Proc. Natl. Acad. Sci. USAVol. 89, pp. 8847-8851, September 1992Biochemistry

A distinct picornavirus group identified by sequence analysisTIMo HYYPIA*t, CHRISTINE HORSNELLt, MARITA MAARONEN*, MAHBOOB KHANS, NISsE KALKKINEN§,PETRI AUVINEN*, LEENA KINNUNEN¶, AND GLYN STANWAYtDepartment of *Virology, University of Turku, Kiinamyllynkatu 13, SF-20520 Turku, Finland; tDepartment of Biology, University of Essex, Wivenhoe Park,Colchester C04 3SQ, United Kingdom; §Institute of Biotechnology, University of Helsinki, Karvaamonkuja 3, SF 00380 Helsinki, Finland; and NMolecularBiology Unit, National Public Health Laboratory, Mannerheimintie 166, SF 00300 Helsinki, Finland

Communicated by Robert H. Purcell, June 5, 1992

ABSTRACT Although echovirus 22 is presently classifiedas a member of the enterovirus group in the family of picor-naviruses, it has been reported to have exceptional biologicalproperties when compared with other representatives of thegroup. We have determined the complete nucleotide sequenceof the echovirus 22 (Harris strain) genome, which appears to besignificantly different from all the other studied picornavi-ruses. However, the organization of the genome [7339 nucle-otides, excluding the poly(A) tract] is similar to that of previ-ously sequenced picornaviruses. This genome includes a 5'untranslated region, relatively well-conserved when comparedwith aphtho- and cardioviruses, followed by an open readingframe coding for a 2180-amino acid-long polyprotein. Theamino termini of capsid polypeptides VP1 and VP3 weredetermined by direct sequencing, and the other proteolyticcleavage sites in the polyprotein were predicted by comparisonwith other picornavirus proteins. The amino acid identities ofechovirus 22 polypeptides with the corresponding proteins ofother picornaviruses are in the 14-35% range, similar to thosepercentages seen when representatives of the five picornavirusgroups (entero-, rhino-, cardio-, aphtho-, and hepatoviruses)are compared. Our results suggest that echovirus 22 belongs toan independent group of picornaviruses.

Picornaviruses are small, naked, icosahedral particles con-taining a single-stranded RNA genome with mRNA polarity.This group includes a number of important human and animalpathogens and has been studied extensively. Currently pi-cornaviruses are divided into five main groups, largely on thebasis of physicochemical properties and pathogenesis in thehost. These groups are rhinoviruses, aphthoviruses [foot-and-mouth disease viruses (FMDVs)], cardioviruses (e.g.,encephalomyocarditis virus), hepatoviruses [hepatitis A vi-rus (HAV)], and enteroviruses. The latter are subgroupedinto polioviruses, coxsackie A and B viruses, echoviruses,and enteroviruses 68-71. Recently echoviruses have beenshown to contain genetically distinct subgroups, the majorone closely resembling coxsackie B viruses and the minorone, including echovirus 22, exhibiting specific molecularcharacteristics (1, 2). Echovirus 22 is also known to haveother exceptional biological properties when compared withmembers of the enterovirus group (3-7).

Nucleotide-sequence analysis of picornaviruses has castlight on the classification of the family (8). In most cases, thenucleotide and amino acid identities among the group mem-bers correspond well with the previous divisions. For in-stance, polioviruses and coxsackie B viruses have beenshown to be quite homogeneous subgroups and also rela-tively closely related. On the other hand, HAV, previouslyclassified as enterovirus 72, shows only minimal sequencehomology with enteroviruses and, indeed, any picornavirusstudied so far (9). Classification ofpicornaviruses on the basis

of genetic properties may soon become essential becausetheir detection in the disease context can be expected to bedone increasingly by the PCR, which uses conserved andspecific regions ofthe genome (10). Molecular characteristicsare also essential when detailed pathogenetic mechanisms ofviral diseases are concerned. We show here that echovirus 22has a distinctive genome when compared with the presentlyknown picornaviruses, suggesting that it should be classifiedin an independent group among these viruses. 11

MATERIALS AND METHODSEchovirus 22 (EV22; Harris strain) was obtained from theAmerican Type Culture Collection (ATCC). It was plaque-purified three times and shown to be neutralized by aEV22-specific antiserum (ATCC). The propagated virus waspurified in successive sucrose and CsCl gradients, as de-scribed (2). Standard methods, including negative staining by2% potassium phosphotungstate or 1% aqueous uranyl ace-tate, were used in electron microscopy. Virus RNA waspurified and cloned as described (2), and two cDNA clones,together representing almost the entire genome, were se-quenced by the dideoxynucleotide chain-terminationmethod. Three strategies were used: double-stranded se-quencing after subclone generation by the ExoIII deletionmethod (11, 12), direct sequencing with oligonucleotide prim-ers (13), and sequencing of templates generated by cloningrestriction fragments into M13mp18/19 vectors (Pharmacia).Approximately 98% of the sequence, including the entirecapsid region, was determined on both strands, and all thenucleotides were sequenced at least twice. Sequencing of 75nucleotides (nt) at the 5' terminus was done by primerextension with virus RNA as a template (14). For directamino-terminal sequence analysis, the capsid polypeptidesfrom purified virus were separated in a SDS/12.5% poly-acrylamide gel, electroblotted on a poly(vinylidene difluo-ride) membrane (15), and subjected to sequence analysis in agas/pulsed liquid sequencer equipped with an on-line phen-ylthiohydantoin-amino acid analyzer (16). Sequences wereassembled by using Genetics Computer Group (17) andStaden-Plus (Amersham) software; the analysis was carriedout by programs GAP, PRErrY, and WORDSEARCH from theGenetics Computer Group package. Picornavirus sequencesused in the computer comparisons have been presented in arecent review article (8). Dendrograms were derived by usingthe KITSCH package of the PHYLIP suite of programs (18).

RESULTSStructure and Organization of the Genome. Electron mi-

croscopy of purified EV22 revealed spherical particles 28 ±

Abbreviations: HAV, hepatitis A virus; UTR, untranslated region;nt, nucleotide(s); FMDV, foot-and-mouth disease virus.tTo whom reprint requests should be addressed.IThe sequence reported in this paper has been deposited in theGenBank data base (accession no. L00675).

8847

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Proc. Natl. Acad. Sci. USA 89 (1992)

1 nm (n = 58) in diameter with the appearance of a picorna-virus (Fig. 1). Complete nucleotide-sequence analysisshowed that the length of the EV22 genome (Fig. 2), exclud-ing the poly(A)-tract, is 7339 nt, which is in the range foundfor rhinoviruses and enteroviruses. The base composition(adenine = 32%, cytosine = l9o, guanine = 20%, and uracil= 29o) is relatively similar to rhinoviruses (in HRV14, G+C= 41%) and HAV (G+C = 38%) rather than to enteroviruses(G+C = -46%). As seen in most other picornaviruses, theoccurrence of the dinucleotide CG is very low. The predicted5' untranslated region (UTR) extends to nt 709 and is fol-lowed by an open reading frame coding for a 2180-aminoacid-long polyprotein. Codon usage analysis shows a pre-

ponderance of XXU and XXA (65%), similar to that ofrhinoviruses and HAV. The length of the 3' UTR is 90 nt.The codon that initiates the open reading frame was

identified by two main criteria: the occurrence of a 9-nucle-otide stretch of pyrimidine residues located 19 nt upstreamand the methionine codon being in an optimal Kozak context(AXXAUGG). A polypyrimidine motif is found in all studiedpicornaviruses, and that seen in EV22 is very similar insequence, and also in spacing from the AUG, to that ofcardioviruses, aphthoviruses, and HAV. Picornavirus VP4polypeptides, studied so far, have an amino-terminal glycine,and they are myristoylated (8). The first glycine in the EV22polyprotein is located 13 residues from the predicted aminoterminus and appears within a myristoylation consensus

motif. It is, therefore, possible a short leader is present inEV22. The putative leader sequence is more reminiscent ofthe predicted one in HAV, where apparently the true aminoterminus of VP4 is seven amino acids downstream, than ofthe long leader polypeptides found in aphthoviruses andcardioviruses (19).The genome organization of EV22 is analogous to that of

other picornaviruses because amino acid identities can beobserved throughout the polyprotein, and this makes itpossible to suggest most of the cleavage sites in the polypro-tein. For direct detection of some of the cleavage sites, thecapsid proteins in the purified virus were separated bySDS/PAGE, and the proteins were electroblotted on a poly-(vinylidene difluoride) membrane. Protein bands correspond-ing to apparent molecular masses of 30 kDa, 30.5 kDa, and 38kDa were subjected to amino-terminal sequence analysis.The 30-kDa and 30.5-kDa proteins gave the results APNGK-KKN and NSWGSQMDL, respectively, unambiquously lo-calizing these structures on the deduced sequence (VP3 and

FIG. 1. Electron micrograph of purified echovirus 22.(x300,000.)

VP1). The largest polypeptide repeatedly gave no result inEdman degradation, and no signal representing the predictedamino terminus of VP2 was seen in any bands analyzed.Tryptic peptide analysis of the 38-kDa band generated se-quences localized in the predicted VP4 region (VESVG-NEIGGNLLTK), in the VP2 region (NVVQATTTVNT-TNL), and spanning the predicted VP2/VP4 cleavage site(VADDASNILGPNXFATTA). Together with the inabilityto find any trace of VP2 sequences in the other capsidpolypeptide bands, this result strongly suggests that noVP4/VP2 cleavage occurs, and VP0 is the predominantspecies in mature virions.

In the polypeptide region, homologous to the VP2 proteinin other picornaviruses, there is an apparent deletion in the"puff" region (20) when compared with entero-, rhino-, andcardioviruses, making the EV22 polypeptide structurallymore similar to those of aphthoviruses and HAV. VP3 islarger than in other picornaviruses, mainly from a hydrophilicextension of "20 amino acids at its amino terminus. Aninteresting finding in the VP1 polypeptide is an RGD se-quence close to its carboxyl terminus. Such a motif is knownto be involved in cell attachment in various systems (21).Of the nonstructural proteins, some (e.g., 2C, 3D) can

easily be aligned with the corresponding polypeptides ofother picornaviruses, whereas others (2A, 3A) exhibit onlyminimal identity (Table 1). 2A is an important protein toconsider because the strategy of its action divides picorna-viruses into two categories: in enteroviruses and rhinovirusesit is responsible for an autocatalytic cleavage activity directedto its amino terminus, whereas in aphthoviruses and cardio-viruses, 2A cleaves at its carboxyl terminus (8). However,deducing the mode of action of EV22 2A from the predictedsequence is not possible. The function ofpolypeptides 2B and3A in virus replication is still poorly understood, and theyhave a relatively low degree of conservation. Although thefunction of polypeptide 2C has not yet been completelyelucidated, it is one of the most conserved proteins amongpicornaviruses. As this protein aligned well with other picor-naviruses, polypeptide 2C was used in detailed comparativeanalyses. The localization of polypeptide 3B (VPg) waspredicted by the genomic position, presence ofthe conservedtyrosine (Y) as the third amino acid, and comparisons withother picornavirus VPgs. The 3C protease contains theconserved sequence GXCGG, proposed to form part of theactive site. Previously observed, highly conserved motifs(KDELR, YGDD, FLKR) are found in the 3D polymerase.

Relationship of Echovirus 22 to Other Picornaviruses. Thenucleotide identities ofthe 5' and 3' UTRs and the amino acididentity of the predicted polypeptides were compared withrepresentatives of picornavirus groups (Table 1). In parts ofthe 5' UTR, EV22 RNA exhibits a high degree of sequenceconservation relative to aphtho- and cardioviruses; for in-stance, there are several blocks of, at least, an identical 10 nt.These viruses, together with HAV, share a similar predicted5' UTR secondary structure, which is distinct from that ofentero- and rhinoviruses (8). The identity of the EV22 poly-peptides with corresponding proteins of other picornavirusesvaried between 14 and 35%; this value is of the same order asthat seen when HAV is compared with other picornavirusgroups (9).Because the most pronounced identity was seen in poly-

peptide 2C, this protein was analyzed in more detail. Whenits amino acid sequence was compared with the NationalBiomedical Research Foundation data base, this EV22 poly-peptide was found to be more similar to the 2C protein of 22other sequenced picornaviruses than to any other proteins inthe data base (data not shown). In a dendrogram, illustratingthe relationships between picornaviruses, EV22 and HAVjoin to the other major groups at an amino acid-differencelevel exceeding 70%o when polypeptide 2C is used for com-

8848 Biochemistry: Hyypid et al.

Biochemistry: Hyypia et al. Proc. Natl. Acad. Sci. USA 89 (1992) 8849

50

STATAACCCGAC1TTCTGA230

6GTGTAAATOATCTTTTCt410

CTAGTOCCAGCGGAACA590

COCAGGTAACAAGAGCAO

770=TCGTTGATrCTACCATCS V D S T I

950rACTACCAACTTACTCANT T N L T Q

1130rACACATSTAGAATSACCl

V E L P1310

rTCAAAACTGAATTTOGS X L E F C

1490kTCTGCTAACCAAGTTGAIS A N a V D

1670rATAGCAGAAGGTCCTGI A E C P C

1650

VCCATCAGAAAATCATGGP S E N G

2030AGTACTTTCAATAGAGS S F N R C

2210M"ATTGTTTCAAATTSUC L F Q I E

2390ATTCAAGASCACACACAJI E D D S

2570WSCATGCTTTTTTATGGAA F F E

2750ACTAGCAATGASCGACTI

S N D R V

2930ATTGAGGTTTATCTTAG

70

G.CTTCTATAGGAAAAAACCCI250

TAACCTGATATGTACAGOO430

ACATCTGOTAACAGATGC=TCI610

ACTCTGGATCTGATCTGOGOC4

790

CAATGCAGTCAATGAAAAOGTJN A V N E V

970

ACATCCACCG0CACCTACGATCH P S A P T

1150

TAAACTTTTTTGGOATCACCAJX V F D

1330

AGCCTTTACAAATCTTCCACAKA F T N L P

1510

FOTCACCATACTGGCTAOCTTCV T I L G S L

1690

TTCAATGAACATGGCAAATGTCS N N Aa V

1870IATTGA:TCAAAAGGTATTTCV D a X Y F

2050

TCOATTGAGGOTAIGCTTCTTCR L R C F F

2230

AGTCTTAAACAGGCTCACATAV L N R L T Y

2410

AA&TTGCAAACAAACAATGTCIN C X T 5

2590

CCATACTTTCACCAATGACGJT F T N E G

2770

CAATTTTCTGTCATCGAACGG'N F L S S N

2950

CCTGAGATCTCCAAATSTCTT'

90

TTCCCAGCCCT0G00CTGTCAA270

OGCAGATGSCGTSCCATAAATCTATT450

rS.GCOCAAAA GCCAAGOTTGACA0J630

AACTACCTCTATCAOSTGAGTTAGTT.

610

CAAAGCGTCGOTAATGAAATTGGAGCE S V C N E I

990

;CCCTTTTCACCAGATTTCTCCAATGTP F S P D F S N V

1170

GACaAGCccGCATATGGACAATCACOD X P A YG Q S R

1350

VTTTTGATGOAATTTGCTGAAACCACV L N LA T T

1530

;TTGCAATTAGACTTCCAAAATCCAAGL Q L D F Q N P A

1710

CTTTGTACAACTOGTGCTCAATCAGIL C T T CA Q S V

1690

:AAGTOGGCACAACAACAOCCCCCAJOSA T A P Q

2070

CCCAATGCCACAACGGACTCAACTTCP N A T T D S T S

2250

:AACAGTTCTACTCCATCOGGOOTTTAN S S S P S E V Y

2430

'CCAAATGAACTAOGACTCACTTCAGCP N E L C L S A

2610

LCAATGGAGAGTGCCATTGGAATTTCCQ R V P L E F P

2790

IGTAATAACTGTACCAGCCGGAGAGCCV I T V P A C E Q

2970

rTCCCTCTTCCTGCCCCTAAGGTTAC

110

rAAAAACCCCCATAG290

1TOGGATACCACGCI470

.CrATTA00ATT0TG650

AAAAACGTCTAGTOC

630

rAATTTTTAACTAAMN L TR

1010

ACAcATTTTCACWTD F S

1190

TACTTTGCGGCTGTAY F A A

1370

LCAOGCGOACTTATGQA D L C

1550

AITTTGCTCAAGAIVF A Q

1730

OGCTTTAGTAGGTGAMA L C

1910

SAGTATTGTCCACAGOS I B

2090

:ACCTTOGACAATGCIT L D NA

2270

rTGTATAGTCCAA0GC I V 0

G

2450

CCAAGATCATGGCCQQ D D C P

2630

LAAACAAGGTCATGGX Q HG

2H10

;ATGACACTTTCAG CIT L S A

2990

ZiATAGTCCTGCACTA

130'AACCAACACCTAAG

310'TOTGACCTTATGC

490T'CAAAACCTGAATTI

670;OCCAAACCCAGGGO

650WTAGCTGATGATGCIV A D D A

1030rATGGCTTATGACATC

a X D I1210IAAGATGTGGCCTTCAR C C F H

1390FTATCCCCTATGTTGC

I P Y V A1570

FGTCAACATATATGAV l I I

1750LAGAGCTTTCTATGEO A F I D

1930LAAIAIIGTCTACTTN I V Y L

2110

AIAITACACAAIAIOI Y T I C

2290'AAATOGGACAAGNX N

C Q D2470

LCTTGGCCAAGAAAAAL C Q E K2650;TCCTTATCACTIGTS L S L L

2830'CCCTACTATTCAAAP Y Y S N

3010LCGGGGTGATATGGC

150;ACAATTTGATCAACCCTATGC

330CCACACAGCCATCCTCTAGTA

510rGTTGTGGAAGATATTCAGTAC

690;GGATCCCTGGTTTCCTTTTAT

670TTCCAATATCCTAGGACCAAA

S N I L G P N1050

CACAACTGGGGACAAAAATCCT T G D1

N P1230

6CTTCAAGTACAAGTTAATGTF Q V V N V

1410TGACACAAACTATGTCAAGAC

D T N Y VIT

1590

ITAATGCACCAAATGGTAAAAIN A P N G X XLVP3 1770

kTCCTAGAACTGCAGGCAGTAAP R T

A G S X1950

IAAGACTATTTCCAAATTTGAAR L F P N L N

2130;TGATATAGGTAGTGACkATAG

D I G0

D N S2310

CTGCCAGGTTCTC0TGCCAACA R F F C P T

2490GCCAAATTATTTTCTCAATTTP N Y F L N

O2670

GTTTGCTTATTTTACTGGTCAF A Y F T G E

2650kCAAACCATTAAGAACTGTCAG

K P L R T V R3030

:AAACCTTACAAATCAGAGTCC

170.CTGGTCCCCACT;rTCGAAGGCAACTT

350IAGTTTGTAAAATGTCTGGTGAGATGTG

530:CTATCAATCTGGTAGTGGTGCAAACAC

710rTGTTAATATTGACATTATGGAGACAAT

HE T IL ?TTGTTTTGCAACGACTGCTGAACCAGAC F A T T A E P E

1070*CAGTAAATTGGTTCGTCTTGAAACCCA

S K L V R L E T B1250

rGAATCAAGGTACTGCAGGCAGTGCCTTN Q G T A G S A L

1430.CGATTCGTCAGACTTAGGGCAGTTAAA

D S S D L G Q L X1610

GAAAAATTGGAAGAAAATTATGACCATX N0

X0

IM T M

1790ATCAAGATTTGATGATCTTGTTAAGAT

S R F D D L V I1970

TGTTTTTGTTAACAGTTATTCATACTTV F V N S Y S Y F

2150,TTTTGAAATTACAATCCCTTACTCTTTF E I T I P Y S F

2330TGGTTCTGTTGTTACATTCCAGAATTCGOS V V T F Q0N0

2510 LyplrTAGGTCGATGAATGTGGACATTTTTACR S M

N V D I F T2690

kACTGAATATCCATGTTCTGTTCCTAAGL N I H V L F L S

2670;AGATAACAATAGTCTTGGTTATTTGAT

D N N S L GY L M

3050-ATATGGTCAACAACCACAGAATCGTAT

CARP F L-T GT M T 01 I B V I L S L RC PMNF FF P L P A P VI OSS R AL ROGD M A NML TON O P IYGQ QP 0009R3070 3090 3110 3130 3150 3170 3190 3210 L A 3230

MRX L AO L DRO R H 0000I V O D VIOQL D10111 F KAlA L TOGRARXF T RIB L T 5 DM VI BBB COE LIDI3250O 3270 3290 3310 3330 3350 3370 3390 3410

F RI RIY LBES A V lOBS OIF O VIDRONC ET I AID I FOGT BI L SQ A0HAIG L V GT I L L TAG LOMS T I IT P3430 3450 3470 3490 3510 3530 3550 3570 3590

VONA V I ORB F F MB8A I D GDB QIGOL S L L V QIXCIT 1FF S S A AT B I LIM I0L VRXF I VAK I L V R I L C I MV3610 3630 3650 1 B 3670 3690 3710 3730 3750 3770

LOYC H R PM I L T T AC LOST L L I MIDVIT55 V L 5 PM CIXA LMOQC L MIO I V RIX L ABE OVALES M000111D3790 3610 3630 3950 3970 3090 3910 3930 3950

I B V RB 00Q C DITV 011010T L SMN 01 XG1 F MBE V S T A FOB I01MM 061B L LAI0D M V L S V F O P S IB3970 3990 4010 LP64050 4070 4090 4110 4130

SIRA I 00V LBE ROAR H V C O IL DIIA M DII V ESA I Q SAMIXTOIFP 100R SIDC L AIKF A PI M A I C F RO4150 4170 4190 4210 4230 4250 4270 4290 4310

CD MOOSIS MIT VI ALF QOB L A R 0 P0000I1001N L I R I B P 00000 QOB P G QOK S F L TBH T L 00R0 L Q4330 4350 4370 4390 4410 4430 4450 4470 4490

O C LON G VF TIMPITA S B F MI 01G D NQI OS9LO IDD LOOQT RI RIOKD EMN L C M CI SOS V P F I V P M AO LBE4510 4530 4550 4570 4590 4610 4630 4650 4670

BIKGIXF 1101S L V VA 11010 OFDP SO TYVLOI S G AL R R FP 10rMOOI AAA IA 10 I A GAK LMNV S Q AMNA4690 4710 4730 4750 4770 4790 4010 4M30 4650O

I MOS TO B COB V S K100GR1DB I L I L RID L VII 01011MBNE RNA V100A 00Q LB 0N Q LII LII A V4670 4690 4910 4930 4950 4970 4990 L A 5030

YII SONF P IA I POYV D E L0N IBM SITLO BEOMO A F I EIPIRP S V F R CF AM 10001001KS A S RABV VIM5050o 5070 5090 5110 5130 5150 5170 5190 5210

F 011010 MNL S F VBERMIRAM LITV VO A VI S A 000 L L L VII I FIIBBE SIXD BR A 10 I-

L P V A A PR5230 5250 5270 5290 5310 5330 5350 i....3 Vi' ) 5390

GITF P V 5 010 F ROBEA P Y 10 OLIE BIOIS QOMA YI T0100110601M SCA 0 Y0001000I LOHGOS III LBE5410 IL C5450 5470 5490 5510 5530 5570

QOBIDE LITLSB 1RMR V FP 0 E QPOSVITQ VITL GO PMIDL A I LAKCIK L PF OF 110011S I T MAO GIB S M5590 5610 5630 5050 5670 5690 5710 5730 5750

L I 0 MI 00000GI TAB VOARV HHSG0000ARBOG SKB T0111000TIV AS C A G M C GOG L L I 0 AVE GON FRI5770 5790 5610 5630 5650o 5670 5690 5910 5930

LOGM 00I A GONGE MOGV A OP F O F LI SI MO 0100I VITE I T P 10oPM 10011NT TO 000HX P VIY G A VE VIKM5950o 5970 5990 6010 L 3D 6050 6070 6090 6110

OP A V LOSXS DIT RLB B P V BC L IAKRS A 010Y AVOI F Q V 4MB LMV QGV IA CVI OAXF R E I F 0MM 00G V6130 6150 6170 6190 6210 6230 6250 6270 6290

DMA TAOI LOITS 0 VS 0 MI LOST S A 010 F V 0011111KK L I C LBE P F O VA P L L EARL VQ OAXF DON L LAX6310 6330 6350 6370 6390 6410 6430 6450 6470

0W00011T I F MIT C LI D EL RIR LIIRI A 001 TARCO B A CE VIDI C I VI A M I MB 01110101KIYQ P C 110

6490 6510 6530 6550 6570 6590 6610 6630 6650

O L A VOG IMNP ORK DOD FM I M A L NDYNYl10YBM1000GB LOSS M L LOB A V EV L AO C BOO. P D LV MQ L6670 6690 6710 6730 6750 6770 6790 6610 6630

DIKP V00IDSD V V F BOOR L ID GOMNP 000 PCI I V LO 0 L CO LMNM C I11110 LOOSP 000 C L P0I V 10G6650s 6670 6690 6910 6930 6950 6970 6990 7010

D DV I L S LI RBX I O P ERK L 000 M AID SFOGA EVITGS0001 PP S LIKPARM E V EF LAXRAKP G I F P E 0 IF7030 7050 7070 7090 7110 7130 7150 7170 7190

I VO R LIIT0MNM I005 LMMMR ONF S I F 100 LOQS Y L M EL C LHBGXD010100010 IAL B P0Y L Q E MOO T V7210 7230 7250 7270 7290 7310 7330

FIG. 2. Nucleotide sequence of cDNA representing echovirus 22 genomic RNA. The predicted amino acid sequence of the polyprotein andsuggested cleavage sites, determined by alignment with known picornavirus polypeptides, are indicated. The amino-terminal sequences of VP3and VP1 were verified by directly sequencing the polypeptides.

10TTTGAAAGGGGTCTCCTAC

190GCAATAAGAACAGTGGAAC

370CCOAACTTATTGGAAACAAC

550TAGTTGTAAGGCCCACGOU

730TAAGAGTATTGCAGATATSX1 I A DI

910GAACAAGAATGTAGTTCAJN X N VV 0

1090TGAGTGGACGCCATCTTGOE0 T P S M

1270OGTAGTATATGAACCTAAV V Y E P X

1450AGTCTATGTCTGGACTCC.V Y V D T P

1630GAGCACAAAATACAAATGS T0 Y K1

1610AGCCCAGTTATTCTCACTA O L F S V

1990TAGSCGTTCATTAGTTTTAR C S L V L

2170TTCCACTTGGATGAGGOAS TMMOR X

2350ATGGGGTTCACAGATGGA!M G S O M D

2530TGTATCACATACTAAAGTAV S H T X V

2710TGIGAGGGGGTTTCTGAGE R C F L R

2690GTGCAAGCCCTTCTTGAC

30AOGAGCTTGCCTAGGGCCI

210:AAGATOCTTAAAGCATJ

390:AATTTICTTAATAOCATI

570LGGATOCCCAGAAGOTACC

750OGCTACTGGTGTTOTAAG'A T CLVVOSGCCCACTACTACCTGTAAIA T T T V N

1110;oCAAGAOGATACCAGATA R G Y1 0

1290CCAGTTGTCACITTATGAP V V T Y D

1470TTOCCAATCCCCACTGGL S I P T G

1650;0CCAAACAAAAATCaT R T1 I1

1630;ATGGCAGITTCAAOCACIM A D S T T

2010kA1TT0AGCGTCTACGCR L S V Y A

2190ACAAATGCCACCCCAT'T V C 0 P I

2370rTAACCOaCCCTCTFTTL T D P L C

2550WATAACCTATTTG10CCD N LF G R

2730OOTTGCACACACATATGAV A S T Y D

2910rCGACCTCTACCGGTAA

G)

M

rc

GC

,TM

I.Tl

ITI

M

rim

NO

NU

IrA

IT

A

A

.AC

Icc

I.CC

LAC

I

c

c

I

2TJ

rcl

W,

CA4

rG4

WI

.IGCI

LAGE

LTGly

I.GII

I.CC

IC

A

A

ff

I

MI

rTjI

MI

AlI

M

rGci

ukci

;GCi

umI

.Al

;Q

I

POLiki

riWsFIGICAA:T(rGI

m.TsCCIrc:Cjra

hiITIPhr.6cmLAMCuu

kcACrclITsLA;TsLr

rc;mCIL;ACkG;arim

hc:TRG;TcrckisFIG

cirMCCQrTXLAJZTI

Proc. Nadl. Acad. Sci. USA 89 (1992)

Table 1. Comparison of nucleotide and amino acid (coding region) identities of echovirus 22 withrepresentatives of other picornavirus groups

Identity with echovirus 22, %

Virus 5'UTR VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D 3'UTRPolio 1 38 24 22 19 22 14 21 30 15 35 24 27 36HRV1B 35 20 22 17 15 15 19 29 19 30 23 26 48FMDV 46 20 23 20 23 - 15 29 14 25 18 25 29EMCV 43 20 24 22 20 18 23 29 20 30 18 25 34HAV 36 19 18 23 21 17 27 25 29 35 19 27 44

These identities were calculated by using the GAP program ofthe Genetics Computer Group package.Polio 1, poliovirus 1; HRV1B, human rhinovirus 1B; FMDV, FMDV strain 01K; EMCV, encephalo-myocarditis virus. In the 5' UTR optimal alignments were used for comparison.

parison (Fig. 3). This is clearly a lower degree of relatednessthan, for example, between the major enterovirus/rhinovirusgroup (51%), and this value also slightly exceeds the valueseen between these two groups, and aphtho- and cardiovi-ruses (68%). Very similar results are obtained when theregion, corresponding to the VP2 polypeptide in other picor-naviruses, is used for comparison (data not shown).

DISCUSSIONClassification of viruses is mainly based on virion morphol-ogy, the nature of the nucleic acid, and replication strategy.Other biochemical and antigenic properties are also used, andthey are important in identification and grouping ofindividualvirus types and strains. Optimal classification should com-bine the essential biological properties of the group and, inaddition, give an adequate tool for diagnosis and treatment ofclinical diseases. Introduction of diagnostic proceduresbased on nucleic acid sequences has recently increased theneed for precise classification of viruses according to theirmolecular characteristics.

Echoviruses were discovered soon after the first tissue-culture techniques were introduced into laboratories (22).These viruses were isolated from human intestinal tract, asare polioviruses and coxsackieviruses, but they did not sharethe major pathogenic properties ofthese groups. Because theassociation with human disease was, at the time of theirdiscovery, unknown, they were given the name ECHO(enteric, cytopathogenic, human, orphan) viruses. It wasoriginally postulated that whenever an echovirus was estab-FMDVO

TMEVEMCVHRV2HRV1BHRV89Pdio3PotiolPa~o2CAV21CAV9CBV1SVDVCBV3CBV1.EV11eV7OBEVHRV14.EV22HAV

5 10 15 20 25 30 35 40 45 50 55 60 65 70 75

PERCENTAGE DIFFERENCE

FIG. 3. Dendrogram of the relationships between picornavirusgroups based on comparisons of amino acid differences of 2Cpolypeptides. FMDVO, FMDV strain 01K; FMDVA, FMDV strainA-10-61; TMEV, Theiler's murine encephalomyocarditis virus;EMCV, encephalomyocarditis virus; HRV, human rhinovirus; Po-lio, poliovirus; CAV, coxsackie A virus; CBV, coxsackie B virus;SVDV, swine vesicular disease virus; EV, echovirus; eV, enterovi-rus; BEV, bovine enterovirus.

fished as the etiologic agent of a clinically distinct disease, itwould be removed from the group (23). Later on, it becameevident that individual echovirus types cannot be directlyassociated with individual illnesses but rather with a widespectrum of clinical manifestations. This is why they still areclassified together, forming the largest of the enterovirussubgroups. However, from the original 33 echovirus sero-types, types 1 and 8 were closely enough related to becombined, type 10 was shown to be a reovirus, and type 28appeared to be a rhinovirus.

Echovirus serotypes 22 (including the prototype 101 Harrisvirus) and 23 were isolated during a study ofsummer diarrheain 1956 (3). Although these viruses shared the general prop-erties found in the group, the cytopathic destruction of cellcultures was incomplete when compared with previouslycharacterized echoviruses. It has been reported that cyto-logic changes caused by echovirus types 22 and 23 involvedistinctive nuclear manifestations (4, 24) and that the repli-cation of these viruses is not inhibited by 2-(a-hydroxyben-zyl)benzimidazole, in contrast to other members of thesubgroup (25). There is also evidence of exceptional second-ary structure ofEV22 RNA (6). Recently EV22 infection hasbeen shown not to cause the host-cell protein synthesisshut-off (7, 26) characteristic of enteroviruses.

In addition to the biological characteristics, the epidemi-ology and pathogenesis of EV22 infections also differ whencompared with the other echoviruses. Perhaps the moststriking difference is that 60%o ofthe EV22 isolations are fromchildren of <1 yr of age (17% in all the echoviruses), andaltogether 92% of the isolations are from children under 5 yrof age (5). From the isolation-positive cases, 29% had gas-trointestinal symptoms (11% in all the echoviruses), and only11% had symptoms indicating involvement of the centralnervous system (56% in the entire echovirus subgroup).

Previous work has, thus, given evidence that EV22 differsbiologically from other echoviruses. Our data indicate themolecular basis for these characteristics. Although clearly apicornavirus in terms of electron microscopy and genomeorganization, EV22 has a very divergent nucleotide andamino acid sequence. These characteristics locate it welloutside the enterovirus group and, together with the otherbiological data, suggest that EV22 belongs to another picor-navirus genus.The diverse nature of the capsid proteins, when compared

with other picornaviruses, made prediction of some cleavagesites relatively difficult, and, therefore, the VP2/VP3 andVP3/VP1 cleavages were established by directly analyzingthe amino-terminal sequence of polypeptides VP3 and VP1.The amino-terminal sequence of VP2 protein could not bedetermined, and because VP4 can be myristoylated in picor-naviruses, one explanation for lack of a signal is that nocleavage occurs between VP4/VP2 during maturation ofEV22 virions. This alternative is strongly supported by theresults from direct amino acid analysis of the tryptic peptidesfrom the 38-kDa band, and these data suggest that capsid

8850 Biochemistry: HyypiA et al.

Proc. Natl. Acad. Sci. USA 89 (1992) 8851

structure of EV22 differs from other picornaviruses in thisrespect. Sequence comparison gives some evidence of con-served amino acids in the capsid proteins of EV22 (data notshown), indicating that they probably share the typical three-dimensional structure found in picornaviruses, an eight-stranded antiparallel (3-barrel. The unique amino-terminalextension of -20 residues in VP3, contains eight positivelycharged amino acids and may have important functions in thevirus structure. The observation of an RGD motif near thecarboxyl terminus of EV22 VP1 may give some indication ofthe type of the cell receptor used by the virus. In aphthovi-ruses (FMDV) the carboxyl terminus of VP1 is known toproject onto the virus surface and participate in receptorbinding in conjunction with an RGD motif located elsewherein the VP1 primary sequence butjuxtaposed structurally (27).An RGD at the carboxyl terminus of VP1 has recently beenimplicated in receptor binding of coxsackievirus A9 (28, 29),and a comparison with strains of this virus and FMDV showsthat the EV22 motif (RGDMANL) conforms well to a con-sensus that can be derived (RGDM/LXXL; ref. 30).The 2A protein, known to be associated with a cis-acting

proteolytic activity in other picornaviruses, does not conformin sequence to either ofthe two well-characterized types (31).In entero- and rhinoviruses, the 2A protein acts autocatalyt-ically at its amino terminus and is also involved in cleavingthe p220 component of the cap-binding complex, shutting offhost-cell protein synthesis. This protein is similar to thepicornaviral 3C proteases, as it is also a member of thetrypsin-like proteases in which the usual active-site serine hasbeen replaced by cysteine. Such proteases have a catalytictriad made up of histidine, aspartate/glutamate, and serine/cysteine residues, the latter being found in the contextGXCG, which is well-conserved between these proteins froma wide range of sources. EV22 2A is similar in size to that ofentero- and rhinoviruses, and there are a few short regions ofamino acid homology. However, the critical GXCG motif isabsent, and the protein is unlikely to function exactly in thesame manner. This protein is also dissimilar in sequence fromthe 2A protein of aphthoviruses and cardioviruses. In theseviruses, 2A protein cleaves at its carboxyl terminus and doesnot participate in host-cell shut-off, which, in aphthovirus-infected cells, is accomplished by the proteolytic activity ofthe leader. The cardiovirus leader protein has no knownproteolytic function, and this virus does not shut-off host-cellsynthesis by inactivating the cap-binding complex, whichalso happens in EV22 infection (7, 26). Further insights intothe role of 2A protein in EV22 replication must await directanalysis of its proteolytic capability.

In view of the highly divergent nature of the rest of thegenome, the similarity of the 5' UTR to cardio- and aphtho-viruses is somewhat unexpected. The observation raises thepossibility that EV22 is a recombinant virus or, alternatively,that the 5' UTR evolves more slowly than other genomeregions. Predictions suggest that similarity of this region isreflected in secondary-structural terms, and this may play arole in the observed viral properties. For instance, becausethe 5' UTR is vital in translating the virus polyprotein, thissimilarity may explain the efficient translation of EV22 RNAin rabbit reticulocyte lysate (7) also seen in cardio- andaphthoviruses, and contrasting with the poor translation ofentero- and rhinovirus RNA in this system. As suggestedpreviously by hybridization experiments (7), the EV22 5'UTR lacks the poly(C) tract seen in aphthoviruses andencaphalomyocarditis virus but absent from Theiler's murineencephalitis virus, another virus that shares the common 5'UTR structure. The 3' UTR has been shown (2) to have anexceptional predicted secondary structure when comparedwith enteroviruses.

In conclusion, our data demonstrate that EV22 has astructure and genome organization corresponding to otherpicornaviruses. However, its similarity to other sequencedmembers of this virus family is low. This similarity is ap-proximately at the same level as that observed for HAV (9),originally classified as an enterovirus but now assigned to thehepatovirus genus of picornaviridae. The molecular datapresented here suggest that classification ofEV22 should alsobe reconsidered. Although this virus is already known to befound in young children and is often associated with gastro-enteritis, the role of EV22 virus in clinical disease needscareful reevaluation against the background of its distinctmolecular properties. As soon as these data and the identityand characteristics of possible relatives of EV22 are known,it would be possible to give the putative additional genus anomenclature that corresponds to that of other picornavirusgroups.

We thank Dr. Carl-Henrik von Bonsdorfffor his advice in electronmicroscopy and Drs. Pekka Halonen and Tuija Poyry for theircomments during manuscript preparation. The study was supportedby grants from the Sigrid Juselius Foundation, the Turku UniversityFoundation, and the Medical Research Council.

1. Auvinen, P., Stanway, G. & Hyypia, T. (1989) Arch. Virol. 104,175-186.

2. Auvinen, P. & Hyypia, T. (1990) J. Gen. Virol. 71, 2133-2139.3. Wigand, R. & Sabin, A. B. (1961) Arch. Gesamte Virusforsch. 11,

224-247.4. Shaver, D. N., Barron, A. L. & Karzon, D. T. (1961) Proc. Soc.

Exp. Biol. Med. 106, 648-652.5. Grist, N. R., Bell, E. J. & Assaad, F. (1978) Prog. Med. Virol. 24,

114-157.6. Seal, L. A. & Jamison, R. M. (1984) J. Virol. 50, 641-644.7. Coller, B.-A. G., Chapman, N. M., Beck, M. A., Pallansch, M. A.,

Gauntt, C. J. & Tracy, S. M. (1990) J. Virol. 64, 2692-2701.8. Stanway, G. (1990) J. Gen. Virol. 71, 2483-2501.9. Cohen, J. I., Ticehurst, J. R., Purcell, R. H., Buckler-White, A. &

Baroudy, B. M. (1987) J. Virol. 61, 50-59.10. Hyypid, T., Auvinen, P. & Maaronen, M. (1989) J. Gen. Virol. 70,

3261-3268.11. Murphy, G. & Kavanagh, T. A. (1988) Nucleic Acids Res. 16, 5198.12. Herikoff, S. (1984) Gene 28, 351-359.13. Zagursky, R. J., Baumeister, K., Lomax, N. & Berman, M. L.

(1985) Gene Anal. Tech. 2, 89-94.14. Huovilainen, A., Kinnunen, L., Ferguson, M. & Hovi, T. (1988) J.

Gen. Virol. 69, 1941-1948.15. Speicher, D. W. (1989) in Techniques in Protein Chemistry, ed.

Hugli, T. E. (Academic, San Diego), pp. 24-35.16. Kalkkinen, N. & Tilgmann, C. (1988) J. Protein Chem. 7, 242-243.17. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids Res.

12, 387-395.18. Felsenstein, J. (1985) Evolution 39, 783-791.19. Palmemberg, A. C. (1989) in Molecular Aspects of Picornavirus

Infection and Detection, eds. Semler, B. L. & Ehrenfeld, E. (Am.Soc. Microbiol., Washington), pp. 211-241.

20. Rossmann, M. G., Arnold, E., Erickson, J. W., Frankenberger,E. A., Griffith, J. P., Hecht, H.-J., Johnson, J. E., Kamer, G., Luo,M., Mosser, A. G., Rueckert, R. R., Sherry, B. & Vriend, G. (1985)Nature (London) 317, 145-153.

21. Ruoslahti, E. & Pierschbacher, M. D. (1987) Science 238, 491-493.22. Melnick, J. L. (1965) in Viral and Rickettsial Infections ofMan, eds.

Horsfall, F. L., Jr., & Tamm, I. (Lippincott, Philadelphia), 4th Ed.,pp. 513-545.

23. Committee on the ECHO viruses (1955) Science 122, 1187-1188.24. Jamison, R. M. (1974) Arch. Gesamte Virusforsch. 44, 184-194.25. Eggers, H. J. & Tamm, I. (1961) J. Exp. Med. 113, 657-682.26. Coller, B.-A. G., Tracy, S. M. & Etchison, D. (1991) J. Virol. 65,

3903-3905.27. Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. & Brown,

F. (1989) Nature (London) 337, 709-716.28. Chang, K. H., Auvinen, P., Hyypia, T. & Stanway, G. (1989) J.

Gen. Virol. 70, 3269-3280.29. Roivainen, M., Hyypia, T., Piirainen, L., Kalkkinen, N., Stanway,

G. & Hovi, T. (1991) J. Virol. 65, 4735-4740.30. Chang, K. H., Day, C., Walker, J., HyypiA, T. & Stanway, G.

(1992) J. Gen. Virol. 73, 621-626.31. Harris, K. S., Hellen, C. U. T. & Wimmer, E. (1990) Sem. Virol. 1,

323-333.

Biochemistry: Hyypid et al.