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Nucleotide sequence of the glycoprotein gene and intergenic region of the Lassa virus S genome RNA
VIROLOGY
154,155-167 (1986)
Nucleotide Sequence of the Glycoprotein Gene and lntergenic Region of the Lassa Virus S Genome RNA DAVID D. AUPERIN,l DONNA R. SASSO, AND JOSEPH B. MCCORMICK of Viral Diseases, Center for I@ectious Diseases, Centers for Disease Control, Atlanta, Georgia 303%
Special Pathogens Branch, Divisim
Received March 18, 1986; accepted June 12, 1986 Two overlapping cDNA clones corresponding to the 5’ region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an amhisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5’ terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3’ termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression. Q 19% Academic PEWI, IIIC INTRODUCTION
Lassa virus was first isolated in 1969 associated with a fatal febrile illness in humans in northeast Nigeria (Frame et aL, 1970). Since then, illness or serologic evidence of infection with Lassa virus has been demonstrated in numerous West African countries. The natural host of the virus is the multimammate rat lMastomys natalensis from which isolations have been made in Nigeria and Sierra Leone (Monath et aL, 19’74; Murphy and Walker, 1978; McCormick et aL, 1986; Walker et al, 1975). Morphologic, antigenic, and biochemical properties of the virus have established it as an arenavirus (Casals et uL, 1975; Clegg and Lloyd, 1983; Kiley et aL, 1980; Murphy et al, 1970). The arenavirus genome consists of two molecules of single-stranded RNA designated L (large) and S (small) (Rawls and Leung, 1979; Vezza et CAL,1978). The sizes of the L and S RNAs are approximately 2.5 X lo6 and 1.1 X lo6 Da, respectively. Genetic i To whom correspondence should be addressed.
and biochemical analyses have established that the S RNA encodes the nucleoprotein (N) and the precursor to the glycoproteins (GPC) (Harnish et cd, 1983; Vezza et aL, 1980). The L RNA codes for a large polypeptide presumed to be the RNA-dependant, RNA polymerase component of the virus (Harnish et aL, 1983). Recent nucleotide sequence analyses on the S genome RNA segments of Pichinde and lymphocytic choriomeningitis (LCMV) viruses have demonstrated that the N and GPC genes are encoded in nonoverlapping reading frames originating from opposite ends of the S RNA (Auperin et aL, 1984a, 1984b; Romanowski and Bishop, 1985; Romanowski et aL, 1985). The N gene is encoded by viral complimentary (vc) RNA sequence corresponding to the 3’-half of the molecule and the GPC gene is encoded by viral (v) RNA sequence corresponding to the 5’-half of the molecule. The term “ambisense RNA” has been used to describe this coding strategy. The reading frames encoding the N and GPC genes are separated by a short, noncoding, intergenic region, the nueleotide sequence of which is such that a hair-
155
0042~6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
156
AUPERIN,
SASSO, AND
pin structure of some 18-22 bp can form. In view of the location of this structure between the termination codons for the N and GPC genes, and the size of the subgenomic mRNA species for each gene, it is postulated that the hairpin functions as a transcription terminator (Auperin et al, 1984b). The nucleotide sequence of the 3’ region of the S genome RNA of Lassa virus (Nigerian strain) has recently been reported (Clegg and Oram, 1985). Like Pichinde arenavirus and LCMV, the viral nucleoprotein gene was found to be encoded in vcRNA sequence from this region of the molecule. We have initiated studies to molecularly clone the GPC gene of Lassa virus for its possible use in a genetically engineered vaccine. Here we report the nucleotide sequence of the 5’ region of the S genome RNA of the Josiah strain of Lassa virus. The sequence contains the complete GPC gene, the intergenic region and the carboxy-terminal position of the N gene.
MC CORMICK
albumen (BSA) and stored in liquid nitrogen vapors. Virus purification was achieved by successive rounds of centrifugation in gradients of 30% glycerol/50% potassium tartrate (Vezza et a& 1978) and 20-60% sucrose. Virions banding in the sucrose gradient were harvested, diluted with TE (10 mil4 Tris-HCl, pH 7.4, 1 mM EDTA), and pelleted through a cushion of 30% glycerol (in TE). Purified virus was gently resuspended in low salt buffer (LSB) (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.14 M NaCl), and stored in liquid nitrogen vapors. RNA extraction, To purified virus in LSB, sodium dodecyl sulfate (SDS) was added to a final concentration of 1%. The mixture was extracted two times with an equal volume of phenol:chloroform (1:l). To the final aqueous phase, NaCl was added to 0.4 M and the RNA precipitated with the addition of 3 vol of ethanol. RNA was collected by centrifugation, redissolved in high salt buffer (HSB) (10 mMTris-HCl, pH 7.4,0.4 M NaCl, 1 mM EDTA) and reprecipitated with ethanol. Final RNA pellets were MATERIALS AND METHODS rinsed in absolute ethanol, lyophilized, and Virus and cells. The Josiah strain of resuspended in sterile deionized water at Lassa virus (reference no. 800593) was iso- 1 fldcll. For cytoplasmic RNA, confluent monolated from human serum taken in Sierra Leone in 1976. The virus was plaque puri- layers of BHK-21 cells were washed two fied three times on monolayers of Vero E6 times in chilld phosphate-buffered saline cells (ATCC no. CRL1586) and a high titer (PBS) followed by one wash in TNE (10 mM stock prepared by passage in BHK-21 cells Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA). Cells were then scraped up in TNE (ATCC no. CCLlO). Virus growth and pur$cation. All ma- (5 ml/150 cm2 of cells) and transferred to nipulations with infectious material were 30-ml corex centrifuge tubes. The cells were performed within the maximum contain- collected by centrifugation and washed ment laboratory at the Centers for Disease once more in TNE. Following centrifugaControl. Lassa virus was grown on tion the cell pellet was resuspended in TNE monolayers of BHK-21 cells nourished plus 1% Nonidet-P40 (four times packed with Dulbecco’s modified Eagle’s medium cell volume) and incubated on ice for 15 (GIBCO laboratories) supplemented with min. Nuclei and mitochondria were re10% fetal calf serum. Cell monolayers were moved by centrifugation and SDS was infected with virus (m.o.i. = 0.1) at 70% added to a final concentration of 1% . The confluency. Following adsorption, media resulting lysate was then extracted with was added and the cells incubated at 37”. phenol:chloroform (1:l) two times and the Virus was harvested by withdrawing the RNA precipitated by the addition of NaCl media from cell cultures, and clarifying by to 0.4 M and three vol of ethanol. The cylow speed centrifugation. Virus was then toplasmic RNA was collected by centrifucollected by ultracentrifugation at 25K gation, rinsed in ethanol, and lyophilized. RPM for 90 min in an SW-27 rotor. Crude Final RNA pellets were redissolved in virus pellets were resuspended in Dulbec- sterile deionized water at 1 pg/pl. co’s medium containing 1% bovine serum cDNA synthesis. An initial preparation
LASSA
VIRUS
of cDNA was synthesized using the synthetic oligodeoxyribonucleotide (5’pCGCACAGTGGATCCTAGGC) that is complementary to the 3’ terminal 19 nucleotides of the arenavirus S genome RNA (Auperin et a& 1984a). The first strand reaction contained vRNA (100 ng/pl); 5’phosphorylated oligonucleotide (4 ng/pl); 50 mM Tris-HCI (pH 8.3); 70 mM KCl; 10 mM MgClz; 10 mM DTT, 1 mM each dATP, dGTP, dCTP, and dTTP; 0.4 &i/p1 rH]dGTP (26.5 Ci/mM) and 0.75 U/p1 AMV reverse transcriptase (Life Sciences) in a volume of 250 yl. Prior to starting the reaction, the oligonucleotide primer was annealed to vRNA templates by incubation at 42’ for 15 min in the presence of the Tris-HCl and KC1 components of the reaction. Following annealing, the remaining components of the reaction were added, and first strand cDNA was synthesized at 42’ for 40 min. The reaction was terminated by the addition of EDTA to 20 mM and the RNA hydrolyzed in 0.1 NNaOH at 70” for 20 min. Following hydrolysis, the reaction was neutralized with HCl, extracted with phenol:chloroform (l:l), and chromatographed over a column of Sephadex G-50 (fine) in TE. First-strand cDNA eluting in the void volume was collected by ethanol precipitation and lyophilized. Second-strand cDNA was synthesized relying on the ability of the 3’ end of first-strand cDNA to form hairpin structures. The reaction conditions were similar to those used for first-strand synthesis except that 5fa-32P]dATP (1 &i/Fl, 32 Ci/ mM) was used to monitor the synthesis and incubation was at 42” for 90 min. The double-strand cDNA products were extracted, chromatographed as before, and collected by ethanol precipitation. The hairpin structures present in the cDNAs were removed by digestion with Sl nuclease (BRL), and the resulting double-stranded molecules repaired with the Klenow fragment of DNA polymerase I (New England Biolabs) prior to cloning. A second preparation of cDNA was synthesized using, as a primer, the singlestrand of DNA complimentary to the viral S genome RNA obtained by strand separating the 298-bp DdeI restriction frag-
GPC GENE
157
ment of clone LS13 (position 995-1293 in Fig. 1). The single-strand DNA primer was annealed to vRNA template in Tris-HCl and KC1 as before except that the hybridization temperature was initially 65” and allowed to slowly cool to room temperature over 4 hr. The reaction conditions were identical to those described above except that 51a-32P]dTTPwas used to monitor the synthesis. The products of the reaction were then electrophoresed on a 3% polyacrylamide gel in 1X TBE containing 7 M urea. Autoradiography of the gel revealed a single species of first-strand cDNA approximately 1300 nucleotides long. This material was eluted from the gel and precipated with ethanol. Deoxyadenosine tails were then added to the 3’ termini in a 30~1reaction containing 120 &sodium cacodylate (pH 6.8), 0.1 mMDTT, 1 mMCoC12, 0.25 mM dATP, and 27 U terminal deoxynucleotidyl transferase (P.L. Biochemicals). The reaction was incubated at 37” for 5 min then extracted with phenol:chloroform and ethanol precipitated. Secondstrand synthesis was primed with 5’ phosphorylated oligo (dT)12-1eand performed as described above. Finally, the doublestranded cDNAs were treated with Sl nuclease and repaired with the Klenow fragment of DNA polymerase I to generate blunt ends prior to cloning. Molecular cloning. Blunt-end cDNAs were ligated into a pUC18 vector at the unique SwzuI site in the poly-linker region using T4 DNA ligase (New England Biolabs). The vector was prepared by first restricting the plasmid DNA with SmaI, then removing the 5’ terminal phosphates with calf intestinal alkaline phosphatase (Boehringer Mannheim). The products of the ligation reaction were transfected into competent Escherichia coli MC1061 cells and transformants selected by their resistance to ampicillin. Plasmid-containing colonies were then replica-plated onto BA85 nitrocellulose filters (Schleicher & Schuell) and screened by colony hybridization (Grunstein and Hogness, 1975) for Lassa cDNA inserts using either a short copy probe made by extending the synthetic oligonucleotide against vRNA (Bishop et aL, 1982) or nick translating the
158
AUPERIN,
SASSO, AND
two times for 15 min each in 2X SSC + 0.5% SDS at 65”; and two times for 15 min each in 0.1X SSC at room temperature, then dried and autoradiographed. DNA sequence analysis. DNA nucleotide sequences were determined on strand-separated, end-labeled, restriction fragments by the method of Maxam and Gilbert (1980) using the formic acid protocol for the A + G reactions as described previously (Bishop et a& 1982).
298-bp DdeI restriction fragment form clone LS13. Hybridization-positive clones were picked and their cDNA inserts sized by comparing their H&f1 restriction fragments to those of pUC18 and pBR322. Large cDNA clones were then verified as S RNA specific by northern blot analyses to vRNA. Northern blot analpzs. Preparations of RNA were denatured by reacting with 1 M glyoxal in 50% demethyl sulfoxide and 10 mM sodium phosphate (pH 7.0) at 50” for 60 min. (McMaster and Carmichael, 1977). Following denaturation, the RNAs were electrophoresed on 1.0% agarose gels cast in 10 mM sodium phosphate (pH 7.0) at a constant voltage of 50 V for 53 hr. The gel was stained and prepared for blotting by soaking in 50 mM NaOH containing 1 pg/ ml ethidium bromide for 30 min followed by four successive 15 min washes in 25 mM sodium phosphate (pH 6.5). The RNA was osmotically transferred to “Genescreen” hybridization membranes in 25 n&sodium phosphate (pH 6.5). After transfer the membranes were dried, baked at 80” for 2 hr, and prehybridized in 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M Na citrate), 1% SDS, 0.04% polyvinyl-pyrrolidone, 0.04% BSA, 0.04% Ficoll, and 100 pg/ml denatured salmon sperm DNA for 16 hr at 42”. 32P-Labeled cDNA probes were then added and hybridization continued for at least 16 hr. Following hybridization, the membranes were washed two times for 5 min each in 2~ SSC at room temperature;
LS13
I
Nucleotide Sequence of the 5’ Region of the Lmsa VimLsS RNA Two overlapping cDNA clones were isolated and used to determine the nucleotide sequence of the 5’ region of the Lassa virus S genome RNA. Figure 1 illustrates the position of the two clones in relation to the S RNA and the strategy used to determine their sequence. Clone LS13 was isolated from a cDNA library generated by priming first-strand cDNA synthesis with the synthetic oligonucleotide known to be complimentary to the 3’ terminus of the arenavirus S RNA. The clone was positioned along the viral S RNA based on the coding capability of its sequence and noting that one end of the cDNA encoded the carboxy terminus of the nucleoprotein gene, the sequence of which had been reported for the Nigerian strain of Lassa Virus (Clegg and Oram, 1985). The cDNA was found to orig-
I-
H,nf
EcoAl
RESULTS
I
LS37
Ddr
MC CORMICK
I I
c---c
J ---
-+,
--
----
f -
l
‘
BarnHI Xho I
-
>
FIG. 1. Overlapping cDNA clones corresponding to the 5’ region of the Lassa virus S RNA and the strategy used to determine their nucleotide sequence. Clone LS13 was derived from a preparation of cDNA generated by priming first-strand synthesis with a synthetic DNA oligonucleotide that is complimentary to the 3’ terminus of the arenavirus S RNA. Clone LS37 was derived from a cDNA preparation generated by initiating first-strand synthesis with the vc strand of DNA obtained from a DbeI restriction fragment (position 995-1293) of clone LS13. Arrows indicate the positions of the 3’terminally labeled, single-strand DNAs that were sequenced by the method of Maxam and Gilbert.
LASSA
VIRUS
inate with the nucleotide sequence encoding the 21 amino acids of the carboxy-terminal portion of the N gene and extend toward the 5’ terminus of the S RNA for 798 nucleotides. The observation that this clone was selected by hybridization to a short copy probe made by extending the oligonucleotide that is complimentary to the 3’ terminus of the viral S RNA indicates that under our conditions of probe synthesis (a large molar excess of primer to RNA template and relatively nonstringent hybridization prior to cDNA synthesis) the oligonucleotide is able to prime cDNA synthesis at positions other than the 3’ terminus. Homology searches of our sequence have failed to indicate any close matches for sequences that are complimentary to the oligonucleotide however, while Northern blot analyses performed prior to colony hybridization confirmed that the probe was specific for S RNA sequences. From the known size of the S RNA species of Pichinde and LCM viruses (Auperin et al, 1984b; Romanowski et ah, 1985), it was reasoned that clone LS13 terminated approximately 1300 nucleotides from the 5’ end of the Lassa virus S RNA. A second preparation of cDNA was then made, using as a primer in a first-strand synthesis reaction, the vc strand of DNA obtained from a DdeI restriction fragment of clone LS13 (position 995-1293). Clone LS37 was isolated from the resulting cDNA library, and nucleotide sequence analysis confirmed that the clone originated with the first nucleotide of the DdeI restriction fragment (position 1293) and correctly terminated at the 5’ end of the viral S RNA, based on sequence homology to Pichinde and LCM viruses. Northern blot analyses shown in Fig. 2 demonstrate that nick translated probes of LS13 and LS37 cDNAs specifically hybridize to Lassa virus S genome RNA. The nucleotide sequence of the 5’ region of the Lassa S genome RNA is given in Fig. 3. In agreement with Pichinde and LCM viruses, a reading frame encoding the GPC gene product is present in vRNA sequence originating at position 56 and terminating at position 1529from the 5’ end of the RNA.
159
GPC GENE LS37
LS13
*
18s
(18s
FIG. 2. Northern blot analyses of BHK-21 cell cytoplasmic RNA and Lassa viral RNA resolved by agarose gel electrophoresis (see under Materials and Methods) and osmotically transferred to “Genescreen” hybridization transfer membrane. Positions of the viral L and S genome RNAs as well as the 28 S and 18 S ribosomal RNAs were visualized by ethiduim bromide staining and ultraviolet illumination following transfer to the membrane. The probes, as indicated, include the cDNAs of clones LS13 and LS3’7.
Assuming the first methionine in this reading frame is the initiating methionine, the glycoprotein precursor contains 491 amino acids and has a calculated molecular mass of 55,820 Da. This compares with 503 amino acids (57,279 Da) and 498 amino acids (56,293 Da) for the corresponding proteins of Pichinde and LCM (WE strain) viruses, respectively. The amino acid composition and net charge of the GPC gene product of Lassa virus is given in Table 1. This protein is comparatively rich in cysteine (3.7%) and histidine (3.9%), similar to Pichinde and LCM viruses. The coding regions of the N and GPC genes do not overlap but are separated by a short, noncoding, intergenic region of 61 nucleotides, which, although not very homologous to the corresponding sequences in Pichinde and LCM viruses, can form a hairpin structure of 16 G-C and 3 A-U base pairs. Figure 4 illustrates the genetic organization of the Lassa virus S RNA and the nucleotide sequence within the inter-
160
AUPERIN,SASSO,AND MC CORMICK
IO 20 30 40 50 60 70 80 90 100 GffiCACCGGGGATCCTAGGCATmTCGTTGCG~~~GT~CCTA~T~T~AC~TAGTGACA~C~C~~MGTGCCT~T~MT~MGAGGTGA~MCA~T HETGlyClnIleValThrPhePheGlnCluValProHisV~lIleGl~Gl~V~l~TA~nlleVa
110
120
130 140 150 160 170 160 190 200 210 TCTCATTGCACTGTCTGTACAGCAGTGCTG~~TCTGTA~~TTG~M~TGTGGC~TGTTG~~~T~A~~CCTCCTGTTGTGTGGTAGGTCTTG~CMCC~T~A 1LeuIleAlaLeuSerValLeuAlbValLeuLysGlyLeuTyrAsnPheAl~ThrCysGlyLeuVslGlyLeuValThrPheLeuLeuLeuCysGlyArgSerCysThrThrSerLeuTy
230
240
250 260 270 260 290 300 310 320 330 340 TMAGGGGTTTATGAGCTTCAGACTCTGGAACTAAACAT~AGACACTC~TATGACCA~CCT~TCTCC~CAC~G~CMC~TCATCATTATATMT~T~GCM~AGACAGG rLysGlyValTyrGluLeuGlnThrLeuGluLeuAsn~TGluThrLeuAsn~TThrHETProLeuSerCysThrLysAsnAanSerHisHisTyrIleNETVa1G1yAsnGluThrGl
350
360
370 380 390 400 410 420 430 440 450 460 ACTAGAACTGACCTTGACCAACACtiAGCATTATTMTCAC~T~TGC~TCTGTCTGATGCC~C~G~CCTCTATGACCA~CTC~ATGAGCAT~TCTC~CTTTCCA~~T yLeuGluLeuThrLeuThrAsnThrSerllelleAsnHisLysPheCysAsnLeuSerAspAlaHisLysLysAsnLeuTyrA~pHi~AlaLe~TSerlleIleSerThrPheHisLe
47u
480
490 500 510 520 530 540 550 560 570 580 GTCCATCCCCAACTTCAATCAGTATGAGGCAATCAGCTGAGCTGCGATT~MTG~~~~TTAGTGTGCAGTA~CCTGAGT~ACAGCTATG~~ffiAT~ffiC~CCA~G~~AC uSerlleProAsnPheAsnGlnTyrGl~ls~TSe~CysAspPheAsnGl~lyLyslleS~rValGl~TyrAsnLeuSerHisSerTyrAlaGlyAspAl~Al~s~~isCysGlyTh
5Y0
600
610 620 630 640 650 660 670 680 690 700 TGTTGCAAATGGTGTGTTACAGA~*TTAT(;ACGAGGAT~~~~G~GT~GAG~TA~AT~~T~TTGA~T~AtiG~~tiTGG~~~TGtiGA~T~TATTATtiA~TAGTTAT~~TAT~TGAT~T rValAlaAsnGlyValLeuGlnThrPheEtETArgMETAlaTrpGlyGlySerTyrIleAlaLeuAspSerG1yAr~lyAsnTrpAspCysIlellEl~hrSerTyrGlnTyrLeuIleI1
710
720
730 740 75u 760 770 760 790 800 610 n2u CCAAAATACAACCT(;GGAATCACTGCCAATTCTCGAGA~~T~r~C~AT~GGTTATCTCG~TCCT~CA~~GGACTAGAGATA~TATATTAtiTAG~ti~rTG~TA~~ACATT eGlnAnnThrThrTrpGl,dspHisCysClnPheSerArgProSerProlleGlyTyrLeuGlyLeuLe~SerGlnArgThrArgAsplleTyrlleSerArgArgLe~Le~GlyThrPh
830
840
B50 860 870 nno BY0 YOO YIO 920 930 '140 CACATGCACACTGTCAGATTCTGAAGGTAAAGACACAC~AG~GGATA~GT~TCACCAGGTGGATGCT~l~GAGGCTtiMCT~TGCTT~GGG~CA~AGCTGTGGC~TGT~ eThrTrpThrLeuSerAspSerCluClyLysAspThrProGlyClyTyrCysLe"ThrArgTrp~TL~~IleGl~Al~Gl~L~"LysCy~Ph~GlyAs"~h~Al~V~lAl~Ly~~ysA~
Y50
Y60
970 YMO YYlJ LO"" 1010 1020 1030 1040 I"5U 1060 TGAGAAGCATGATGAGGAATTTTGTGACATGCTGAGGCTGTTTGACTTC~C~C~G~CATT~~GGTTti~GCTGMGCAC~TG~CATT~A~~GAT~~GCAGT~ nGluLysHisAspGluGluPheCysAspt~~TLeuArgLeuPheAspPheAsnLysGlnAlaIletilnArgLeuLysAlaGluAlaGln~ETSerIleGlnLeulleAsnLysAlaValAs
1070
1OMlJ
109" 1100 ,110 1120 1130 1140 1150 116U 117u 1180 T6CTTTGATAAATCACCMCTTATAATGAA(;AACCATCTAC~GACATCATG~~~CCATA~tiT~TTACAGCMGTA~r~TACCT~~CCACA~~CTA~CG~AG~~ATCACT nAlaLeuIleAsnAspGlnLeuIle~TLysAsnHisLeuArgAspIleMETGlylleProTyrCysAsnTyrSerLysTyrTrpTyrLeuAsnHisThrThrThr(;lyArgThrSerLe
llY0
1200
220
1210 LZL" 123u 124u IL50 lL6U 127u 1280 1ZY" 1300 1310 GCCCAAATGTTGGCTTGTATCAAATGG~CATACTTGMCGAGA~CCACTTTT~TGATGATA~GM~CMGCTGACAATATGATCACTGAGAT~CTAC~~GGAGTATAT~AGAG uProLysCysTrpLeuVa1SerAsnGlySerTyrLeuAsnGluThrHisPheSerAspAspIletiluGlnl:lnAlaAspAsnMETIleThrGluMETLeuClnLysGluTyrHETGl~r
132U
1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 GCAGGGGAAGACACCATTGGCTCTACTTCACCTCTTTGTG~CAGTAC~GTTTCTAT~TATTAGCATCTT~C~~ACCTAGTC~TACC~~TCATAGG~ATATTGTA~C~GTC gGlnClyLysThrProLeuGlyLeuValAspLeuPheValPheSerThrSerPheTyrLeulleSe~llePheLeu~isLe~ValLysll~ProThrHlsArgH~sll~V~lGlyLysS~
1440
1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560 GTGTCCWVUCCTCACAGATTGAATCATATGGCCATATG~CATTTGTTCCTGTGGA~T~TAC~CAGCCTtiGTGTG~CTGTG~TGGMGAG~rGAGACCClTGT~GGGC~CCCGTGACCCACC rCysProLysProHisArgLeuAsnHist~TGlylleCysSerCysGlyLeuTyrLysGlnPr~tilyValProValLysTrpLysArg~~d 1570 1580 1590 1600 1610 1620 1630 1640 1650 GCCTATT(;GCGGTGGGTCACGGGGGCGT~C~~~~A~G~CGACTCTAGGTGTC~~TT~CG~CACCATATCTCTG~CAG~CTGCTCTC~C
FIG. 3. Nucleotide sequence and encoded gene product (glycoprotein precursor) of the 5’ region of the s genome RNA of Lassa virus. The sequence originates at the 5’ terminus and is given in the DNA form. The sequence presented is that of the viral-sense RNA. corresponding
genie region. The size and base composition of the hairpin structure is very similar to those of Pichinde and LCM viruses. The 19bp hairpin of Lassa virus compares with 21 bp (14 G-C and 7 A-U) for LCMV and 18 bp (14 G-C and 4 A-U) for Pichinde arenavirus. The nucleotide sequence homology between the 5’ region of the S RNA of Lassa virus and the corresponding region of the S RNA of LCMV (WE) is shown in Fig. 5. Excluding the 5’ terminal G nucleotide on the Lassa virus S RNA, the sequences are identical for 19 nucleotides and contain
only three mismatches in the first 33 positions. Overall, the Lassa virus nucleotide sequence is 60% homologous to LCMV which compares to only 48% homology to the corresponding sequence of Pichinde virus (data not shown). Comparison of the Amino Acid Sequences of the GPC Gene Products of Lassa Virus, LCMV (WE Strain) and Pichinde Arenavirus The amino acid sequence of the glycoprotein precursor of Lassa virus was
LASSA TABLE
VIRUS
1
AMINO ACID COMPOSITION OFTHE GPC GENE PRODUCTOFLASSA VIRUS
Amino acid Ala Arg Asn Asp CYS Gln Glu GUY His Ile Leu LYS Met Phe Pro Ser Thr Trp Tyr Val
Number of residues
Percentage by weight
21 17 32 19 18 20 22 33
4.3 3.5 6.5 3.9 3.7 4.1 4.5 6.7 3.9 6.7 11.4 5.1 4.1 4.1 2.9 6.9 7.1 1.6 4.5 4.7
19 33 56 25 20 20 14 34 35 8 22 23
Net charge = +10.5”
161
GPC GENE
arg-arg residues at position 262-263 in the glycoprotein precursor of LCMV have been shown to react with glycoproteins Gl and G2, respectively, thereby localizing the point of cleavage within GPC to this region of the molecule (Buchmeier et aL, manuscript in preparation). A hydrophatic analysis (Kyte and Doolittle, 1982) of the GPC gene product of Lassa virus indicates that there are two hydrophobic domains similar to those previously noted in the corresponding proteins of Pichinde and LCM viruses (Fig. 7). One of the domains is located near the amino terminus (amino acid residues 18-54) and is interrupted by a single lysine residue at position 33. This hydrophobic region ends at position 55 with the occurrence of another positively charged amino acid, arginine. It is noteworthy that these two positively charged residues are conserved in the glycoproteins of all three viruses. The second hydrophobic domain is located near the carboxy terminus (amino acid residues 426-450) and, as with Pichinde and LCM viruses, is followed by a hydrophilic region containing several charged amino acids.
Mel wt = 55,820 “The net charge was calculated assuming that arginine and lysine residues are each +l, aspartic acid and glutamic acid residues are each -1, and histidine residues are each +1/2 at neutral pH.
aligned by computer to those of LCMV and Pichinde arenavirus as shown in Fig. 6. The data illustrate that the Lassa virus GPC gene product is significantly more homologous to that of LCMV with 290 matched amino acids than to that of Pichinde arenavirus with 222 matched amino acids. In both comparisons however, there is a greater degree of homology present in the carboxy-terminal region of the molecule, beginning with the conserved dibasic amino acid residues at position 256-257 in Lassa virus, position 262-263 in LCMV, and position 270-2’71 in Pichinde arenavirus. Antibodies raised to synthetic peptides corresponding to locations on either the amino- or carboxy-terminal sides of the
DISCUSSION
The nucleotide sequence data presented here concerning the 5’ region of the S genome RNA of the Josiah strain of Lassa virus compliments the sequence data presented by Clegg and Oram (1985), which covers the 3’ region of the S RNA of the Nigerian strain of Lassa. Taken together, these data demonstrate that Lassa virus, like Pichinde arenavirus and LCMV, has an ambisense S RNA. The nucleoprotein gene is encoded by vcRNA sequence corresponding to the 3’ half of the S RNA, whereas the GPC gene is encoded by vRNA sequence corresponding to the 5’ half of the S RNA. The coding regions for the N and GPC genes are separated by a short, intergenie region of 61 nucleotides. The sequence of nucleotides within this region is able to form a 19-bp hairpin structure now characteristic of the arenavirus S RNA. It was originally noted that there ap-
AUPERIN,
162
NUCLEOPROTEIN
Thr
MC CORMICK
j
N”CLEOPROTEfN
“CRNA 5’.....ACA
SASSO, AND
ser Thr Pro Arg “al “al “CG ACA CC” AGA GUC G””
-
A” A A c---G c---G G---C C---G C---G A---” C---G C---G c---G A---” G---C “---A 6---C C---G C---G C---G C---G C---G Le” OCh CUB “AA AUGGACG---ccC”GAcAAGGG”c
UGU AGC UGU GGA UCU CAG CAA GAC AW UACCUGC---GCGACUGUUCCCAG G---t G---c G---C G---C G---C C---G A---U C---G U---A G---C G---C G---C "---A G---C G--C C---G G---c G---C u u "A
GLYCOPROTEIN
“CA UC” C””
CCA W”
AW AGA GAA GGU AM opt Arg LYS Trp Lyr
fi
5’
CAC AGG CAC ACC GUG ucc GUG UCG.....~' VaL Pro "al GLy
VRNA
GL"CcPROTEIN
FIG. 4. Genetic organization of the Lassa virus S RNA and the nucleotide sequences of the vRNA and vcRNA within the intergenic region. Diagonally lined areas indicate the untranslated regions of the genome. Arrows indicate the 5’ to 3’ orientations of the coding sequences for the N and GPC genes in vcRNA and vRNA sequences, respectively.
peared to be an extra G nucleotide on the 5’ terminus of the S RNA of Pichinde arenavirus (Auperin et al, 198513).This nucleotide was thought to be a cloning artifact since its presence interferred with the exact alignment of the complimentary termini. We have now observed that the S genome RNA of Lassa virus also contains an extra G nucleotide on the 5’ terminus suggesting that this nucleotide may actually be present on the S RNA of both viruses. The ambisense coding strategy is thought to provide a means for the independent regulation of the expression of the N and GPC genes. The messages for these genes must each be transcribed from S sized v and vc RNA templates, respectively. Presumably, the viral RNA polymerase
recognizes the conserved sequence on the 3’ termini of the viral specific RNAs in order to initiate RNA synthesis. A comparison of the terminal nucleotide sequences of the S RNA species of Pichinde arenavirus, LCMV and Lassa virus is shown in Fig. 3A. The data illustrate that the 3’ sequences of all three vRNAs are identical for 19 nucleotides as are their 5’ sequences, excluding the extra G nucleotide present on the 5’ termini of the S RNAs of Lassa and Pichinde viruses. In addition, for all three viruses, the nucleotide sequence on the 3’ termini of the vc S RNAs differs from that on the vRNAs in two positions (Fig. 8B). The observation that these differences are conserved in both type and position among three different arenaviruses sug-
x
10
LO
30
41,
5”
60
~“CAC~CCGATCCTAC~A~T~~~~CC~I.TCMI;TI;TCCT~~”~TAM-----------------------~~~~ACAMTA~~TCACATTCT~.CC~C~~CTCAT”TA ::::::::::::::::::: :::: : :::::: :: : ::: . . .. .
70 . .
. .
CGCACCCCGGATCCTA~CTTmCCATTCCGCTTTCCTTTCCT~A~A~CTGGGTG~GGATTCT~CCCA~~GG~GTCAGATT~G~~CG~GAGG‘~TG~T~~TC x I” 2” 3” 4” 5” 60 70 80
8”
.. . .. .
:
::
90
::::: : :::
210
.. .. .. .. ..
450 460 47” 480 490 S”” 51” 52” 440 TATGACCACCCTCTTATCAGCAT~TCTC~CCTTCCA~,~GTC~T~CC~C~C~TCAGTATGAGGC~TGAGCTGCGATT~~---T~~G~GA~~TGTG~GT~C :::::::::::::::::::::: ::: : :: : ::: :: :: . . . . . .. .. .. : :::::: TTTGACCATACACTCATGA(iTATAGTCTCGAGTCTACAC~C~TATCAGA~~~CC~CTAC~GCAGTGTCTTGTGAT~~C~TG~ATCACCATT~----T~~C 500 51” 53” 55” 560 48” 49” 520 54”
300
310
34”
350 430 ::
530 :
: :::
470
54”
550 ::::::
58” 650
7,!” 67” 69” 7”” 73” 74” 75” 68” II” CACTCAGGCCCTGGCAACT~GA~GTATTATCACTACTAGTTATC~TATCTG~C~TCC-TAC~CCTG~~CATCACTGCC~~~C~AGACCATCTCCCATCGG~ATC~~ : :::: : :: ::: :: :: ::::: :: :: :: :: .....’ . .. . : :: :::::: : ::::: :: : CGTTCAGATGCCMGACCACTTCCTCCAGCC~C~CCT~CCAGTACCT~TCATAC-CAGGAC~G~-CCACTGTAGATAT~A~CCCTT~GGGATGTCT~~TCCTC 7L” 75” 78” 73” 74” 76” 79” no0
770
700
:::::
::
:
88”
890 ::
:::::
:
::::::
IL60
1170
124”
1250
,640 ,650 1620 1630 ----TCTGMCACCATATCTCCGGCAGCACTCTCAAM :::::: : :: ::: ::::: : CCTCCTCTGMGATCAMTCATCTCCCA~~ATGTTGTG~C~TC~ 1670 1680 1690 1660 NUnBER
OF “ATCHED
BASES
-
:
::::::::
:
:::::: ,,I30 1260
::::
:
129”
136”
::
,300
137”
:::::: :: :: : :: :: :::: :: :: :: : :: : :: :: i: ::::
,311”
:::
:: : ::
,400
,410
,420
148”
,490
150”
:::
:::::
:
,520
:::
::::
1530
,600
1630
1140
:::::
:::
,280
:
,060
,130
: ::
:::
:::
LOS”
,I50 ,170 ,160 ILt)” 119” LLUO ,Zl” ,220 1230 TCTMTIACAGCMCTA~TCCTACCT~C~CACMC~‘ACT~G~~CATCA~GCCC~TG~~CTTGTATC~T~TTCAT~~~~AGACCCA‘~T~~CTGATGATATT .. .. .. .. .. .. .. .. .. .. .. ::::::::: :::: ::::: :: : :: : : .. .. .. .. .. :: : ::::: : ::::: :: :::::::: ~MTTACTCAMG~CTCGT~TCGMCATGCTM(;ACT~~CTG~~T~~~~~~T~~~~~~~~MG~G~~G~~~~~~~A~~~~~~~C~~~~~GMTGAGAC~~~~~T~~~TGAT~TC 1190 121” 120” 12LO 1230 124” ,250 126” 127”
15,” 15LO 153” 154” 1 155” 156” 157” ,560 1 1590 CCTCTCCCT~CAMTCC~A~TCACACC~TCTC~~CCCCCT~CCC~CCC~A~~C~T~~TCACti~~CCTCCATTTACAC~CA~CT~~TCTC~T~---. . . .. . ::::: ::: : : ::: ::: :: :::: : :: ::: ::::: :::::: CCTCTAAAMCTATCTW;AACGCT~TCAG-~A~~CCTCCCTGACTCTCCACCTCG~GAGGTGGAGAGTCA~G~GCC-----CAG~~CTTAGAGTGT~C~A~G L 550 ,560 1570 I 58 Y ,590 ,610 ,620 1
,020
1010
:::: 1040
,390
::: 940
93”
100”
1270 ,280 119” 13”” ,310 132” 135” 133” I34” GMCMCMGCTGACMTATGATCACTGAGATGTTACAG~GAGTATATGGAG~GCA~GG~GACACCATTGG‘~CTAGT~A~TCT~GTG~C~TAC~GT~‘~ATCTTATT
900 :::::::
92”
::
::
820
1120
1390 140” 1410 142” 143” 144” 145” 147” 146” AGCATCTTCCTTCACCTACTCAAMTACCMCTCATAC~CTCATAGGCATATTGTA~~GT~TGTCC-CCTCACAGA~MTCATAT~CA~T~~C~~~~ACTCTA-~GC~C :::::::: :: :: : :: : :::::::: ::::: :: :: ::: :: ::::: :: :: :: : :::: : : ::: :: AGCATCTTTCTGCATTTTGTCAGGATACCUCACC~CACATAGACACAT~~GC~*CAT~CC~‘C~CATCG~T~ACC~C~~GATCTGTAG~GT~TC~TT~~~~ 143” 144” 1450 ,460 147” 148” 149” ,500 15,”
780
810
l”5” ,060 l”8” ,030 I”40 L”7” I”90 I LO” II,0 CAMGGTTGMAGCTGMGCACAMTGACCATTCAGCATTCAGTTGATC~-G~T~TGCT~GAT~TGACC~CTTAT~TG~G~CCATCTA~~ACATCATG~~~CCATAC :::::: ::::: :::: : ::::::::::::::::::: :::::: : :::: ::: :::::: AGTAACTTCAAGCMGATTCT~AGTCTGCCTT(;CATGTATTC-C~CA=~=CTCTGA~TCCGATCAGCTG=GATGA~~TCAT~~AGATCT~T~G~TACCATAC ,090 107” ,080 ,100 ,I,” ,120 1130 ,150 ,140
GMC~MGCAGATMCATCATCACAGAGATC~G~M~A~ACAT~~ACM~GAtiTACTCC~TA~CTTMT~ATC~C~~A~~~CMCATCA~ATA~TGATC 13LO 132” 133” 134” 135” I36” 137” 138”
660
:::
760
:::::
:
::
460
620 560 57” 580 590 60” 6,” 63” 640 CTCAGTWCA1;CTATCCTGUCAT(;CAGCCMCCATIGT---------TCTTACAGACL‘---TTTATGAGGATGCCTTCGCCTGCT :::::::::::: : ::: : : : :: :::: ::: ::: ::: ::: :: TTGTCATCTTC~ACCCACAGAGCGCCATCAGCCAGT~CA~ACTTTCAGAGCTAGAGT~CTGGACAT~TTA~ACTGCCT~GGA~~GTACATGAG~GTGG~~CT~ACA 63” 64” 66” 67” 680 690 600 610 62”
91” 920 93” 94” 95” 96” 97” 99” 9nu ATCCTMTTGAGG~CAACTAAAATGCTTCGCGMCACAUJTT ::: : ::: :: :: :: ::::: :: ::::: :::::::: ::::::::::::: :: :::::::: :: :: ::::::::::: ATGATCC~GCTGCAGAGCTCAMTCTT~~G~TACA~TG~GC~TGT~TGTC~TCATGATG~GAGTT~GTGACAT~TACGACT~~GATTAC~~G~~GCCCTG 950 960 97” 98” I”“0 99” LOlO ,020 l”30
::
: ::
57”
8,” 84” 870 790 BOO n2” 0,” (15” ll6” ~CCTCTCAC~~ACTAGAGATATTTATATA~AGTAG~‘A~~GCT~GC~A~CACATGGACA~TC~ATTCTC*GGT~GACA~C~~GGATA~~GTCTGACCAGGTGG : : : :: : : ::::::: :: ::: ::: : :::::::::::: ::::: . .. . . . . .. . .. .. TTTCCTCAG~~AMCTTTCTCAC------TAGCM;ACTTTCA(%CACACACCTGGACCCT 850 86” tit)” 87” 119” 900 YLO
::::
: ::: ::
42” :
: : :
:: 23”
: ::: :::: :::::
36” 37” 3nu 390 400 410 330 34u 350 320 MCAACAGTCATCATTATATMTGCTUIGCMTCACACAG~ACTAGMCTGACCTTGACCMCA~AGCA~A~MTCAC~TTTTGCMTCTGTCTG~CGCC~C-GMCCT~ .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. :::::: :::::: :: :: : :::: : ::::: :: : :: :: :::::: ::: : AACMCTCTC:T~~~~---~ACT-~T 42” 43” 44” 38” 4”O 41” 450 36”
:::::: :::: :: :: :::::::: ::::::::
:
120
AGCTCCTGTC(;CATGTAC~CCTTMTGCTCCCCGATAC~~AiI’MAGL;(;(;T’CTACCAC’LTCAMTCA(jT66AL;TTTGATAT~CCTCA~~T~GACGATGCC~~CGTGCTCAG~ IId” 29” 31” 31” L6” 27” 300 33” 250 240
:
11”
2”O
29”
. . . ...* . ..*....... . . . . . . . :: : : : :::: : : :::
: : :::
. ..*... .......
LOO
170 I40 180 190 I L” I50 16” 12” 13” loo ATAGMGA~TCATGMCATTGTTCTCATTGCACTCCA~~~C’L’CTACTAGCAGTCCTGAMC(jlCTGTACMTL‘TTCCT . . . . . . . . . . . . . . . . . . :: :: ::::: :: :::::::: : :::: :: : :: : : : :::: : .. .. .. . . . . . . . .. . . . : : : :::::: ATTCATCACGTCATCMCATTLTCATTATTCTCCTCATTAT~TCA~ACCATC~CCTCTC~AC~T=~CCCCAC~~CT~CA~ATTACCA~CCTCAC~TC~TTT~~~C~ 19” 130 14” 160 17” 180 200 2,” 12” 15” 240 22” 250 26” 27” 28” .‘,o AGCGTCTT---GCACAACCA(;TCTT---------------TAT~~~TATG~CTTCAGACTCTGG~CT~CAT~AGACAC~~TATGACCATGC~CT~CCT~AC-
90
:::
1540
,610
:::
::::: 1640
1650
X
L7OOX
987
FIG. 5. Alignment of the nucleotide sequences corresponding to the 5’ regions of the S RNA’s of Lassa virus and LCMV (WE strain). The sequences were aligned by the NUCALN program (Wilbur and Lipman, 1933) with a K-tuple size = 3, window size = 10, and a gap penalty = 5. Horizontal lines indicate the positions of the initiation and termination codons for the reading frame encoding GPC. Vertical lines denote the boundaries of the hairpin structures within the intergenic regions.
AUPERIN,
164
SASSO, AND
MC CORMICK
Q”&“WGSYW(B~HPYNMhYSQ~“:::: :::::::::: : ::: ::: :::::::: 4w
410
420
: :
: :: : ::: : :::::::
: :
430
FIG. 6. Alignment of the amino acid sequence of the glycoprotein precursor of Lassa virus to that of LCMV (WE strain) (A) and Pichinde arenavirus (B). The PRTALN program (Wilbur and Lipman, 1963) was used to align the sequences with a K-tuple size = 1, window size = 10, and gap penalty = 5.
LASSA
VIRUS PICHXNDE
-40
+*
--+++++
0
--
-++
-----++ .I+ *+
GPC
-----*+
LCM
YY YY
Y
--+ ++
+u++
300
SEOUENCE
4
+*++
208
100
165
GPC GENE
+**
400
500
NUMBER
GPC
Y
Y
w
2
-2 --
--
--
-+.I-?
0
----++++++
200
100
300
SEOUENCE
LASSA
0
-_------_ ***cc,+*+++
100
200 SEOUENCE
4.
-*
400
NUMBER
GPC
----_
--
300
400
NUMBER
FIG. 7. Comparison of the hydropathic profiles of the GPC gene products of Lassa virus, LCMV (WE strain), and Pichinde arenavirus. Regions with a net hydrophobicity appear above the horizontal center line, and regions with a net hydrophilicity appear below the center line. Y structures indicate the positions of the potential asparagine-linked glycosylation sites along the polypeptide chain. + and - symbols denote the positions of positively (arginine and lysine) and negatively (aspartic acid and glutamic acid) charged amino acids.
A
LCMVvRNA
3' HOGCGUGUCACCUACGAUCCG.........................CGGAUCCUAGGGGCCACG
Lassa vRNA
3'HoIoo~oooo~
Pichinde
3'Hooooooomrroo~.........................mrrmrmmr
vRNA
Arenavirus
vF.NA
Arenavirus
vcRNA
.........................
C 5'
omomnmnwm5' oo.0
5'
3'HOGCGUGUCACCIlAGGAUCCG......................... 3',,(c)o8@oeC~r.........................
FIG. 8. (A) Comparison of the terminal nucleotide sequences of the viral S RNAs of LCM, Lassa, and Pichinde viruses. Closed circles indicate homologous nucleotides in the Lassa and Pichinde sequences compared to LCMV. (B) Nucleotide sequence homology between the 3’ termini of the viral and viral-complimentary S RNA species of arenaviruses.
166
AUPERIN,
SASSO, AND
MC CORMICK
gests that they are functional and have, CLEGG,J. C. S., and ORAM, J. D. (1985). Molecular cloning of Lassa virus RNA: Nucleotide sequence therefore, been maintained through evoand expression of the nucleocapsid protein gene. lution. One possibility is that the 3’ terVirdogy 144,363-372. minal nucleotide sequence present on the FRAME, J. D., BALDWIN,J. M., GOCKE,D. J., and TROUP, v and vc S RNA species functions in deterJ. M. (1970).Lassa fever, a new virus disease of man mining the efficiency with which RNA synfrom West Africa. I. Clinical description and thesis is initiated from either template, pathological findings. Am J. Trap. Med Hz/s- 19, thereby providing the basis for the genetic 670-676. regulation inferred by the ambisense cod- GRUNSTEIN,J. M., and HOGNESS,D. S. (1975). Colony ing strategy. hybridization: A method for isolation of cloned DNA’s that contain a specific gene. Proc Nati Acm! The regulation of arenavirus RNA repSci. USA 72,3961-3965. lication, gene transcription and expression are areas for further analyses. The avail- HARNISH, D. G., DIMOCH,K., BISHOP,D. H. L., and RAWLS,W. E. (1933). Gene mapping in Pichinde viability of cDNA probes to the S RNA rus: Assignment of viral polypeptides to genomic L should allow these processes to be followed and S RNA%. J. Viral 46,633~641. during productive as well as persistent inKILEY, M. P., TOMORI,O., REGNERY,R. L., and JOHNSON, fections. The determination of the exact 3’ K. M. (1980). Characterization of the arenaviruses termini of the mRNA’s transcribed from Lass and Mozambique. In “Replication of Negative the N and GPC genes will undoubtedly Strand Viruses” (D. H. L. Bishop and R. W. Comprovide further insight into the function pans, eds.), pp. 1-19. Elsevier North-Holland, Amof the intergenic region. Finally, cDNA sterdam. clones of the N and GPC genes of Lassa KYTE,J., and DOOLITIXE,R. F. (1982).A simple method for displaying the hydropathic character of a provirus can now be expressed in a number of tein. J. Mel Bid 157,105-132. eukaryotic systems for use as reagents in the diagnosis of Lassa infections, in the MAXAM, A., and GILBERT,W. (1980). Sequencing end labelled DNA with base specific chemical cleavages. study of the cell-mediated immune reIn “Methods in Enzymology” (L. Grossman and K. sponse to Lassa infection, and in genetiMoldave, eds.), Vol. 65, pp. 499-560.Academic Press, cally engineered vaccines for the prophyOrlando, Fla. laxis of Lassa fever in areas of the world MCCORMICK,J. B., KING, I. J., WEBB,P. A., SCRIBNER, where the virus is endemic. C. L., CRAVEN, R. B., JOHNSON,K. M., E~~~crrr, REFERENCES
AUPERIN, D. D., GALINSKI, M., and BISHOP,D. H. L. (1984a). The sequence of the N protein gene and intergenie region of the S RNA of Pichinde arenavirus. Virobgp 134,208-219. AUPERIN,D. D., ROMANOWSKI,V., GALINSKI, M., and BISHOP,D. H. L. (1984b). Sequencing studies of Pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA. J. Vid 52,
L. H., and WILLIAMS,B. (1986).Lassa fever: Effective therapy with Ribavirin. N. En& J. Med 314, 2026. MCMASTER, G. K., and CARMICHAEL.,G. G. (1977). Analysis of single- and double-stranded nucleic acids on polyacylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74,4835-4838.
MONATH,T. P., NEWHOUSE,V. F., KEMP,G. E., SETTER, H. W., and CACCIAPUOTI,A. (1974). Lassa virus isolation from Masbmys natolensis rodents during an epidemic in Sierra Leone. science 185,263-265. 897-904. BISHOP,D. H. L., GOULD,K. G., AKASHI, H., and CLERX- MURPHY,F. A., and WALKER, D. H. (1978). Arenaviruses: Persistent infection and viral survival in resVAN IIAASTER,C. (1982).The complete sequence and ervoir hosts. In “Viruses and Environment” (E. coding content of snowshoe hare bunyavirus small Kurstak and K. Maramorosch, eds.), pp. 155-180. (S) viral RNA species. Nucleic Acids I&s. 10,3703Academic Press, New York. 3713. CASALS,J., BUCKLEY,S. M., and CEDENO,R. (1975). MURPHY,F. A., WEBB, P. A., JOHNSON,K. M., WHITFIELD, S. G., and CHAPPJZLL,W. A. (1970). ArenaAntigenic properties of the arenaviruses. Bull WHO viruses in Vero cells: Ultrastructural studies. J. VZ52,421~427. rol 6,507-513. CLEGG,J. C. S., and LLOYD,G. (1983). Structural and RAWLS,W. E., and LEUNG,W.-C. (1979).Arenaviruses. cell associated proteins of Laasa virus. J. Cen vird In “Comprehensive Virology” (H. Frankel-Conrat 64,1X7-1136.
LASSA VIRUS GPC GENE and R. R. Wagner, eds.), Vol. 14, pp. 157-192. Plenum, New York. ROMANOWSKI,V., and BISHOP,D. H. L. (1985). Conserved sequences and coding of two strains of lymphocytic choriomeningitis virus (WE and ARM) and Pichinde arenavirus. V&us Res. 2,35-51. ROMANOWSKI, V., MATSUURA,Y., and BISHOP,D. H. L. (1985). Complete sequence of the S RNA of lymphocytic choriomeningitis virus (WE strain) compared to that of Pichinde arenavirus. virus Res. 3, 101-114. VEZZA, A. C., CASH, P., JAHRLING,P., EDDY, G., and BISHOP,D. H. L. (1980). Arenavirus recombination: The formation of recombinants between Pichinde
167
and Pichinde Munchique viruses and evidence that arenavirus S RNA codes for N polypeptide. Vi* 106.250-260. VEZZA,A. C., CLEWLEY,J. P., GARD, G. P., ABRAHAM, N. Z., COMPANS,R. W., and BISHOP,D. H. L. (1978). Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami. J. I&-o1 26,485-497. WALKER,D. H., WULFF,H., LANGE,J. V., and MURPHY, F. A. (1975). Comparative pathology of Lassa virus infection in monkeys, guinea-pigs, and Mastays nu-
Bull
WHO 52,523-534.
WILBUR,W. J., and LIPMAN, D. J. (1983). Rapid similarity searches of nucleic acid and protein data banks. Proc. Nat1 Acad Sci. USA 30,726-730.
154,155-167 (1986)
Nucleotide Sequence of the Glycoprotein Gene and lntergenic Region of the Lassa Virus S Genome RNA DAVID D. AUPERIN,l DONNA R. SASSO, AND JOSEPH B. MCCORMICK of Viral Diseases, Center for I@ectious Diseases, Centers for Disease Control, Atlanta, Georgia 303%
Special Pathogens Branch, Divisim
Received March 18, 1986; accepted June 12, 1986 Two overlapping cDNA clones corresponding to the 5’ region of the Lassa virus S genome RNA were isolated and their nucleotide sequences determined. Similar to Pichinde and lymphocytic choriomeningitis viruses (LCMV), Lassa virus has an amhisense S RNA. The precursor to the viral glycoproteins (GPC) is encoded in viral RNA sequence originating at position 56 and terminating at position 1529 from the 5’ terminus of the S RNA. A short, noncoding, intergenic region capable of forming a hairpin structure separates the termination codons of the nucleoprotein (N) and GPC genes. Hydropathic analysis of the GPC gene product of Lassa virus indicates the presence of hydrophobic domains near the amino and carboxy termini as previously noted in the corresponding proteins of Pichinde and LCM viruses. A comparison of the nucleotide sequences on the 3’ termini of the viral and viral-complimentary S RNA species of Lassa, LCM, and Pichinde viruses reveals slight sequence differences that may possibly be involved in the regulation of RNA synthesis and gene expression. Q 19% Academic PEWI, IIIC INTRODUCTION
Lassa virus was first isolated in 1969 associated with a fatal febrile illness in humans in northeast Nigeria (Frame et aL, 1970). Since then, illness or serologic evidence of infection with Lassa virus has been demonstrated in numerous West African countries. The natural host of the virus is the multimammate rat lMastomys natalensis from which isolations have been made in Nigeria and Sierra Leone (Monath et aL, 19’74; Murphy and Walker, 1978; McCormick et aL, 1986; Walker et al, 1975). Morphologic, antigenic, and biochemical properties of the virus have established it as an arenavirus (Casals et uL, 1975; Clegg and Lloyd, 1983; Kiley et aL, 1980; Murphy et al, 1970). The arenavirus genome consists of two molecules of single-stranded RNA designated L (large) and S (small) (Rawls and Leung, 1979; Vezza et CAL,1978). The sizes of the L and S RNAs are approximately 2.5 X lo6 and 1.1 X lo6 Da, respectively. Genetic i To whom correspondence should be addressed.
and biochemical analyses have established that the S RNA encodes the nucleoprotein (N) and the precursor to the glycoproteins (GPC) (Harnish et cd, 1983; Vezza et aL, 1980). The L RNA codes for a large polypeptide presumed to be the RNA-dependant, RNA polymerase component of the virus (Harnish et aL, 1983). Recent nucleotide sequence analyses on the S genome RNA segments of Pichinde and lymphocytic choriomeningitis (LCMV) viruses have demonstrated that the N and GPC genes are encoded in nonoverlapping reading frames originating from opposite ends of the S RNA (Auperin et aL, 1984a, 1984b; Romanowski and Bishop, 1985; Romanowski et aL, 1985). The N gene is encoded by viral complimentary (vc) RNA sequence corresponding to the 3’-half of the molecule and the GPC gene is encoded by viral (v) RNA sequence corresponding to the 5’-half of the molecule. The term “ambisense RNA” has been used to describe this coding strategy. The reading frames encoding the N and GPC genes are separated by a short, noncoding, intergenic region, the nueleotide sequence of which is such that a hair-
155
0042~6822/86 $3.00 Copyright All rights
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
156
AUPERIN,
SASSO, AND
pin structure of some 18-22 bp can form. In view of the location of this structure between the termination codons for the N and GPC genes, and the size of the subgenomic mRNA species for each gene, it is postulated that the hairpin functions as a transcription terminator (Auperin et al, 1984b). The nucleotide sequence of the 3’ region of the S genome RNA of Lassa virus (Nigerian strain) has recently been reported (Clegg and Oram, 1985). Like Pichinde arenavirus and LCMV, the viral nucleoprotein gene was found to be encoded in vcRNA sequence from this region of the molecule. We have initiated studies to molecularly clone the GPC gene of Lassa virus for its possible use in a genetically engineered vaccine. Here we report the nucleotide sequence of the 5’ region of the S genome RNA of the Josiah strain of Lassa virus. The sequence contains the complete GPC gene, the intergenic region and the carboxy-terminal position of the N gene.
MC CORMICK
albumen (BSA) and stored in liquid nitrogen vapors. Virus purification was achieved by successive rounds of centrifugation in gradients of 30% glycerol/50% potassium tartrate (Vezza et a& 1978) and 20-60% sucrose. Virions banding in the sucrose gradient were harvested, diluted with TE (10 mil4 Tris-HCl, pH 7.4, 1 mM EDTA), and pelleted through a cushion of 30% glycerol (in TE). Purified virus was gently resuspended in low salt buffer (LSB) (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.14 M NaCl), and stored in liquid nitrogen vapors. RNA extraction, To purified virus in LSB, sodium dodecyl sulfate (SDS) was added to a final concentration of 1%. The mixture was extracted two times with an equal volume of phenol:chloroform (1:l). To the final aqueous phase, NaCl was added to 0.4 M and the RNA precipitated with the addition of 3 vol of ethanol. RNA was collected by centrifugation, redissolved in high salt buffer (HSB) (10 mMTris-HCl, pH 7.4,0.4 M NaCl, 1 mM EDTA) and reprecipitated with ethanol. Final RNA pellets were MATERIALS AND METHODS rinsed in absolute ethanol, lyophilized, and Virus and cells. The Josiah strain of resuspended in sterile deionized water at Lassa virus (reference no. 800593) was iso- 1 fldcll. For cytoplasmic RNA, confluent monolated from human serum taken in Sierra Leone in 1976. The virus was plaque puri- layers of BHK-21 cells were washed two fied three times on monolayers of Vero E6 times in chilld phosphate-buffered saline cells (ATCC no. CRL1586) and a high titer (PBS) followed by one wash in TNE (10 mM stock prepared by passage in BHK-21 cells Tris-HCl, pH 7.4, 0.1 M NaCl, 1 mM EDTA). Cells were then scraped up in TNE (ATCC no. CCLlO). Virus growth and pur$cation. All ma- (5 ml/150 cm2 of cells) and transferred to nipulations with infectious material were 30-ml corex centrifuge tubes. The cells were performed within the maximum contain- collected by centrifugation and washed ment laboratory at the Centers for Disease once more in TNE. Following centrifugaControl. Lassa virus was grown on tion the cell pellet was resuspended in TNE monolayers of BHK-21 cells nourished plus 1% Nonidet-P40 (four times packed with Dulbecco’s modified Eagle’s medium cell volume) and incubated on ice for 15 (GIBCO laboratories) supplemented with min. Nuclei and mitochondria were re10% fetal calf serum. Cell monolayers were moved by centrifugation and SDS was infected with virus (m.o.i. = 0.1) at 70% added to a final concentration of 1% . The confluency. Following adsorption, media resulting lysate was then extracted with was added and the cells incubated at 37”. phenol:chloroform (1:l) two times and the Virus was harvested by withdrawing the RNA precipitated by the addition of NaCl media from cell cultures, and clarifying by to 0.4 M and three vol of ethanol. The cylow speed centrifugation. Virus was then toplasmic RNA was collected by centrifucollected by ultracentrifugation at 25K gation, rinsed in ethanol, and lyophilized. RPM for 90 min in an SW-27 rotor. Crude Final RNA pellets were redissolved in virus pellets were resuspended in Dulbec- sterile deionized water at 1 pg/pl. co’s medium containing 1% bovine serum cDNA synthesis. An initial preparation
LASSA
VIRUS
of cDNA was synthesized using the synthetic oligodeoxyribonucleotide (5’pCGCACAGTGGATCCTAGGC) that is complementary to the 3’ terminal 19 nucleotides of the arenavirus S genome RNA (Auperin et a& 1984a). The first strand reaction contained vRNA (100 ng/pl); 5’phosphorylated oligonucleotide (4 ng/pl); 50 mM Tris-HCI (pH 8.3); 70 mM KCl; 10 mM MgClz; 10 mM DTT, 1 mM each dATP, dGTP, dCTP, and dTTP; 0.4 &i/p1 rH]dGTP (26.5 Ci/mM) and 0.75 U/p1 AMV reverse transcriptase (Life Sciences) in a volume of 250 yl. Prior to starting the reaction, the oligonucleotide primer was annealed to vRNA templates by incubation at 42’ for 15 min in the presence of the Tris-HCl and KC1 components of the reaction. Following annealing, the remaining components of the reaction were added, and first strand cDNA was synthesized at 42’ for 40 min. The reaction was terminated by the addition of EDTA to 20 mM and the RNA hydrolyzed in 0.1 NNaOH at 70” for 20 min. Following hydrolysis, the reaction was neutralized with HCl, extracted with phenol:chloroform (l:l), and chromatographed over a column of Sephadex G-50 (fine) in TE. First-strand cDNA eluting in the void volume was collected by ethanol precipitation and lyophilized. Second-strand cDNA was synthesized relying on the ability of the 3’ end of first-strand cDNA to form hairpin structures. The reaction conditions were similar to those used for first-strand synthesis except that 5fa-32P]dATP (1 &i/Fl, 32 Ci/ mM) was used to monitor the synthesis and incubation was at 42” for 90 min. The double-strand cDNA products were extracted, chromatographed as before, and collected by ethanol precipitation. The hairpin structures present in the cDNAs were removed by digestion with Sl nuclease (BRL), and the resulting double-stranded molecules repaired with the Klenow fragment of DNA polymerase I (New England Biolabs) prior to cloning. A second preparation of cDNA was synthesized using, as a primer, the singlestrand of DNA complimentary to the viral S genome RNA obtained by strand separating the 298-bp DdeI restriction frag-
GPC GENE
157
ment of clone LS13 (position 995-1293 in Fig. 1). The single-strand DNA primer was annealed to vRNA template in Tris-HCl and KC1 as before except that the hybridization temperature was initially 65” and allowed to slowly cool to room temperature over 4 hr. The reaction conditions were identical to those described above except that 51a-32P]dTTPwas used to monitor the synthesis. The products of the reaction were then electrophoresed on a 3% polyacrylamide gel in 1X TBE containing 7 M urea. Autoradiography of the gel revealed a single species of first-strand cDNA approximately 1300 nucleotides long. This material was eluted from the gel and precipated with ethanol. Deoxyadenosine tails were then added to the 3’ termini in a 30~1reaction containing 120 &sodium cacodylate (pH 6.8), 0.1 mMDTT, 1 mMCoC12, 0.25 mM dATP, and 27 U terminal deoxynucleotidyl transferase (P.L. Biochemicals). The reaction was incubated at 37” for 5 min then extracted with phenol:chloroform and ethanol precipitated. Secondstrand synthesis was primed with 5’ phosphorylated oligo (dT)12-1eand performed as described above. Finally, the doublestranded cDNAs were treated with Sl nuclease and repaired with the Klenow fragment of DNA polymerase I to generate blunt ends prior to cloning. Molecular cloning. Blunt-end cDNAs were ligated into a pUC18 vector at the unique SwzuI site in the poly-linker region using T4 DNA ligase (New England Biolabs). The vector was prepared by first restricting the plasmid DNA with SmaI, then removing the 5’ terminal phosphates with calf intestinal alkaline phosphatase (Boehringer Mannheim). The products of the ligation reaction were transfected into competent Escherichia coli MC1061 cells and transformants selected by their resistance to ampicillin. Plasmid-containing colonies were then replica-plated onto BA85 nitrocellulose filters (Schleicher & Schuell) and screened by colony hybridization (Grunstein and Hogness, 1975) for Lassa cDNA inserts using either a short copy probe made by extending the synthetic oligonucleotide against vRNA (Bishop et aL, 1982) or nick translating the
158
AUPERIN,
SASSO, AND
two times for 15 min each in 2X SSC + 0.5% SDS at 65”; and two times for 15 min each in 0.1X SSC at room temperature, then dried and autoradiographed. DNA sequence analysis. DNA nucleotide sequences were determined on strand-separated, end-labeled, restriction fragments by the method of Maxam and Gilbert (1980) using the formic acid protocol for the A + G reactions as described previously (Bishop et a& 1982).
298-bp DdeI restriction fragment form clone LS13. Hybridization-positive clones were picked and their cDNA inserts sized by comparing their H&f1 restriction fragments to those of pUC18 and pBR322. Large cDNA clones were then verified as S RNA specific by northern blot analyses to vRNA. Northern blot analpzs. Preparations of RNA were denatured by reacting with 1 M glyoxal in 50% demethyl sulfoxide and 10 mM sodium phosphate (pH 7.0) at 50” for 60 min. (McMaster and Carmichael, 1977). Following denaturation, the RNAs were electrophoresed on 1.0% agarose gels cast in 10 mM sodium phosphate (pH 7.0) at a constant voltage of 50 V for 53 hr. The gel was stained and prepared for blotting by soaking in 50 mM NaOH containing 1 pg/ ml ethidium bromide for 30 min followed by four successive 15 min washes in 25 mM sodium phosphate (pH 6.5). The RNA was osmotically transferred to “Genescreen” hybridization membranes in 25 n&sodium phosphate (pH 6.5). After transfer the membranes were dried, baked at 80” for 2 hr, and prehybridized in 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M Na citrate), 1% SDS, 0.04% polyvinyl-pyrrolidone, 0.04% BSA, 0.04% Ficoll, and 100 pg/ml denatured salmon sperm DNA for 16 hr at 42”. 32P-Labeled cDNA probes were then added and hybridization continued for at least 16 hr. Following hybridization, the membranes were washed two times for 5 min each in 2~ SSC at room temperature;
LS13
I
Nucleotide Sequence of the 5’ Region of the Lmsa VimLsS RNA Two overlapping cDNA clones were isolated and used to determine the nucleotide sequence of the 5’ region of the Lassa virus S genome RNA. Figure 1 illustrates the position of the two clones in relation to the S RNA and the strategy used to determine their sequence. Clone LS13 was isolated from a cDNA library generated by priming first-strand cDNA synthesis with the synthetic oligonucleotide known to be complimentary to the 3’ terminus of the arenavirus S RNA. The clone was positioned along the viral S RNA based on the coding capability of its sequence and noting that one end of the cDNA encoded the carboxy terminus of the nucleoprotein gene, the sequence of which had been reported for the Nigerian strain of Lassa Virus (Clegg and Oram, 1985). The cDNA was found to orig-
I-
H,nf
EcoAl
RESULTS
I
LS37
Ddr
MC CORMICK
I I
c---c
J ---
-+,
--
----
f -
l
‘
BarnHI Xho I
-
>
FIG. 1. Overlapping cDNA clones corresponding to the 5’ region of the Lassa virus S RNA and the strategy used to determine their nucleotide sequence. Clone LS13 was derived from a preparation of cDNA generated by priming first-strand synthesis with a synthetic DNA oligonucleotide that is complimentary to the 3’ terminus of the arenavirus S RNA. Clone LS37 was derived from a cDNA preparation generated by initiating first-strand synthesis with the vc strand of DNA obtained from a DbeI restriction fragment (position 995-1293) of clone LS13. Arrows indicate the positions of the 3’terminally labeled, single-strand DNAs that were sequenced by the method of Maxam and Gilbert.
LASSA
VIRUS
inate with the nucleotide sequence encoding the 21 amino acids of the carboxy-terminal portion of the N gene and extend toward the 5’ terminus of the S RNA for 798 nucleotides. The observation that this clone was selected by hybridization to a short copy probe made by extending the oligonucleotide that is complimentary to the 3’ terminus of the viral S RNA indicates that under our conditions of probe synthesis (a large molar excess of primer to RNA template and relatively nonstringent hybridization prior to cDNA synthesis) the oligonucleotide is able to prime cDNA synthesis at positions other than the 3’ terminus. Homology searches of our sequence have failed to indicate any close matches for sequences that are complimentary to the oligonucleotide however, while Northern blot analyses performed prior to colony hybridization confirmed that the probe was specific for S RNA sequences. From the known size of the S RNA species of Pichinde and LCM viruses (Auperin et al, 1984b; Romanowski et ah, 1985), it was reasoned that clone LS13 terminated approximately 1300 nucleotides from the 5’ end of the Lassa virus S RNA. A second preparation of cDNA was then made, using as a primer in a first-strand synthesis reaction, the vc strand of DNA obtained from a DdeI restriction fragment of clone LS13 (position 995-1293). Clone LS37 was isolated from the resulting cDNA library, and nucleotide sequence analysis confirmed that the clone originated with the first nucleotide of the DdeI restriction fragment (position 1293) and correctly terminated at the 5’ end of the viral S RNA, based on sequence homology to Pichinde and LCM viruses. Northern blot analyses shown in Fig. 2 demonstrate that nick translated probes of LS13 and LS37 cDNAs specifically hybridize to Lassa virus S genome RNA. The nucleotide sequence of the 5’ region of the Lassa S genome RNA is given in Fig. 3. In agreement with Pichinde and LCM viruses, a reading frame encoding the GPC gene product is present in vRNA sequence originating at position 56 and terminating at position 1529from the 5’ end of the RNA.
159
GPC GENE LS37
LS13
*
18s
(18s
FIG. 2. Northern blot analyses of BHK-21 cell cytoplasmic RNA and Lassa viral RNA resolved by agarose gel electrophoresis (see under Materials and Methods) and osmotically transferred to “Genescreen” hybridization transfer membrane. Positions of the viral L and S genome RNAs as well as the 28 S and 18 S ribosomal RNAs were visualized by ethiduim bromide staining and ultraviolet illumination following transfer to the membrane. The probes, as indicated, include the cDNAs of clones LS13 and LS3’7.
Assuming the first methionine in this reading frame is the initiating methionine, the glycoprotein precursor contains 491 amino acids and has a calculated molecular mass of 55,820 Da. This compares with 503 amino acids (57,279 Da) and 498 amino acids (56,293 Da) for the corresponding proteins of Pichinde and LCM (WE strain) viruses, respectively. The amino acid composition and net charge of the GPC gene product of Lassa virus is given in Table 1. This protein is comparatively rich in cysteine (3.7%) and histidine (3.9%), similar to Pichinde and LCM viruses. The coding regions of the N and GPC genes do not overlap but are separated by a short, noncoding, intergenic region of 61 nucleotides, which, although not very homologous to the corresponding sequences in Pichinde and LCM viruses, can form a hairpin structure of 16 G-C and 3 A-U base pairs. Figure 4 illustrates the genetic organization of the Lassa virus S RNA and the nucleotide sequence within the inter-
160
AUPERIN,SASSO,AND MC CORMICK
IO 20 30 40 50 60 70 80 90 100 GffiCACCGGGGATCCTAGGCATmTCGTTGCG~~~GT~CCTA~T~T~AC~TAGTGACA~C~C~~MGTGCCT~T~MT~MGAGGTGA~MCA~T HETGlyClnIleValThrPhePheGlnCluValProHisV~lIleGl~Gl~V~l~TA~nlleVa
110
120
130 140 150 160 170 160 190 200 210 TCTCATTGCACTGTCTGTACAGCAGTGCTG~~TCTGTA~~TTG~M~TGTGGC~TGTTG~~~T~A~~CCTCCTGTTGTGTGGTAGGTCTTG~CMCC~T~A 1LeuIleAlaLeuSerValLeuAlbValLeuLysGlyLeuTyrAsnPheAl~ThrCysGlyLeuVslGlyLeuValThrPheLeuLeuLeuCysGlyArgSerCysThrThrSerLeuTy
230
240
250 260 270 260 290 300 310 320 330 340 TMAGGGGTTTATGAGCTTCAGACTCTGGAACTAAACAT~AGACACTC~TATGACCA~CCT~TCTCC~CAC~G~CMC~TCATCATTATATMT~T~GCM~AGACAGG rLysGlyValTyrGluLeuGlnThrLeuGluLeuAsn~TGluThrLeuAsn~TThrHETProLeuSerCysThrLysAsnAanSerHisHisTyrIleNETVa1G1yAsnGluThrGl
350
360
370 380 390 400 410 420 430 440 450 460 ACTAGAACTGACCTTGACCAACACtiAGCATTATTMTCAC~T~TGC~TCTGTCTGATGCC~C~G~CCTCTATGACCA~CTC~ATGAGCAT~TCTC~CTTTCCA~~T yLeuGluLeuThrLeuThrAsnThrSerllelleAsnHisLysPheCysAsnLeuSerAspAlaHisLysLysAsnLeuTyrA~pHi~AlaLe~TSerlleIleSerThrPheHisLe
47u
480
490 500 510 520 530 540 550 560 570 580 GTCCATCCCCAACTTCAATCAGTATGAGGCAATCAGCTGAGCTGCGATT~MTG~~~~TTAGTGTGCAGTA~CCTGAGT~ACAGCTATG~~ffiAT~ffiC~CCA~G~~AC uSerlleProAsnPheAsnGlnTyrGl~ls~TSe~CysAspPheAsnGl~lyLyslleS~rValGl~TyrAsnLeuSerHisSerTyrAlaGlyAspAl~Al~s~~isCysGlyTh
5Y0
600
610 620 630 640 650 660 670 680 690 700 TGTTGCAAATGGTGTGTTACAGA~*TTAT(;ACGAGGAT~~~~G~GT~GAG~TA~AT~~T~TTGA~T~AtiG~~tiTGG~~~TGtiGA~T~TATTATtiA~TAGTTAT~~TAT~TGAT~T rValAlaAsnGlyValLeuGlnThrPheEtETArgMETAlaTrpGlyGlySerTyrIleAlaLeuAspSerG1yAr~lyAsnTrpAspCysIlellEl~hrSerTyrGlnTyrLeuIleI1
710
720
730 740 75u 760 770 760 790 800 610 n2u CCAAAATACAACCT(;GGAATCACTGCCAATTCTCGAGA~~T~r~C~AT~GGTTATCTCG~TCCT~CA~~GGACTAGAGATA~TATATTAtiTAG~ti~rTG~TA~~ACATT eGlnAnnThrThrTrpGl,dspHisCysClnPheSerArgProSerProlleGlyTyrLeuGlyLeuLe~SerGlnArgThrArgAsplleTyrlleSerArgArgLe~Le~GlyThrPh
830
840
B50 860 870 nno BY0 YOO YIO 920 930 '140 CACATGCACACTGTCAGATTCTGAAGGTAAAGACACAC~AG~GGATA~GT~TCACCAGGTGGATGCT~l~GAGGCTtiMCT~TGCTT~GGG~CA~AGCTGTGGC~TGT~ eThrTrpThrLeuSerAspSerCluClyLysAspThrProGlyClyTyrCysLe"ThrArgTrp~TL~~IleGl~Al~Gl~L~"LysCy~Ph~GlyAs"~h~Al~V~lAl~Ly~~ysA~
Y50
Y60
970 YMO YYlJ LO"" 1010 1020 1030 1040 I"5U 1060 TGAGAAGCATGATGAGGAATTTTGTGACATGCTGAGGCTGTTTGACTTC~C~C~G~CATT~~GGTTti~GCTGMGCAC~TG~CATT~A~~GAT~~GCAGT~ nGluLysHisAspGluGluPheCysAspt~~TLeuArgLeuPheAspPheAsnLysGlnAlaIletilnArgLeuLysAlaGluAlaGln~ETSerIleGlnLeulleAsnLysAlaValAs
1070
1OMlJ
109" 1100 ,110 1120 1130 1140 1150 116U 117u 1180 T6CTTTGATAAATCACCMCTTATAATGAA(;AACCATCTAC~GACATCATG~~~CCATA~tiT~TTACAGCMGTA~r~TACCT~~CCACA~~CTA~CG~AG~~ATCACT nAlaLeuIleAsnAspGlnLeuIle~TLysAsnHisLeuArgAspIleMETGlylleProTyrCysAsnTyrSerLysTyrTrpTyrLeuAsnHisThrThrThr(;lyArgThrSerLe
llY0
1200
220
1210 LZL" 123u 124u IL50 lL6U 127u 1280 1ZY" 1300 1310 GCCCAAATGTTGGCTTGTATCAAATGG~CATACTTGMCGAGA~CCACTTTT~TGATGATA~GM~CMGCTGACAATATGATCACTGAGAT~CTAC~~GGAGTATAT~AGAG uProLysCysTrpLeuVa1SerAsnGlySerTyrLeuAsnGluThrHisPheSerAspAspIletiluGlnl:lnAlaAspAsnMETIleThrGluMETLeuClnLysGluTyrHETGl~r
132U
1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 GCAGGGGAAGACACCATTGGCTCTACTTCACCTCTTTGTG~CAGTAC~GTTTCTAT~TATTAGCATCTT~C~~ACCTAGTC~TACC~~TCATAGG~ATATTGTA~C~GTC gGlnClyLysThrProLeuGlyLeuValAspLeuPheValPheSerThrSerPheTyrLeulleSe~llePheLeu~isLe~ValLysll~ProThrHlsArgH~sll~V~lGlyLysS~
1440
1450 1460 1470 1480 1490 1500 1510 1520 1530 1540 1550 1560 GTGTCCWVUCCTCACAGATTGAATCATATGGCCATATG~CATTTGTTCCTGTGGA~T~TAC~CAGCCTtiGTGTG~CTGTG~TGGMGAG~rGAGACCClTGT~GGGC~CCCGTGACCCACC rCysProLysProHisArgLeuAsnHist~TGlylleCysSerCysGlyLeuTyrLysGlnPr~tilyValProValLysTrpLysArg~~d 1570 1580 1590 1600 1610 1620 1630 1640 1650 GCCTATT(;GCGGTGGGTCACGGGGGCGT~C~~~~A~G~CGACTCTAGGTGTC~~TT~CG~CACCATATCTCTG~CAG~CTGCTCTC~C
FIG. 3. Nucleotide sequence and encoded gene product (glycoprotein precursor) of the 5’ region of the s genome RNA of Lassa virus. The sequence originates at the 5’ terminus and is given in the DNA form. The sequence presented is that of the viral-sense RNA. corresponding
genie region. The size and base composition of the hairpin structure is very similar to those of Pichinde and LCM viruses. The 19bp hairpin of Lassa virus compares with 21 bp (14 G-C and 7 A-U) for LCMV and 18 bp (14 G-C and 4 A-U) for Pichinde arenavirus. The nucleotide sequence homology between the 5’ region of the S RNA of Lassa virus and the corresponding region of the S RNA of LCMV (WE) is shown in Fig. 5. Excluding the 5’ terminal G nucleotide on the Lassa virus S RNA, the sequences are identical for 19 nucleotides and contain
only three mismatches in the first 33 positions. Overall, the Lassa virus nucleotide sequence is 60% homologous to LCMV which compares to only 48% homology to the corresponding sequence of Pichinde virus (data not shown). Comparison of the Amino Acid Sequences of the GPC Gene Products of Lassa Virus, LCMV (WE Strain) and Pichinde Arenavirus The amino acid sequence of the glycoprotein precursor of Lassa virus was
LASSA TABLE
VIRUS
1
AMINO ACID COMPOSITION OFTHE GPC GENE PRODUCTOFLASSA VIRUS
Amino acid Ala Arg Asn Asp CYS Gln Glu GUY His Ile Leu LYS Met Phe Pro Ser Thr Trp Tyr Val
Number of residues
Percentage by weight
21 17 32 19 18 20 22 33
4.3 3.5 6.5 3.9 3.7 4.1 4.5 6.7 3.9 6.7 11.4 5.1 4.1 4.1 2.9 6.9 7.1 1.6 4.5 4.7
19 33 56 25 20 20 14 34 35 8 22 23
Net charge = +10.5”
161
GPC GENE
arg-arg residues at position 262-263 in the glycoprotein precursor of LCMV have been shown to react with glycoproteins Gl and G2, respectively, thereby localizing the point of cleavage within GPC to this region of the molecule (Buchmeier et aL, manuscript in preparation). A hydrophatic analysis (Kyte and Doolittle, 1982) of the GPC gene product of Lassa virus indicates that there are two hydrophobic domains similar to those previously noted in the corresponding proteins of Pichinde and LCM viruses (Fig. 7). One of the domains is located near the amino terminus (amino acid residues 18-54) and is interrupted by a single lysine residue at position 33. This hydrophobic region ends at position 55 with the occurrence of another positively charged amino acid, arginine. It is noteworthy that these two positively charged residues are conserved in the glycoproteins of all three viruses. The second hydrophobic domain is located near the carboxy terminus (amino acid residues 426-450) and, as with Pichinde and LCM viruses, is followed by a hydrophilic region containing several charged amino acids.
Mel wt = 55,820 “The net charge was calculated assuming that arginine and lysine residues are each +l, aspartic acid and glutamic acid residues are each -1, and histidine residues are each +1/2 at neutral pH.
aligned by computer to those of LCMV and Pichinde arenavirus as shown in Fig. 6. The data illustrate that the Lassa virus GPC gene product is significantly more homologous to that of LCMV with 290 matched amino acids than to that of Pichinde arenavirus with 222 matched amino acids. In both comparisons however, there is a greater degree of homology present in the carboxy-terminal region of the molecule, beginning with the conserved dibasic amino acid residues at position 256-257 in Lassa virus, position 262-263 in LCMV, and position 270-2’71 in Pichinde arenavirus. Antibodies raised to synthetic peptides corresponding to locations on either the amino- or carboxy-terminal sides of the
DISCUSSION
The nucleotide sequence data presented here concerning the 5’ region of the S genome RNA of the Josiah strain of Lassa virus compliments the sequence data presented by Clegg and Oram (1985), which covers the 3’ region of the S RNA of the Nigerian strain of Lassa. Taken together, these data demonstrate that Lassa virus, like Pichinde arenavirus and LCMV, has an ambisense S RNA. The nucleoprotein gene is encoded by vcRNA sequence corresponding to the 3’ half of the S RNA, whereas the GPC gene is encoded by vRNA sequence corresponding to the 5’ half of the S RNA. The coding regions for the N and GPC genes are separated by a short, intergenie region of 61 nucleotides. The sequence of nucleotides within this region is able to form a 19-bp hairpin structure now characteristic of the arenavirus S RNA. It was originally noted that there ap-
AUPERIN,
162
NUCLEOPROTEIN
Thr
MC CORMICK
j
N”CLEOPROTEfN
“CRNA 5’.....ACA
SASSO, AND
ser Thr Pro Arg “al “al “CG ACA CC” AGA GUC G””
-
A” A A c---G c---G G---C C---G C---G A---” C---G C---G c---G A---” G---C “---A 6---C C---G C---G C---G C---G C---G Le” OCh CUB “AA AUGGACG---ccC”GAcAAGGG”c
UGU AGC UGU GGA UCU CAG CAA GAC AW UACCUGC---GCGACUGUUCCCAG G---t G---c G---C G---C G---C C---G A---U C---G U---A G---C G---C G---C "---A G---C G--C C---G G---c G---C u u "A
GLYCOPROTEIN
“CA UC” C””
CCA W”
AW AGA GAA GGU AM opt Arg LYS Trp Lyr
fi
5’
CAC AGG CAC ACC GUG ucc GUG UCG.....~' VaL Pro "al GLy
VRNA
GL"CcPROTEIN
FIG. 4. Genetic organization of the Lassa virus S RNA and the nucleotide sequences of the vRNA and vcRNA within the intergenic region. Diagonally lined areas indicate the untranslated regions of the genome. Arrows indicate the 5’ to 3’ orientations of the coding sequences for the N and GPC genes in vcRNA and vRNA sequences, respectively.
peared to be an extra G nucleotide on the 5’ terminus of the S RNA of Pichinde arenavirus (Auperin et al, 198513).This nucleotide was thought to be a cloning artifact since its presence interferred with the exact alignment of the complimentary termini. We have now observed that the S genome RNA of Lassa virus also contains an extra G nucleotide on the 5’ terminus suggesting that this nucleotide may actually be present on the S RNA of both viruses. The ambisense coding strategy is thought to provide a means for the independent regulation of the expression of the N and GPC genes. The messages for these genes must each be transcribed from S sized v and vc RNA templates, respectively. Presumably, the viral RNA polymerase
recognizes the conserved sequence on the 3’ termini of the viral specific RNAs in order to initiate RNA synthesis. A comparison of the terminal nucleotide sequences of the S RNA species of Pichinde arenavirus, LCMV and Lassa virus is shown in Fig. 3A. The data illustrate that the 3’ sequences of all three vRNAs are identical for 19 nucleotides as are their 5’ sequences, excluding the extra G nucleotide present on the 5’ termini of the S RNAs of Lassa and Pichinde viruses. In addition, for all three viruses, the nucleotide sequence on the 3’ termini of the vc S RNAs differs from that on the vRNAs in two positions (Fig. 8B). The observation that these differences are conserved in both type and position among three different arenaviruses sug-
x
10
LO
30
41,
5”
60
~“CAC~CCGATCCTAC~A~T~~~~CC~I.TCMI;TI;TCCT~~”~TAM-----------------------~~~~ACAMTA~~TCACATTCT~.CC~C~~CTCAT”TA ::::::::::::::::::: :::: : :::::: :: : ::: . . .. .
70 . .
. .
CGCACCCCGGATCCTA~CTTmCCATTCCGCTTTCCTTTCCT~A~A~CTGGGTG~GGATTCT~CCCA~~GG~GTCAGATT~G~~CG~GAGG‘~TG~T~~TC x I” 2” 3” 4” 5” 60 70 80
8”
.. . .. .
:
::
90
::::: : :::
210
.. .. .. .. ..
450 460 47” 480 490 S”” 51” 52” 440 TATGACCACCCTCTTATCAGCAT~TCTC~CCTTCCA~,~GTC~T~CC~C~C~TCAGTATGAGGC~TGAGCTGCGATT~~---T~~G~GA~~TGTG~GT~C :::::::::::::::::::::: ::: : :: : ::: :: :: . . . . . .. .. .. : :::::: TTTGACCATACACTCATGA(iTATAGTCTCGAGTCTACAC~C~TATCAGA~~~CC~CTAC~GCAGTGTCTTGTGAT~~C~TG~ATCACCATT~----T~~C 500 51” 53” 55” 560 48” 49” 520 54”
300
310
34”
350 430 ::
530 :
: :::
470
54”
550 ::::::
58” 650
7,!” 67” 69” 7”” 73” 74” 75” 68” II” CACTCAGGCCCTGGCAACT~GA~GTATTATCACTACTAGTTATC~TATCTG~C~TCC-TAC~CCTG~~CATCACTGCC~~~C~AGACCATCTCCCATCGG~ATC~~ : :::: : :: ::: :: :: ::::: :: :: :: :: .....’ . .. . : :: :::::: : ::::: :: : CGTTCAGATGCCMGACCACTTCCTCCAGCC~C~CCT~CCAGTACCT~TCATAC-CAGGAC~G~-CCACTGTAGATAT~A~CCCTT~GGGATGTCT~~TCCTC 7L” 75” 78” 73” 74” 76” 79” no0
770
700
:::::
::
:
88”
890 ::
:::::
:
::::::
IL60
1170
124”
1250
,640 ,650 1620 1630 ----TCTGMCACCATATCTCCGGCAGCACTCTCAAM :::::: : :: ::: ::::: : CCTCCTCTGMGATCAMTCATCTCCCA~~ATGTTGTG~C~TC~ 1670 1680 1690 1660 NUnBER
OF “ATCHED
BASES
-
:
::::::::
:
:::::: ,,I30 1260
::::
:
129”
136”
::
,300
137”
:::::: :: :: : :: :: :::: :: :: :: : :: : :: :: i: ::::
,311”
:::
:: : ::
,400
,410
,420
148”
,490
150”
:::
:::::
:
,520
:::
::::
1530
,600
1630
1140
:::::
:::
,280
:
,060
,130
: ::
:::
:::
LOS”
,I50 ,170 ,160 ILt)” 119” LLUO ,Zl” ,220 1230 TCTMTIACAGCMCTA~TCCTACCT~C~CACMC~‘ACT~G~~CATCA~GCCC~TG~~CTTGTATC~T~TTCAT~~~~AGACCCA‘~T~~CTGATGATATT .. .. .. .. .. .. .. .. .. .. .. ::::::::: :::: ::::: :: : :: : : .. .. .. .. .. :: : ::::: : ::::: :: :::::::: ~MTTACTCAMG~CTCGT~TCGMCATGCTM(;ACT~~CTG~~T~~~~~~T~~~~~~~~MG~G~~G~~~~~~~A~~~~~~~C~~~~~GMTGAGAC~~~~~T~~~TGAT~TC 1190 121” 120” 12LO 1230 124” ,250 126” 127”
15,” 15LO 153” 154” 1 155” 156” 157” ,560 1 1590 CCTCTCCCT~CAMTCC~A~TCACACC~TCTC~~CCCCCT~CCC~CCC~A~~C~T~~TCACti~~CCTCCATTTACAC~CA~CT~~TCTC~T~---. . . .. . ::::: ::: : : ::: ::: :: :::: : :: ::: ::::: :::::: CCTCTAAAMCTATCTW;AACGCT~TCAG-~A~~CCTCCCTGACTCTCCACCTCG~GAGGTGGAGAGTCA~G~GCC-----CAG~~CTTAGAGTGT~C~A~G L 550 ,560 1570 I 58 Y ,590 ,610 ,620 1
,020
1010
:::: 1040
,390
::: 940
93”
100”
1270 ,280 119” 13”” ,310 132” 135” 133” I34” GMCMCMGCTGACMTATGATCACTGAGATGTTACAG~GAGTATATGGAG~GCA~GG~GACACCATTGG‘~CTAGT~A~TCT~GTG~C~TAC~GT~‘~ATCTTATT
900 :::::::
92”
::
::
820
1120
1390 140” 1410 142” 143” 144” 145” 147” 146” AGCATCTTCCTTCACCTACTCAAMTACCMCTCATAC~CTCATAGGCATATTGTA~~GT~TGTCC-CCTCACAGA~MTCATAT~CA~T~~C~~~~ACTCTA-~GC~C :::::::: :: :: : :: : :::::::: ::::: :: :: ::: :: ::::: :: :: :: : :::: : : ::: :: AGCATCTTTCTGCATTTTGTCAGGATACCUCACC~CACATAGACACAT~~GC~*CAT~CC~‘C~CATCG~T~ACC~C~~GATCTGTAG~GT~TC~TT~~~~ 143” 144” 1450 ,460 147” 148” 149” ,500 15,”
780
810
l”5” ,060 l”8” ,030 I”40 L”7” I”90 I LO” II,0 CAMGGTTGMAGCTGMGCACAMTGACCATTCAGCATTCAGTTGATC~-G~T~TGCT~GAT~TGACC~CTTAT~TG~G~CCATCTA~~ACATCATG~~~CCATAC :::::: ::::: :::: : ::::::::::::::::::: :::::: : :::: ::: :::::: AGTAACTTCAAGCMGATTCT~AGTCTGCCTT(;CATGTATTC-C~CA=~=CTCTGA~TCCGATCAGCTG=GATGA~~TCAT~~AGATCT~T~G~TACCATAC ,090 107” ,080 ,100 ,I,” ,120 1130 ,150 ,140
GMC~MGCAGATMCATCATCACAGAGATC~G~M~A~ACAT~~ACM~GAtiTACTCC~TA~CTTMT~ATC~C~~A~~~CMCATCA~ATA~TGATC 13LO 132” 133” 134” 135” I36” 137” 138”
660
:::
760
:::::
:
::
460
620 560 57” 580 590 60” 6,” 63” 640 CTCAGTWCA1;CTATCCTGUCAT(;CAGCCMCCATIGT---------TCTTACAGACL‘---TTTATGAGGATGCCTTCGCCTGCT :::::::::::: : ::: : : : :: :::: ::: ::: ::: ::: :: TTGTCATCTTC~ACCCACAGAGCGCCATCAGCCAGT~CA~ACTTTCAGAGCTAGAGT~CTGGACAT~TTA~ACTGCCT~GGA~~GTACATGAG~GTGG~~CT~ACA 63” 64” 66” 67” 680 690 600 610 62”
91” 920 93” 94” 95” 96” 97” 99” 9nu ATCCTMTTGAGG~CAACTAAAATGCTTCGCGMCACAUJTT ::: : ::: :: :: :: ::::: :: ::::: :::::::: ::::::::::::: :: :::::::: :: :: ::::::::::: ATGATCC~GCTGCAGAGCTCAMTCTT~~G~TACA~TG~GC~TGT~TGTC~TCATGATG~GAGTT~GTGACAT~TACGACT~~GATTAC~~G~~GCCCTG 950 960 97” 98” I”“0 99” LOlO ,020 l”30
::
: ::
57”
8,” 84” 870 790 BOO n2” 0,” (15” ll6” ~CCTCTCAC~~ACTAGAGATATTTATATA~AGTAG~‘A~~GCT~GC~A~CACATGGACA~TC~ATTCTC*GGT~GACA~C~~GGATA~~GTCTGACCAGGTGG : : : :: : : ::::::: :: ::: ::: : :::::::::::: ::::: . .. . . . . .. . .. .. TTTCCTCAG~~AMCTTTCTCAC------TAGCM;ACTTTCA(%CACACACCTGGACCCT 850 86” tit)” 87” 119” 900 YLO
::::
: ::: ::
42” :
: : :
:: 23”
: ::: :::: :::::
36” 37” 3nu 390 400 410 330 34u 350 320 MCAACAGTCATCATTATATMTGCTUIGCMTCACACAG~ACTAGMCTGACCTTGACCMCA~AGCA~A~MTCAC~TTTTGCMTCTGTCTG~CGCC~C-GMCCT~ .. .. .. .. .. .. .. .. .. .. .. . .. .. .. .. .. :::::: :::::: :: :: : :::: : ::::: :: : :: :: :::::: ::: : AACMCTCTC:T~~~~---~ACT-~T 42” 43” 44” 38” 4”O 41” 450 36”
:::::: :::: :: :: :::::::: ::::::::
:
120
AGCTCCTGTC(;CATGTAC~CCTTMTGCTCCCCGATAC~~AiI’MAGL;(;(;T’CTACCAC’LTCAMTCA(jT66AL;TTTGATAT~CCTCA~~T~GACGATGCC~~CGTGCTCAG~ IId” 29” 31” 31” L6” 27” 300 33” 250 240
:
11”
2”O
29”
. . . ...* . ..*....... . . . . . . . :: : : : :::: : : :::
: : :::
. ..*... .......
LOO
170 I40 180 190 I L” I50 16” 12” 13” loo ATAGMGA~TCATGMCATTGTTCTCATTGCACTCCA~~~C’L’CTACTAGCAGTCCTGAMC(jlCTGTACMTL‘TTCCT . . . . . . . . . . . . . . . . . . :: :: ::::: :: :::::::: : :::: :: : :: : : : :::: : .. .. .. . . . . . . . .. . . . : : : :::::: ATTCATCACGTCATCMCATTLTCATTATTCTCCTCATTAT~TCA~ACCATC~CCTCTC~AC~T=~CCCCAC~~CT~CA~ATTACCA~CCTCAC~TC~TTT~~~C~ 19” 130 14” 160 17” 180 200 2,” 12” 15” 240 22” 250 26” 27” 28” .‘,o AGCGTCTT---GCACAACCA(;TCTT---------------TAT~~~TATG~CTTCAGACTCTGG~CT~CAT~AGACAC~~TATGACCATGC~CT~CCT~AC-
90
:::
1540
,610
:::
::::: 1640
1650
X
L7OOX
987
FIG. 5. Alignment of the nucleotide sequences corresponding to the 5’ regions of the S RNA’s of Lassa virus and LCMV (WE strain). The sequences were aligned by the NUCALN program (Wilbur and Lipman, 1933) with a K-tuple size = 3, window size = 10, and a gap penalty = 5. Horizontal lines indicate the positions of the initiation and termination codons for the reading frame encoding GPC. Vertical lines denote the boundaries of the hairpin structures within the intergenic regions.
AUPERIN,
164
SASSO, AND
MC CORMICK
Q”&“WGSYW(B~HPYNMhYSQ~“:::: :::::::::: : ::: ::: :::::::: 4w
410
420
: :
: :: : ::: : :::::::
: :
430
FIG. 6. Alignment of the amino acid sequence of the glycoprotein precursor of Lassa virus to that of LCMV (WE strain) (A) and Pichinde arenavirus (B). The PRTALN program (Wilbur and Lipman, 1963) was used to align the sequences with a K-tuple size = 1, window size = 10, and gap penalty = 5.
LASSA
VIRUS PICHXNDE
-40
+*
--+++++
0
--
-++
-----++ .I+ *+
GPC
-----*+
LCM
YY YY
Y
--+ ++
+u++
300
SEOUENCE
4
+*++
208
100
165
GPC GENE
+**
400
500
NUMBER
GPC
Y
Y
w
2
-2 --
--
--
-+.I-?
0
----++++++
200
100
300
SEOUENCE
LASSA
0
-_------_ ***cc,+*+++
100
200 SEOUENCE
4.
-*
400
NUMBER
GPC
----_
--
300
400
NUMBER
FIG. 7. Comparison of the hydropathic profiles of the GPC gene products of Lassa virus, LCMV (WE strain), and Pichinde arenavirus. Regions with a net hydrophobicity appear above the horizontal center line, and regions with a net hydrophilicity appear below the center line. Y structures indicate the positions of the potential asparagine-linked glycosylation sites along the polypeptide chain. + and - symbols denote the positions of positively (arginine and lysine) and negatively (aspartic acid and glutamic acid) charged amino acids.
A
LCMVvRNA
3' HOGCGUGUCACCUACGAUCCG.........................CGGAUCCUAGGGGCCACG
Lassa vRNA
3'HoIoo~oooo~
Pichinde
3'Hooooooomrroo~.........................mrrmrmmr
vRNA
Arenavirus
vF.NA
Arenavirus
vcRNA
.........................
C 5'
omomnmnwm5' oo.0
5'
3'HOGCGUGUCACCIlAGGAUCCG......................... 3',,(c)o8@oeC~r.........................
FIG. 8. (A) Comparison of the terminal nucleotide sequences of the viral S RNAs of LCM, Lassa, and Pichinde viruses. Closed circles indicate homologous nucleotides in the Lassa and Pichinde sequences compared to LCMV. (B) Nucleotide sequence homology between the 3’ termini of the viral and viral-complimentary S RNA species of arenaviruses.
166
AUPERIN,
SASSO, AND
MC CORMICK
gests that they are functional and have, CLEGG,J. C. S., and ORAM, J. D. (1985). Molecular cloning of Lassa virus RNA: Nucleotide sequence therefore, been maintained through evoand expression of the nucleocapsid protein gene. lution. One possibility is that the 3’ terVirdogy 144,363-372. minal nucleotide sequence present on the FRAME, J. D., BALDWIN,J. M., GOCKE,D. J., and TROUP, v and vc S RNA species functions in deterJ. M. (1970).Lassa fever, a new virus disease of man mining the efficiency with which RNA synfrom West Africa. I. Clinical description and thesis is initiated from either template, pathological findings. Am J. Trap. Med Hz/s- 19, thereby providing the basis for the genetic 670-676. regulation inferred by the ambisense cod- GRUNSTEIN,J. M., and HOGNESS,D. S. (1975). Colony ing strategy. hybridization: A method for isolation of cloned DNA’s that contain a specific gene. Proc Nati Acm! The regulation of arenavirus RNA repSci. USA 72,3961-3965. lication, gene transcription and expression are areas for further analyses. The avail- HARNISH, D. G., DIMOCH,K., BISHOP,D. H. L., and RAWLS,W. E. (1933). Gene mapping in Pichinde viability of cDNA probes to the S RNA rus: Assignment of viral polypeptides to genomic L should allow these processes to be followed and S RNA%. J. Viral 46,633~641. during productive as well as persistent inKILEY, M. P., TOMORI,O., REGNERY,R. L., and JOHNSON, fections. The determination of the exact 3’ K. M. (1980). Characterization of the arenaviruses termini of the mRNA’s transcribed from Lass and Mozambique. In “Replication of Negative the N and GPC genes will undoubtedly Strand Viruses” (D. H. L. Bishop and R. W. Comprovide further insight into the function pans, eds.), pp. 1-19. Elsevier North-Holland, Amof the intergenic region. Finally, cDNA sterdam. clones of the N and GPC genes of Lassa KYTE,J., and DOOLITIXE,R. F. (1982).A simple method for displaying the hydropathic character of a provirus can now be expressed in a number of tein. J. Mel Bid 157,105-132. eukaryotic systems for use as reagents in the diagnosis of Lassa infections, in the MAXAM, A., and GILBERT,W. (1980). Sequencing end labelled DNA with base specific chemical cleavages. study of the cell-mediated immune reIn “Methods in Enzymology” (L. Grossman and K. sponse to Lassa infection, and in genetiMoldave, eds.), Vol. 65, pp. 499-560.Academic Press, cally engineered vaccines for the prophyOrlando, Fla. laxis of Lassa fever in areas of the world MCCORMICK,J. B., KING, I. J., WEBB,P. A., SCRIBNER, where the virus is endemic. C. L., CRAVEN, R. B., JOHNSON,K. M., E~~~crrr, REFERENCES
AUPERIN, D. D., GALINSKI, M., and BISHOP,D. H. L. (1984a). The sequence of the N protein gene and intergenie region of the S RNA of Pichinde arenavirus. Virobgp 134,208-219. AUPERIN,D. D., ROMANOWSKI,V., GALINSKI, M., and BISHOP,D. H. L. (1984b). Sequencing studies of Pichinde arenavirus S RNA indicate a novel coding strategy, an ambisense viral S RNA. J. Vid 52,
L. H., and WILLIAMS,B. (1986).Lassa fever: Effective therapy with Ribavirin. N. En& J. Med 314, 2026. MCMASTER, G. K., and CARMICHAEL.,G. G. (1977). Analysis of single- and double-stranded nucleic acids on polyacylamide and agarose gels by using glyoxal and acridine orange. Proc Natl Acad Sci USA 74,4835-4838.
MONATH,T. P., NEWHOUSE,V. F., KEMP,G. E., SETTER, H. W., and CACCIAPUOTI,A. (1974). Lassa virus isolation from Masbmys natolensis rodents during an epidemic in Sierra Leone. science 185,263-265. 897-904. BISHOP,D. H. L., GOULD,K. G., AKASHI, H., and CLERX- MURPHY,F. A., and WALKER, D. H. (1978). Arenaviruses: Persistent infection and viral survival in resVAN IIAASTER,C. (1982).The complete sequence and ervoir hosts. In “Viruses and Environment” (E. coding content of snowshoe hare bunyavirus small Kurstak and K. Maramorosch, eds.), pp. 155-180. (S) viral RNA species. Nucleic Acids I&s. 10,3703Academic Press, New York. 3713. CASALS,J., BUCKLEY,S. M., and CEDENO,R. (1975). MURPHY,F. A., WEBB, P. A., JOHNSON,K. M., WHITFIELD, S. G., and CHAPPJZLL,W. A. (1970). ArenaAntigenic properties of the arenaviruses. Bull WHO viruses in Vero cells: Ultrastructural studies. J. VZ52,421~427. rol 6,507-513. CLEGG,J. C. S., and LLOYD,G. (1983). Structural and RAWLS,W. E., and LEUNG,W.-C. (1979).Arenaviruses. cell associated proteins of Laasa virus. J. Cen vird In “Comprehensive Virology” (H. Frankel-Conrat 64,1X7-1136.
LASSA VIRUS GPC GENE and R. R. Wagner, eds.), Vol. 14, pp. 157-192. Plenum, New York. ROMANOWSKI,V., and BISHOP,D. H. L. (1985). Conserved sequences and coding of two strains of lymphocytic choriomeningitis virus (WE and ARM) and Pichinde arenavirus. V&us Res. 2,35-51. ROMANOWSKI, V., MATSUURA,Y., and BISHOP,D. H. L. (1985). Complete sequence of the S RNA of lymphocytic choriomeningitis virus (WE strain) compared to that of Pichinde arenavirus. virus Res. 3, 101-114. VEZZA, A. C., CASH, P., JAHRLING,P., EDDY, G., and BISHOP,D. H. L. (1980). Arenavirus recombination: The formation of recombinants between Pichinde
167
and Pichinde Munchique viruses and evidence that arenavirus S RNA codes for N polypeptide. Vi* 106.250-260. VEZZA,A. C., CLEWLEY,J. P., GARD, G. P., ABRAHAM, N. Z., COMPANS,R. W., and BISHOP,D. H. L. (1978). Virion RNA species of the arenaviruses Pichinde, Tacaribe, and Tamiami. J. I&-o1 26,485-497. WALKER,D. H., WULFF,H., LANGE,J. V., and MURPHY, F. A. (1975). Comparative pathology of Lassa virus infection in monkeys, guinea-pigs, and Mastays nu-
Bull
WHO 52,523-534.
WILBUR,W. J., and LIPMAN, D. J. (1983). Rapid similarity searches of nucleic acid and protein data banks. Proc. Nat1 Acad Sci. USA 30,726-730.