingacoiled-coil or asimilarstructure.Finally, sincethe carboxy-terminal region of this protein is apparently not seen either antigenically orbiochemically when the protein
is forming part of the virion, it is also tempting to propose that the region downstream from the long a-helical stretch might contain the site(s) of VP4 thatinteracts more closely with VP7. This hypothesis might be tested by analyzing the amino acid changes that occur in reassortant viruses in which either the VP4 or the VP7 of one virus is replaced by another; inthis case, one would expect that VP4and/or VP7 will have to change in the region(s) where the proteins are in close contact in order toadapt better to one another.
Theinfluenza virus hemagglutinin, which forms the spikes ontheinfluenzavirus virions, has been extensively studied. Fromcrystallographic methods it is known that this protein is assembled into trimers, in which there is a globular region ofantiparallel a-sheet which forms the knob of the spike, and atriple-stranded coiled-coil of a-helices which forms the stem of the spike (54). In reovirus, the
acr
hemagglutinin protein has a similar structure in which theamino-terminal part of the protein has a long stretch of ax-helix which is predicted to form an a-helical coiled-coil and the carboxy-terminal third is predicted to form a structurally complex globular domain (40).The secondary structure predictions for VP4 made in this work resemble, in general, those observed for the HA of
influenza andaci ofreovirus, eventhoughtheseproteinsdo not share any significative amino acid homology (data not shown). The possible structure of VP4 predicted in this
study might serve as a working model to design further
experiments directed to understand thefolding of this pro-tein in relation to its function and its interaction with the other surface protein, VP7.
ACKNOWLEDGMENTS
We are grateful to Jose Amezcua for writingcomputerprograms and for advice and to Alberto Ruiz for excellent technical assis-tance.
This workwaspartially supported by grant RF89088#66 fromthe Rockefeller Foundation.
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