Molecular characterization and expression of glycoprotein gene of Hantavirus R22 strain isolated from Rattus norvegicus in China

Virus Research, 21(1991) 35-52 Q 1991 Elsevier Science Publishers B.V. All rights reserved 0168-1702/91/$03.50 ADONIS 0168170291001115

35

VIRUS 00697

Molecular characterization and expression of glycoprotein gene of Hantavirus R22 strain isolated from Rattus noruegicus in China Xiao Xu *, Suyu L. Ruo, Yiwei Tang *, Susan P. Fisher-Hoch and Joseph B. McCormick Specia! Pathogens Branch, Division of Viral and Rickettsial Diseases, Center for infectious Diseases, Centers for Disease Con&of, Atlanta, GA 30333, U.S.A.

(Received 20 February 1991; revision received and accepted 4 June 1991)

Summary A cDNA containing the complete open reading frame of the M genome segment of Hantavirus R22 strain isolated from Rattus noruegicus in China, was amplified by polymerase chain reaction (PCR), and then cloned. The M segment is 3656 nucleotides in length with a predicted region of 3402 bases encoding a precursor glycoprotein of 1134 amino acids subsequently processed into viral glycoproteins 1 and 2 (Gl and G2). A strain comparison between R22 and SRll (isolated from a rat in Japan), and Hantaan 76-118 (isolated from Apodemus in Korea), and Hallnas Bl (isolated from a bank vole in Sweden) revealed 95%, 74%, and 53% homologies at the deduced amino acid sequence level respectively. This suggests that the rodent host species may be a more important determinant of genetic relationships than geographic proximity. Six potential asparagine linked glycosylation sites (five in Gl and one in G2) were identified, and among them all are conserved in SRll, five in Hantaan virus and four in Hallnas Bl virus. Although different degrees of homology exist among these four viruses at amino acid sequence level, more than 90% of the cysteine residues are conserved, suggesting that structural homology may be very strong between the Hantaviruses. Genetic differences in the M segment genome of R22 and SRll viruses, within the same serotype viruses, were found as random coding changes; some limited to

* Visiting scientists from Zhejiang Medical Universi~ and Shanghai Medical University, P.R. China. Correspondence to: S.P. Fisher-Hoch, Special Pathogens Branch, Division of Viral and Rickettsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta, GA 30333, U.S.A.

36

single amino acids, others in clusters. A recombinant vaccinia virus that contained the fully activated M segment cDNA of R22 was constructed. This recombinant virus expressed two glycoproteins Gl and G2 identical to R22 virus Gl and G2 in molecular weight, cleavage pattern and cellular immunofluorescent patterns. Polymerase chain reaction; Hantavirus R22; Glycoprotein;

Sequence; Expression

Introduction

Hantaviruses, the causative agents of hemorrhagic fever with renal syndrome (HFRS), comprise a new genus of the family Bunyaviridae including at least four serotypes: Hantaan, Seoul, Puumala, and Prospect Hill, which differ in their antigenic properties and host rodent species (Sugiyama et al., 1987; Lee, et al., 1989). As with the other members of the family Bunyaviridae, these viruses have a single-stranded, negative sense, tripartite RNA genome, of which the large CL, 8.2 kb), medium (M, 3.6 kbl, and small (S, 1.7 kb) segments code the viral polymerase, glycoproteins, and nucleocapsid protein respectively, (Elliott et al., 1984; Schmaljohn et al., 1983, 1986, 1987). Two glycoproteins designated as Gl and G2 encoded by the M segment gene in a single, continuous open reading frame, contain neutralizing sites, partially conserved among different serotypes (Sugiyama et al., 1987; Arikawa et al., 1989). Recent studies have demonstrated genetic divergences between Hantaan 76-118 (HTN) and Puumala/Hallnas Bl (Hallnas Bl), and Sapporo rat virus (SRll, antigenically related to Seoul virus) by comparing the M genome segments (Giebel et al., 1989; Arikawa et al., 1990). However, only the M segment of HTN, the prototype strain, has been hitherto cloned and expressed as an entire gene using vaccinia virus or baculovirus recombinants (Pensiero et al., 1988; Schmaljohn et al., 19901. HFRS is mainly prevalent in Asia and Europe. Approximately 200,000 cases of HFRS occur annually with the case fatality varying from 2% to 10% (Yanagihara, 1990). No vaccine is available for Hantavirus infections today. However, in China, HFRS has been a major public health problem in recent years with both the reported incidence and geographic regions rapidly increasing. 115,600 hospitalized HFRS cases with 2,600 deaths (more than half of the total incidence in the world) were reported in 1986 (Disease Surveillance in China, 1987). Two disease syndromes are associated with viruses from Apodemus and Rattus antigenically related to the Korean Hantaan and Seoul viruses respectively, each with distinct geographic distribution, clinical manifestations and epidemiological features (Song et al., 1984). Although much effort has been expended on serological analyses of the Chinese isolates and development of an inactive Hantavirus vaccine to control the disease in China, none of these Chinese isolates has yet been well characterized at the genome level. In this paper, utilizing the polymerase chain reaction (PCR) procedure, we amplified a cDNA of the entire M segment genome of Hantavirus R22 strain

37

(R22), which was isolated from a Ruttus ~~ruegicus in China. This virus is antigenically related to Seoul virus, and apparently causes a more moderate disease than the Apodemus associated virus in this area. The M segment genome of R22 was then well characterized and expressed in a vaccinia vector.

Materials and Methods viruses and cells Hantavirus R22 strain, isolated from lung tissue of Ruttus norvegicus in China (Song et al., 1984), was plaque-purified and passaged in Vero E6 cells as previously described (McCo~ick et al., 1982; Schmaljohn et al., 1983). Vaccinia virus was the New York Board of Health (NYBH) strain obtained from a vial of Wyeth smallpox vaccine (Buller et al., 1985). Plaque-purified vaccinia virus and vaccinia virus recombinant RMV9 were propagated in CV-1 cells or Human TK-143 cells as described (Mackett et al., 1985; Morrison et al., 1989). RNA extraction, PCR amplification and molecular cloning

R22 virus was concentrated and partially purified from the supernatant of infected cells by PEG precipitation and centrifugation twice at 25,000 rpm for 3 h (Beckman, SW41 rotor) on a 30% sucrose cushion. Viral RNA was extracted from the virion as previously described (Schmaljohn and Dalrymple, 1983). An oligonucleotide was synthesized ~ntaining 16 bases at the 5’ end of virion RNA of R22 M segment and a BamHI site (5’ CCGGATCCTAGTAGTAGACTCCGA) as the primer for synthesis of first-strand cDNA and PCR amplification. First-strand cDNA synthesis was primed with this oligonucleotide using AMV reverse transcriptase (Life Sciences Inc.). An RNA-DNA hybrid was used for PCR amplification (Perkin-Elmer Cetus Instruments), undergoing 25 cycles. PCR products were digested with BarnHI, and the residue electrophoresed in 10% agarose gel containing 10% DMSO and purified by electroelution onto EDTA-nitrocellulose (Auperin et al., 1988). The purified PCR products were ligated into the BumHI site of the plasmid pGem 3 zf(+ ), then cloned and identified (Grunstein and Hogness, 1975; Maniatis et al., 1982). Nu&leotide sequence analysis

The plasmid recombinant (RC3), with an approximate 3.6 kb insert, was selected for sequencing. The insert, digested with BamHI and purified as above, was further cut by Sau3A1, S&I and JfinfI respectively and then subcloned into the corresponding sites of Ml3 bacteriophage. DNA sequences were determined by the dideoxy chain termination method with the Sequenase kit (United States Biochemical Corporation). Sequence data including potential open reading frames, translation of a DNA sequence, secondary structure of the RNA, comparison of

38

I

pGem3zf (+)

e _ - . . . . Lacz ~ i

v

\ BamHl

T4 D‘NA

I

P

we

P

T

‘r \

LIGASE -3

LacZ =&lp

Fig. 1. Derivation of chimeric vector, RC3-XN3. R22 M segment gene was cleaved from RC3 and ligated into the vaccinia virus transfer vector pNVV3. TK, and TK, denote the left and right positions of the vaccinia virus thymidine kinase gene. The arrows indicate the 5’ to 3’ orientations of the Lac Z and R22 virus glycoprotein gene. Relevant restriction enzyme sites are indicated.

the deduced amino acid sequences, and hydropathy plots were analyzed with Genetics Computer Group’s Sequence Analysis Software Package (Version 6.1989, Biotechnology Center, University of Wisconsin, 1710 University Avenue, Madison, WI 53705). Construction of vaccinia virus recombinant

The cloned M genome segment was excised from the plasmid recombinant RC3 (Fig. l), and purified. The purified gene was blunt-end ligated into the SmaI site of the transfer vector pNVV3 (a gift of Dr. J.L. Whitton), fused downstream of P7.5. The orientation of the gene in the chimeric vector, RC3-XN3, was determined by restriction enzyme digestion and sequencing through the ligation junctions (Maxam and Gilbert, 1980). Recombination by cotransfection of vaccinia-infected CV-1 cells with recombinant plasmid (RC3-XN3) and Wyeth vaccinia DNA followed established protocols (Mackett et al., 1985). The recombinants were plaque-purified three times and tested for insertion of M-segment cDNA into the TK locus by Southern blot hybridization. Northern and Southern blot hybridization

Viral RNA was fractionated on 1.0% agarose glyoxal, transferred onto a GeneScreen membrane

gel after denaturation with (New England Nuclear, No

39

NEF972), and analyzed by Northern blot using a PCR amplified cDNA probe. The cDNAs from PCR amplification, cloned plasmid RC3 and the vaccinia virus recombinant were electrophoresed in 1.0% agarose gel. After denaturation of the gel with 0.3 M NaOH, DNA was blotted onto GeneScreen membranes. Hybridization was under stringent conditions (50% formamide). Radio-immunoprecipitation

[35S]methionine-labeled polypeptides were immunoprecipitated from cytoplasmic extracts derived from R22 virus and vaccinia virus recombinant infected E6 cells using rabbit anti-R22 serum. The polypeptides were separated by electrophoresis on 12.5% polyacrylamide gels (Elliott et al., 1984). Immunofluorescence

Gl and G2 expression in acetone-fixed CV-1 cells infected with vaccinia virus recombinant was assayed by indirect immunofluorescence using mouse monoclonal antibodies FB03 (for Gl) and HC02 (for G2) (Rue et al., 1991).

Results PCR amplified cDNA and molecular cloning

The choice of primer was based on the 3’ terminal sequence data of R22 viral M segment RNA obtained from standard molecular cloning techniques (data not shown), using the hypothesis that R22 virus M segment RNA shares terminal complementary structures with HTN (Schmaljohn et al., 1987). A cDNA fragment about 3.6 kb in length was amplified with this single primer using the PCR technique (Fig. 2). To confirm the viral specificity of the PCR product, the virion RNAs were hybridized by the nick translated PCR amplified cDNA probe, showing that only M-segment RNA was specifically hybridized (Fig. 2). The apparent fragment size and ability to hybridize to R22 virion RNA indicates that this PCR amplified cDNA fragment contains the complete reading frame for R22 Gl and G2. We, therefore, inserted the fragment into the plasmid. The identity of the cloned insert was confirmed by Southern blot hybridization (Fig. 2). Nucleotide and deduced amino acid sequences

The nucleotide sequence of R22 M segment was determined by the sequencing strategy shown in Fig. 3. The complete sequence of the 3’ end of R22 M segment which consisted of 3656 nucleotides, presented as viral complementary RNA, is shown in Fig. 4. The base composition of the complete sequence of R22 M segment contained 30.7% A, 30.0% U, 20.6% G and 18.7% C. At the 3’ and 5’

40

M

A

S

D

E

28s

23.13Kb 9.42Kt 6.56Kb

_,\

4.36Kb

18s

Fig. 2. Identification of PCR amplified and cloned cDNA. (MI, molecular weight, lambda DNA digested with HindHI; (A) PCR amplified M segment cDNA. (B) DNAs from RC3 clone digested with EarnHI, the lower band is the plasmid DNA; PCR amplified M segment cDNA (0 and RC3 cloned DNA (D) probed by a synthetic internal primer (nucleotides 721-736: 5’ ATCAGCAATGGGCTCC) selected according to the partial sequence data obtained by the standard molecular cloning technique (data not shown). (E) R22 virion RNA probed by PCR ampli~ed M segment cDNA.

terrnini of R22 M genome segment RNA, 20 bases were perfectly matched, and were the same as those of HTN and Hallnas Bl. The free energy for the first 50 bases of this complementary structure was - 60.4 kcal/mol.

-4Fig. 3. DNA sequence strategy. The arrows represent the direction and extent of individual DNA fragments obtained by the digestion of restriction enzyme, HinfI, Suu3A1, and $9~1. Asterisk indicates the DNA fragment was sequenced by a synthetic primer (nucleotides, 721-736).

41

The 3402 bp single open reading frame of the R22 M segment extends from the AUG translation initiation codon at nucleotide 47 to a UAA translation termination codon at nucleotide 3449 with 3’ 46 base and 5’ 205 base untranslated regions. An 1134 amino acid precursor protein is encoded by this open reading frame with a molecular weight of 126.4 kDa (Fig. 4). No additional ORF was found with initiation and stop codon encoding 60 or more amino acids, consistent with results previously reported for HTN, Hallnas Bl and SRll (data not shown) (Schmaljohn et al., 1986; Giebel et al., 1989; Arikawa et al., 1990). As in HTN, the gene order of the R22 M segment is 5’-Gl-G2-3’, and the cleavage signal sequence for Gl and G2 located at amino acids 641-648: 6-L5-W4-A3-A2-S’-A*A+lE+2. The mature G2 should originate at amino acid 647 (Ala), according to the previously described patterns of the amino acids near the signal-sequence of cleavage site (Van Heijne, 1983) and the cleavage sites of HTN (Schmaljohn et al., 1987). On basis of the above prediction, 646 and 488 amino acid polypeptides with 72206 Da and 54178 Da would be encoded in Gl and G2 regions; in reasonable agreement with those estimated (80 kDa for Gl and 62 kDa for G2) from SDS-PAGE, if the potential asparagine linked sugars were included. Six potential asparagine-linked glycosylation sites were found in the deduced R22 glycoproteins, and among them four of five in Gl region and one in G2 region were conserved with HTN and Hallnas Bl. The hydropathic plot of R22 was similar to that of HTN. The hydrophobic N and C termini as well as the short hydrophobic sequence preceding Gl and G2 are likely to serve as signal peptide or transmembrane anchor regions (Fig. 5). Comparison of A4 segment

Before comparing the sequence of R22 M segment to those of SRll, HTN and Hallans Bl, we estimated the accuracy of this PCR amplification since the frequency of errors for Taq polymerase during a 30 cycle amplification has recently been reported from 0.03% to 0.25% (Saiki et al., 1990; Murphy et al., 1990). The nucleotide sequence of the first 1660 bases in 5’ viral complementary sense cDNA amplified by PCR was matched to the sequence in the same region obtained by the standard molecular cloning technique, and no mismatched base and no additions or deletions were found (data not shown), suggesting that the error frequency, if any, is very low in this PCR amplified cDNA. The homologies for entire deduced amino acid sequences between R22 and HTN, and Hallnas Bl were 74% and 53%, and for Gl region were 70% and 45%, whereas the homologies for G2 region were 81% and 60% respectively, indicating that the G2 of Hantaviruses has relatively higher degree of conservation than the Gl. This result also agrees with the recent comparison of SRll to these two viruses (Arikawa et al., 1990). Precise analysis of amino acid substitution sites between R22 and HTN, and between SRll and HTN revealed that R22 and SRll, two independent Rattus-derived isolates, share amino acid subsititutions at 247 common sites with the HTN glycoprotein. However, between R22 and HTN, there are 37 more amino acid changes, which are conserved between SRll and HTN,

42

U"AC"GGCCGC"""AG""GGCCAAGGC""UGCAUU I. LAALVGQGFALKNVFDMRI

AAAAAAUGUUUUUGACAUGAGRAUU

121 25

CAGUG"CCCCACUCAGC"AACUUUGGGGG-C~G"G"GUCAGGC"A"ACAG~U"GCCC QCPHSANFGETSVSGYTELP

18, 45

CCACUCUCAUUACAAGAGGCAGAACAGCUAGCUAGAGAC P L SLQEAEQLVPE

241 65

SSCNMDN

CACCAA"CAC"CUCAACAA"ARAURAAUUAACCAAGGUCGUAUGGCG HQSLSTINKLTKVVWRKKAN

AAAAAAAGCAAA"

101 85

CAGGAAUCAGCAAACCAGRAUUCA"""G~G""G"GGAGAG"G~G"CAGCUU"AAAGGG QESANQNSFEVVESEVSFKG

361 -35

""AUGUAUGUURRAGCAUAGAAUGGUVGRAGAAUCAUAUAUC LCMLKHRMVEE SYRNRRSVI

121 iZ>

UGUUA"GA"C"AGCCUG"AA"AG"ACAAC"GU""ACA"GA"~G""CC" C Y D LACNSTFCKP T"YMI"P

481 145

AAACAUGCUUGCRACAUGAUGAAAAGUUGUUGUUUGGCCUUCAG K HACNMMK s c I. I GLVPYRIQ

541 165

GUUGUCUAUGRILAGGACAUACUGCACUACGGGUAUAUUG V" Y E R T Y C T T G I

L

T

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G

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c

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601 185

CCCGACAAGGC"G"UG"CAGUGCAUUGAAGAGAGAGGCA"G"A"GCCA"AGC~GCA"AGAG P DKAVVSALKRGMYAIASIE

661 205

ACAA"C"GC""U"""A""CA"CAGGGAAUACAUAUA"AG"GAC"GCCA~CACA T I CFFIHQKGNTYKIVTAIT

721 225

"CAGCRA"GGGC"CCAAG"GCAAUAAUACAGAUACAGGGGA"A""A"A"C~G~ SAMGSKCNNTDTKVQGYYIC

781 245

AUUAUUGGUGGAAACUCUGCUCCUGUAUAUGCCCCCGCUGUG I I G SAPVYAPAGEDF G N

R

A

M

54: 165

GAGG"""""UCUGGGA""A""ACCGCAUGGRGAAGG EVFSGIIT SPHGEDHDLPAK

301 285

ARA"UCGCAACAUACCAGA"""CAGGGCAAUCCC"CACACAG"GAGC K FATYQI SGQIEAKIPHTVS

961 305

"C"AAAAAC""GAAA""GA""GCCU~~GCAGG"A"CCA"CAUAC"CAUC~C"AG"AUA AFAGIP SYSSTSI SKNLKLI

1021 325

""GGCUGCUUCAGAAGACGGUCGUUUCAUAUUUAGUCCUG" LAASEDGRFIF SPGLFPNLN

1081 145

CAAUCAGUCUGUGACAACAAUGCACUCCCUUUAAUCUGGACG ~S"CDNNALPLIWRGL1DT.T

1141 365

GGAUACUAUGAGGCAGUCCACCCUUGCAAUGURUUCUGUAUUCUGUGUCUUAUCAGGACCAGGUGCU GYYEAVHPCNVFCVLSGPGA

1201 385

UCAUGUGAAGCCUUUUCAGAGGGAGGUAUUUUCAACAUUACUUCUCC~UGUGUCUGG~G SCEAFSEGGIFNITSPMCL"

1261 495

"CCAAGCAAAA"AGGU""AGAGCAGCAGCAAAUCAGC~~CAGC""UAU~~GCC~GGG"" SFICQR" SKQNRFRAAEQQI

1321 425

AAAAAMGACAAUAUUAACAAAAACRUUA GAUAUGGAUAUUAUAGVGUACUGUAAUGGUAAUGGUC D MD I IVYCNGQKKTILTKTL

:x8?

GUUAUGGCCAGUGCAUUUAURCUAUUACAAGUCUCUUUUC "MA S A F I LLQVSFHCY

,441 <6,

Q

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.50! I 8 '>

UUGAUCACAUUUUGCUUUGGCUGGGUAUUGAUUCCUGCAUGCACAUUAGCUAUUCUUUU.~ L I T F CFGWVLIPACTLAILL

1361 105

GVCC"AAAG"""""UGCAAA"A"CC""CAAGCRAUCGCC "LKFFANILHTSNQENRFKA

1621 52'

A""C"ACGGAAAA"CAAGGAGGAGU"UG ILRKIKEEF

E

UG"AAGUACGAG"G"GRRACA""-GGCAC L K CKYECET

E

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L,-. 10

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GGGGAGUGCCCAUAUUGCUUUACGCACUGUGAACCGACAG,, Y c F T H c E P T G E C P

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.r>J. 5.i>

43

UAC~GUUU~UC~G~CACCCACCGAUUCAGAG~GAUUU REDLKKTVTP YKVCQATHRF

AAAAAAGACUGUAACUCCU

AAARRAUAVUGGGCCAGGCUGUACCGRACAUu~UCuuuUUUi: KKYWARLYRTLNLF

RYKSRC

!a61 60.: 12)’ ii’

UAUAUUCUAACAAUGUGGACUCUUCUUCUCAUUA~UG~UCUAUC~WCUGGG~GGC~GU IESXLWAAS Y I L T M w T L L I. I

!981 64’

x2 GCAGU\t~“fCCUCUUiT~~~~~~~UGGA~A~U~”G~U~A~~~~UUGGGAG~GU~ AAETFLVPLWTDNAHGVGSV

iO4i 66:.

CCUAUGCAWAGGAAuACUUAVGAAUUAGACUUVUCUUUCCCAUCUA~CUCu~GuACACA PMHRNTYELDFSFPSSSKYT

2101 685

UAURRAAGGCAUCUULCAAACCCAGUUAAUGACCARCAGAGUGUCVCAUUG~AUAUAGM YXRHLTNPVNDQQSVSLHIE

216: 705

AUUG~GUCABGGCAUUtGtrGCUG~G~UGU~A~ChU~UUGGACAUG IGADVHHLGNWYDAR I E S Q G

2221 725

UUAAAVCUGAARACCUCAUVCCAUUGUUAUGGUGCCUGCAUAUCCAUGG LNLKTSFHCYGACTKYQYPW

2281 745

CACACUGC~UGCCAUUWGAG~GAUUAUGAGUAUG~UAGC~GGGCAUGC~C HTAKCHFEKDYEYENSWACN

2341 765

CCCCCAGAUUGUCCAGGGGUUGGUA~GGUUGUACUGCCUGUGGA~WAUAUCUAGAUC~ PPUCFGVGTGCTACGLYLDQ

2401 785

UUARAGCCGGUAGCAACACCCUUUURG~UUAU~G~GU~GAUAUAGUA~GUGUGC SVRYSRKVC LKDVATFFRLI

2461 SO5

GUGCAGWWUGGUGAAGAWUUGCAAAACARUVGAWAUG~U~AUUGCUUUGUGACU “QFEEEYLCKTIOMNDCFVT

2521 825

AGGCAUGCCAAAAUAUGUAUAAVUGGGACUGUAUCUAAGUUUUCW~~~GUGACACUCUA RHAKICIIGTVSXFSQGDTL

2581 845

CUAUUilCWUGGACCCAUGGAAGGAGGUGGUAUAAUCUUC~C LFLGPMEGGGIIFKHWCTST

264’ 365

UGUCACUUUGGAGACCCUGGUGAUGAUAUGGGUCCARAAGUUAUUUGCC~U CHFGDPGDRMGPKDKPFICP

2701 885

GAAWCCCAGGGCAAWCAGGMAAAA KFPGQFRKKCNFATTPTCEY

UGUAACUUUGCCACAACWCCAAUUUGUGAAUAU

2761 905

GAUGGG~CAUUAUCUCAGGUUA~~G~GUCCUUG~~~UW~UUCUUUUC~U~A DGNTISGYKKVLATIDSFQS

2821 925

UUURRCACAAGCAAUAUACACUUCACUGAUGAGRGAAUUG~U~A~~ACCCUGAUGGU FNTSNIHFTDERIEWRDFDG

2881 945

AUGC~CGG~~AUAUU~~AUCGUffAUU~U~~~U~GAUVU~~~UGG~U ISKDIDFENLA M I, R D H I N I V

2941 965

GAGAAUCCWGUAAAGUAGGGCUCCAGGCAGC~CAUAGMGGUGCUUGGGGU~CAGGW IEGAWGSG ENPCKVGLQAAN

3001 985

GUCGGGVWUACACUCACAUGCCAGGUGUCUCUCACAU\UVUCuUACGUCA VGFTLTCQVSLTECPTFLTS

3061 1005

AU~GGCCUG~A~UGGCAAUUVG~UAU~U~~A-G~~V~~~~U~CGA~GA IXACDMAICYGAESVTLSRG

3121 1025

CAARAuACUGUCAGGAUUACUG~~GGUGGCCAUAGUGGUUCUUCAUUU~UGC~GU TGKGGHSGSSFKCC QNTVRI

3181 1045

CAUGGG~GRAUGUUCAUCRACUGGCCUCCRAGCCAGUGCRCGUA TGLQASAPWLDKV HGKECSS

3241 1065

RAUGGUAUCUCUGAGUUAGAAAACGAGAAAGUUUAUGAUGGUGGC NGLSELENEKVYDDGAPECG

3301 1085

GUUACU~G~GGUUU~ VTCWfKXSGEWVMGlINGNW

UCAGGUG~UGGGUUAUGGGUAU~U~~&GGG~CUGG

3361 lit35

GUUGUCCilAAUUGUUUUGUGWGVCCUGUUACUCUUUUCU~UUAUCCUGUU~GCAWU~UG VVLIVLCVLLLFSLILLSIL

3421 1125

UGUCCUGWOAGAAAGCAU~UCAUAAAUCCUACCCAVU~W~~VCACACCAUGUAV CFVRKHKKS CGAAUUU~~CACUUUACCAUUU~CAACUUAACCUGGCUCUAAUAUCU~U~CU~

3481 1134

CVVVCAUU~UUAUVVVUAVAVGGACURRULTACV ALICUU~~~VUGAVV~ACCGGGGUGIIUGUCVUGACAtl

~VACUCUCUUCVAUCUCCU 3656

Fig. 4. Nucleotide sequence representing the virus implemental sense RNA of entire M segment gene of R22 virus, and the deduced amino acids through the open reading frame shown under their codons. The deep printing amino acids indicate the potential glycosylation sites.

~22 GP

30 1) Gl

40

1

-1-I

1

--rf--cl-l-*l-l-~I-1-1-l--l~-lT17-l--I--1-l---l~l-t, 101

20,

301

401

5ot

601

701

901

901

ICI01

,,O,

Amino AckISoquenco Numbor

Fig. 5. Hydropathic plots (Kyte and Doolittle, 1982) of amino acids predicated from HTN 76-i18 and R22 (B) M segments. The potential glycosylation sites (N x S/T) are labeled with asterisks.

(A)

whereas there are only 6 amino acid changes vice versa. This indicates that more amino acid substitutions occur in glycoproteins of the Chinese rat isolate R22 than in Japanese rat isolate SRll compared to HTN, the prototype Hantavirus. Very high homologies of 94% and 95% for nucleotide and putative glycoprotein sequences were found between two intragroup viruses, R22 and SRll. In addition, all six potential asparagine-linked glycosylation sites (five in Gl, one in G2), cleavage signal sequence for Gl and G2, and 95% cysteine were also conserved (Fig. 6). However, some intragroup divergence exists: R22 M segment is five nucleotides longer than SRll. Two of them are in the 5’ untranslated region, and another three in the reading frame located at the nucleotides 2058-2060 resulting in one more codon in G2. Additionally, although there are 21 substitutions of amino acids throughout the Gl and G2 limited to single amino acid changes, some

45 40

Xl P.22 (R): M--WSL------LLLAALVGQGFALKN"FDMRIQ------CPHSANFGETS"SG SpJl(S): *__*++-___--+r*t******************______L********~**** nm (a): *GI*K~-_____*~'S**~PVLT*R"Y'*K*E--------***T"S****N**~ B1 (8): *--GE*SP"CLY***----**LL*C=TGAA*NLNELKME***TIRL*QGL*"*

97 FL:

S: 8: 8:

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157 R:

ESEVSFKGLCMLKHRMVEESYRNRRSVICYDLACNSTFCKPTVYMI~KHACNMMKSCLI

s: ****t****tt*****tt******l********",*******.*"*~.*,*********.* 8: *: It:

s: 8:

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B:

S*GDQ****N**K***"S'D*V*'I"N*IHTMALSaPSH**L"~K*---

R:

YKIVTAITSRMGSKC-DTK"Q---------GYYICIINS" *********tt****t~~t****__________***rt*******~*~********,*****

267

s: S: S:

325 R:

FSGIITSPHGEDHDLPAKKF--ATYQISGQIE~IPHTVSSKNLKLIAFAGIPSYSSTSI

s: ++**********t***GESI__*****"*'+*****'*******~********".*** 8: +T**FH****"'*'AGSSI__'S*S*S*"*~~**"**SA**DT*S***YS******,L** B:

L'RMAFA****+'HDIE*NA"S*MR-*A*KVTG*V*S*E**DYVQG**DY"QG***S*S*L*T**G"

Fl:

LAASEDGRFIFSPGLFPNLNQSVCDNNALPLIWKGLIDLTGYYEAVHPCNVFCVLSGPGA *********t*tt**************************t,***"**,*~**********

385

s: 8:

*~S’~~*~~“*+.“**K*‘BTN’*KS*I**“T’M***P*,*******~**~*.*****

S:

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Fig. 6. Continued

on p. 46.

46 It: S: 8: S:

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1134

Fig. 6. Comparison Asterisks indicate

of the predicated amino acid sequences between R22, SRII, HTN, Hallnas Bl. identical amino acids; minuses indicate missing amino acids. Bold printed amino acids show the potential glycosylation sites (NxS/T).

appear to be clustered Whether these random Recombinant

(amino acids: 283-286, 447-467, 606-612 and 669-672). coding changes play a role in viral antigenicity is uncertain.

cirus construction

The insertion of the R22 M shown in Fig. 1. The gene is element and the initiation codon late transcription initiation sites

segment gene into the transfer vector pNVV3 is located downstream of vaccinia P7.5 promoter occurs 93 and 147 nucleotides from the early and respectively. Southern blot analysis (Fig. 7) con-

Fig. 7. Southern blot analysis of the insert of vaccmia virus recombinant RMVY. CM), molecular weight, lambda DNA digested with HindIII; RMVY DNA (A), chimeric transfer vector RC3-XN3 DNA (B), and vaccinia virus DNA (C) digested with BarnHI and Not1 were hybridized with the cloned R22 M segment DNA.

B

C

Fig. 8. Radio-immunoprecipitation with rabbit anti-R22 sera. The ceils were labeled with [%]methionine. (A) R22 infected E6 cell iysates. (B) RMV9 infected E6 cell lysates. (C) Mock infected E6 cell lysates. (D) Wyeth strain infected Efi cell lysates. The positions of the nucleoprotein (N) and mature glycoproteins (Gl and G2) are indicated.

firms that R22 M segment is correctly recombined into the vaccinia TK gene by digestion of the recombinant vaccinia RMV9 with BumHI and Not I. Synthesis of R22 Gl and G2 viral proteins by the recombinant virus RMV9 was observed by radio-immunoprecipitation with high titer rabbit anti-R22 serum, demonstrating that R22 Gl and G2 expressed by the vaccinia virus recombinant RMV9 are comigrant with authentic R22 virus Gl and G2 (Fig. 8). In order to determine the cellular locations of the expressed R22 glycoproteins, indirect immunofluorescen~e assay with monoclonal antibodies FB03 and HC02 was performed on RMV9 infected cells at 36 h postinfection. In contrast to vaccinia virus infected cells, strong fluorescence in the glycoprotein gene construct infected cells was found (Fig. 91, similar to cellular distribution of R22 virus infected cells (data not shown). Discussion

The PCR technique was recently used with Hallnas Bl and SRll viruses for synthesis of several smal1 fragments of cDNA to overlap the incomplete sequences of M genome segments obtained by the standard molecular cloning method. (Giebel et al., 1989; Arikawa et al., 1990). Here we successfully used PCR to amplify the cDNA of the entire R22 M genome RNA segment, containing 3656

48

Fig. 9. Expression of R22 Gl and G2 by vaccinla wrus recombinant in CV-1 cells: RMV9 infecte :d cells detected by monoclonal antibody FB03 (A) and HC02 (B); Wyeth strain infected cells detected by the mixture of FB03 and HC02 (0. The detection was performed by indirect immunofluorescent iIssay.

49

nucleotides, and we subsequently expressed the product in a recombinant vaccinia virus. The identity of the product was confirmed by monoclonal and polyclonal antibodies to the R22 viral glycoproteins, and by size on polyacrylamide gels. This extends the use of PCR beyond facilitating the accumulation of sequence information of Hantavirus M segment gene by obtaining, in one operation, the entire functional gene encoded by the M segment. The M genome segment of R22 virus, a rat isolate from China, resembles those of SRll, a rat isolate from Japan, HTN, the prototype Hantavirus isolated from Apodemus in Korea, and Hallnas Bl isolated from a Swedish bank vole. Common properties include the encoded structure, gene order, hydropathic plots, the cleavage pattern of Gl and G2, and most glycosylation sites. This confirms a high level of major structural conservation in Hantaviruses, though these isolates are from distinct hosts and geographic areas. On the other hand, direct comparison of R22 glycoprotein sequences with the three other Hantaviruses gives homologies of 95% (SRll), 74% (HTN), and 53% (Hallnas Bl), indicating genomic diversity. This provides molecular evidence to support the relatedness among these four viruses previously reported in antigenicity and in pathogenesis. Marked homogeneity was revealed between R22 and SRll from Rattus from different geographic areas, suggesting that Hantaviruses are relatively well conserved in the same host reservoir despite wide geographic separation, whereas strains from different host species in the same geographic area are associated with considerable genetic variability. Oligonucleotide fingerprinting analyses have demonstrated different RNA patterns in the serologically closely related isolates from Rattus in the USA (Schmaljohn et al., 1985). In our experiment, the precise positions of nucleotide or amino acid substitutions and deletions were identified in M segments in geographically disparate isolates of the same serotype. Furthermore, more amino acid substitutions were found in R22 and HTN than in SRll and HTN. Thus, R22 virus may have its own independently evolving process in China. Recent analyses of 35 Rat&s-derived strains from China using monoclonal antibodies binding to 10 distinct epitopes show four different reaction patterns (reviewed by Lee et al., 1989). Comparison of two rat isolates in Japan (SRll and KI-262) revealed that SRll causes fatal infection in suckling mice whereas KI-262 does not (Zhang et al., 1990). Thus, even within the same serogroup, Hantaviruses seem to be composed of variants which may display significant differences in antigenicity and pathogenicity. However, minimal divergence in hydrophobic and highly charged domains, and 95% conservation of cysteine residues on the glycoproteins between R22 and SRll suggest to us close similarities in secondary structure and in antigenicity. Meanwhile, we consider that the further identification of precise functional domains by M segment gene truncation is necessary for better understanding of the biological significance of strain variation. Two eucaryotic virus vectors, vaccinia virus and baculovirus, are popularly used to express the genes of pathogenic viruses. Both vaccinia virus and baculovirus (AcNPV) recombinants expressing HTN virus glycoproteins were recently reported. Unlike the vaccinia virus recombinant, the baculovirus recombinant can

50

only elicit a very low level of antibody response in animal models due to relatively low expression of HTN M segment gene in the infected cells (Schmaljohn et al., 1990). In our hands (unpublished), several attempts to construct an AcNPV baculovirus recombinant for the R22 M gene segment were unsuccessful because of low expression. Low expression of M segment gene also resulted from using cell free translation system for specific transcripts of R22 M segment RNA (data not shown) and HTN M segment RNA (Schmaljohn et al., 1987a). The relationship between structure and low translatability of M gene segment of Hantaviruses is unclear. For this reason, attenuated vaccinia virus is the vector of choice for design of a novel strategy to study the functional role of the R22 virus genes. The expression of the R22 M gene segment in vaccinia virus recombinants produces glycoproteins that are antigenically identical to R22 Gl and G2, and the vaccinia virus recombinant for the R22 S segment also expresses a nucleoprotein identical to that of R22 virus (data not shown here). These vaccinia virus recombinants for Rattus-derived virus glycoproteins and nucleoprotein offer a first opportunity to investigate the function of the differing genomic structure and to evaluate the potential efficacy of a recombinant vaccine for Seoul Hantavirus serotype.

Acknowledgments

We thank Drs David D. Auperin, Anthony Sanchez and Cynthia Warner for their advice regarding this study. We acknowledge Dr. C.S. Hang for supplying R22 virus and Dr. Peter Coyle for supplying the partial sequence data of R22 M segment.

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