The VP35 and VP40 Proteins of Filoviruses

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Volume 322, number 1,4146 FEBS 12389 May 1993 0 1993 Federation of European Biochemical Societies 00145793/93/$6.00

The VP35 and VP40 proteins of filoviruses

Homology between Marburg and Ebola viruses*

A.A. Bukreyev, V.E. Volchkov, V.M. Blinov and S.V. Netesov

Institute of Molecular Biology, 6331.59 Koltsovo, Novosibirsk Region, Russian Federation

Received 9 March 1993

The fragments of genomic RNA sequences of Marburg (MBG) and Ebola (EBO) viruses are reported. These fragments were found to encode the VP35 and VP40 proteins. The canonic sequences were revealed before and after each open reading frame. It is suggested that these sequences are mRNA extremities and at the same time the regulatory elements for mRNA transcription. Homology between the MBG and EBO proteins was discovered.

Marburg virus; Ebola virus; Filoviridae; Nucleotide sequence; Amino acid sequence

1. INTRODUCTION 2. MATERIALS AND METHODS The Popp strain of MBG and the Mayinga strain of EBO were Marburg (MBG) and Ebola (EBO) viruses belong to received from the Belarus Institute of Epidemiology and Microbiology the Filoviridae family [l]. These viruses cause similar (Minsk, Belarus). Purification of the virus, isolation of the genomic hemorrhagic fevers with a high mortality rate [2]. RNA, synthesis, cloning and sequencing of cDNA were carried out Both viruses have seven structural proteins. GP is the as in [lo] for MBG and as in [9] for EBO. only glycosylated protein [3,4]. The gene order determined for MBG by Feldmann et al. [5] is the following: 3’-NP-VP35-VP40-GP-VP3O-VP24-L-5’. The proteins 3. RESULTS AND DISCUSSION are synthesized from mRNAs transcribed from the negative-strand RNA genome [3,4]. The primary structures of the MBG and EBO The nucleotide sequence of the MBG Musoke strain genomic RNAs were determined by sequencing partly NP gene was reported by Sanchez et al. [6] and of the overlapping cDNA-containing recombinant plasmids. L gene by Muhlberger et al. [7]; for the EBO Mayinga We found seven long open reading frames (ORFs) in the strain the sequence of the NP gene was published by cDNA of both viruses which corresponded to the seven Sanchez et al. in [8]. In our previous work we reported known viral proteins. the nucleotide sequence of the EBO Mayinga strain 3’ The fragment of the cDNA MBG genomic RNA se- end of the GP gene [9]. In this work, we present the quence with 0RF2 and 0RF3 encoding the VP35 and nucleotide sequences of MBG and EBO genomic RNA VP40 proteins is shown in Fig. 1. The context of the fragments, which encode the VP35 and VP40 proteins, initiation AUG codon for ORFl is not the most favour- as well as deduced amino acid sequences and the homol- able for translation initiation for eukaryotic ribosomes; ogy between these proteins of both viruses. the AUG codon for 0RF2 corresponds to the Kozak rule [l 11. The length of the putative VP35 polypeptide is 329 amino acids (aa) and that of VP40 is 303 aa. The calculated mol.wt. are 36,149 and 33,734 Da, respectively, which approximates the results of SDS-PAGE [5]. Computer analysis of the full-length cDNA sequence determined canonic regions 3’-CU~C~IJ~- Correspondence address: A.A. Bukreyev, Institute of Molecular Biol- UAAUU-5’ and 3’-UtAUUCUUUUU-5’ (the se- ogy, Koltsovo, 633159, Novosibirsk Region, Russian Federation. quences are given for negative-strand RNA) before and Fax: (7) (3832) 328 831. after each ORF, respectively. The former of the above sequences constitutes a part of transcriptional start sig- *The presented sequences of the fragments of MBG and EBO genomes were published in the EMBL Data Library (X64496, 1992, nal 3’-NNCUNCNUNUAAUU-5’, described in [5] for and X61274, 1991, respectively). MBG Musoke strain and shown to be mRNA extremi-

Published by Elsevier Science Publishers B. V. 41 Volume 322, number I FEBSLETTERS May 1993

~TATTAAAGGTTTTCTTTAATATTCAGAAAAGGTTTTTTATTCTCTTCTTTCT 60 TT~C~CATATTG~TMTMTTTTCACMTGTGGGACT~ATCATATA~CMCM 120 MWDSSYHQQ 9 GTCAGTGAGG~T~AT~C~G~GTTC~CATAGATCMG~~~TGCCMTCCC 180 VSEGLMTGKVPIDQVFGA~ 29 TCAGAGhAGTTACACMGAGAAGGAAACC~GGCACAGTTGGACTACMTGCAGCCCT 240 SEKLHKRRKPKGTAVGLQCSP 49 TGTCTMTGTC~GGCGACACACTGATGATATTGTTTGGGACCMCTGATCGTGMG 300 CLNSKATSTDDIVWDQLIVK 69 AMACACTAGCTGATCTACTTATACCGATAAATAGGCAGATATCGGACATTCAAAGCACT 360 KTLADLLIPINRQISDIQST 89 CT~CG~GT~CMC~~GTCCAT~TT~GCGGCM~ACATGAGATMCCCCA 420 LNEVTTRVHEIERQLHEITP 109 GTGTTAAAAATGGGAAGGACACTGGAAGCP~TTTCCAAGGGGATGTCAGMATGTTAGCC 480 VLKMGRTLEAISKGHSEMLA 129 AAATACGACCACCTCGTMTTTCMCTGGMGMCCACTGCACCAGCTGCTGCCTTTGAT 540 KYDHLVISTGRTTAPAAAFD 149 GCTTACTTAAATGAGCATGGTGTCCCTC~CCCCCMCCTG~~T~TC~GATC~GGG 600 AYLNEHGVPPPQPAIFKDLG 169 G~~TCMCMGC~GTAGT~GGG~~CA~G~~TG~CMCA~TGCAG~C 660 VAQQACSKGTNVKNETTDAA ia GACMGATGTCGAAAGTTCTTGhftCTCAGTGAGGAGACGTTCTCC~GCC~TCTTTCA 720 DKMSKVLELSEETFSKPNLS 209 GCT~GGATTTAGCCCTTTTGTTGTTTACCCATCTACCCGGCMCMCACTCCATTCCAT 780 AKDLALLLFTHLPGNNTPFH 229 ATCCTAGCTCMGTCCTTTCAAAAATTGCTTACAAGTCAG~GTCCGGAGCATTTTTG a40 ILAQVLSKIAYKSGKSGAFL 249 ~TGCA~CACCA~T~T~GT~GGAGA~TGC~AGGCAGCATTGACTC~CTA 900 DAFHQ I L S EG E NAQAAL TR L 269 ~CAGMCA~TGATGCT~CCTCG~GTAG~CCTCCAGTGATMGAGTC~C~C 960 SRTFDAFLGVVPPVIRVKNF 209 CAAACAGTCCCTCGCCCATGTC AMAAAGTCTTCGGGCTGTTCCTCCCAACCCAACMTT 1020 QTVPRPCQKSLRAVPPNPTI 309 W\CAAAGGATGGGTCTGTGTTTATTCATCTGAGCAAGGTGAGACACGGGCCCTG~TC to80 DKGWVCVYSSEQGETRALKI 329 TMTT~TCA~G~M~AG~GCAGGGGGAG~ATC~C~GAG~~TAC~GACACT 1140 " ~~AT~~~GcATATATGTG~~~cGTGA~TAGA~cATc~MTAGMGTAGT 1200 AATTTATTTCTGTCTTAAGTGTGATTTTCACCTTAA 1260 TCCTTGMGTMCTTTTTTATATA~ATAGAGGAACTAATGGGTCT 1320 ACCT~CAGGTATGACTGAGTGATCAGTATATTTTAT~CCMGCM~~CTTCTCAC 1380 TTTTT~~TCMCTMCAACATAGAAAACATATATTTATCCTTGTGTMTTCTCGGCTTA 1440 GTTGGAATTAACTTTTGTTCMTTCMGACGCTTATTCATAGTAGATTATATGA~T~ 1500 TAT~GT~MGATAT~TT~TTATACC~AC~GAGATA~TG~TTM-~~~~ 1560 T~TE~G~CA~MGM~TC~CT~TC~~TG~C~~ACTGGMGGAGTATC~C 1625 M~TCAGC~G~G~~~TTGTTACTTMGTCATTCTT~~TT~TTCACACA 1680 AGGTAGTTTGGGTTTATATCTAGAACAAATTTT~TATGGCCAGTTCCAGC~TTACMC 1740 MASSSNYN a ACATACATGCAATACTTGACCCCCTCCTTATGCTGATCACGGTGC~CCAGTTGATC 1800 TYMQYLNPPPYADHGANQLI 28

CCGGCGGATCAGCTATCAAATCAGCAGGGTATAACTCCAAATTC 1860 PADOLSNQQGITPNYVGDLN 48 CTA~~ATCAG~~~~G~T~~TGCCATGC~TCAC~AGAGGC~T~~GA~ 1920 LDDQFKGNVCHAFTLEAIID 68 ATATCTGCGTATMTGMCCAACAGTCAAAGGTGTGTTCCAGCATGGCTGCCTCTCGGGA~ 1980 ISAYNEPTVKGVPAWLPLGI aa ATGAGCMTTTTGMTATCCTTTAGCTCATACTGTGGCTGCGTTGCTCACAGGCAGCTAT 2040 HSNFEYPLAHTVAALLTGSY 108 ACAATCACCCMTTTACTCATAATGGGCMAAATTCGTCCGTGTMATCGACTCGGTACA 2100 TITQFTHNGQKFVRVNRLGT 128 GGAATCCCAGCACACCCACTCAGMTGTn;CGTGAAGGAB 2160 GIPAHPLRNLREGNQAFIQN 148 ATGGTGATCCCCAGAAATTTTCCACTMTCMTTCACCTACMTCTCACTMCTTAGTA 2220 NVIPRNFSTNQFTYNLTNLV 168 TTGAGTGTGC~GCTTCCTGATGATG~~TGG~G~~~AT~~~GGAC~~T~TTGGG 2280 LSVQKLPDDAWRPSKDKLIG 188 MCACCATGCATCCCGCAGTCTCCATACACCCG~TTTGCCAC~CATTGT~TACC~~A 2340 NTMHPAVSIHPNLPPIVLPT 208 GTCAAGAAGCAGGCTTATCGTCAGCATAAAAATCCCAAC 2400 VKKQAYRQHXNPNNGPLLAI 228 TCTGGCATCCTTCACCMCTGAGGGTCGAG~GTCCCAGAGMGACMGCCTGT~AGG 2460 SGILHQLRVEKVPEKTSLFR 248 ATTTCACTTCCTGCCGATATGTTCTCAGTAAAAGAAGGTATGATGMG~GGGGAGM 2520 ISLPADMFSVKEGHMKKRGE 268 MTTCCCCGGTGGTTTATTTTCMGCACCTGAGMCTTCCCTTTGMTGGCTTC~CMC 2580 NSPVVYFQAPENFPLNGFNN 288 AGACAAGTTGTA~AGCGTATGCGMTCCMCG~TCAGTGCCG~G~T~TGCT~~ 2640 RQVVLAYANPTLSAV* 303 ATWLGACAGGAGTCCATCTGCATMG~GCAn;GCCTAAA 2700 ACMGATTAGTTTGTATTGATTTCAATAATGCTTTAACCTGG 2760 ~M~M~TGATCAGCTTGCMGATGTMTCTCTTTTGGGTCATCAGATCTATMTGG 2820 GTTTACTAGATTATATMAAGAAATAGTMTGTTTTATAAACMTTCTTGCTTAGTTTTA 2880 CTTTGATTTACTMCATATATCATTGTGCCCTTCATTGCTAAGTGATGAT 2940 GATATTCCTTCTGAAATAGTAAGMAAA 2966 42 Volume 322, number 1 FEBSLETTERS May 1993 ties. The latter corresponds to the transcriptional stop Table I signal and mRNAs extremity sequence 3’-UAAUUC- The lengths of the 3’ and 5’ non-coding regions of the VP35 and VP40 UUUUU-5’ [5] (Fig. 1). We suggested that the con- genes of MBG and EBO served sequences from Fig. 1 are mRNA extremities 3’ end 5’ end and signals for initiation and termination of transcription simultaneously. MBG VP35 419 The sequence of the cDNA EBO genomic RNA frag- EBO VP35 1;: (99) 259 ment is shown in Fig. 2. The VP35 gene ORF starts MBG VP40 155 343 from the AUG codon located at positions 100-102 (cal- EBO VP40 91 438 culated mol. wt. of the putative protein is 37,362 Da). Despite the fact that the first AUG codon corresponds better to the consensus sequence for eukaryotic initiation [l l] than the second one located at 157-159, we amino acid homology between the VP35 proteins makes assume that the VP35 synthesis starts from the latter. up 33% (the putative N-end of the EBO VP35 corre- In this case the length of the putative polypeptide is 321 sponds to the second methionine of the ORF); homol- aa; the calculated mol. wt. of 35,277 Da fits better that ogy between the VP40 proteins is 27%. The comparison evaluated by SDS-PAGE analysis [4]. The comparison of hydropathic plots [12] of the amino acid sequences of the putative polypeptide with the corresponding is presented in Fig. 4. It is obvious from the figure that MBG polypeptide (see below; Fig. 3) supports this as- there is similarity between the MBG and EBO proteins. sumption. The AUG codon near the 5’ terminus of the The plot of the MBG VP35 shows a striking hydrophilic VP40 ORF corresponds to the consensus sequence for domain (2842) (Fig. 1); the analogous domain in EBO eukaryotic ribosomes [ll]. The length of the putative is located at 57-76 (Fig. 2). Since no amino acid homol- VP40 polypeptide is 326 aa; the calculated mol. wt. of ogy between these two regions was revealed, the exis- 35,182 Da is less than that estimated by SDS-PAGE tence of the domains may be due to the functional sim- (40,000 Da) [4]. A conceivable reason for this difference ilarity of these regions in both viruses. may be post-translation modification of synthesized The position of the MBG and EBO VP35 and VP40 protein. genes in the genome, and the fact that the VP40 consti- As with MBG, computer analysis of the full-length tutes 39% ohotal virion protein in MBG [3-5,131, sup- cDNA of EBO genomic RNA revealed the canonic se- port the assumption that VP35 is a protein analogous quence 3’-$XJ~CUUCUAAUU-5’ before all the seven to the P proteins of paramyxoviruses, and VP40 is the ORFs and the sequence 3’-UAAUUCUUUUU-5’ after main matrix protein [3,5,8,13]. However, although con- 6 of the 7 ORFs (with the exception of the 7th ORF) siderable homology was revealed earlier between the L (data not shown). Since these sequences are similar to proteins of MBG, EBO and paramyxoviruses [7,14,15], the transcription start signal, 3’-UACUCCUUCUAA- we failed to discover any homology of the MBG and UU-5’ and to the stop signal, 3’-UAAUUCUUUUU- EBO VP35 and VP40 proteins with the protein se- 5’, respectively, which were described for the NP gene quences of paramyxoviruses. of EBO and shown to be NP mRNA extremity se- The significant homology between the MBG and quences [8], we suggest that the canonic sequences from EBO VP35 and VP40 proteins confirms the evolution- Fig. 2 are transcriptional start and stop signals and ary relationship between these two members of the mRNA extremities for the VP35 and VP40 mRNAs Filoviridae family. simultaneously. The VP35 and VP40 genes of EBO share a common interesting trait: the stop signal of VP35 and the start signal of VP40 are overlapping (Fig. 2). A similar feature was found with the MBG VP30 and VP24 genes ([5]; Bukreyev et al., unpublished data). Both pairs of the genes have long 3’ and 5’ untrans- lated regions. Table I shows the lengths of these regions. Significant homology between both polypeptides of the two viruses was revealed. The alignments between the MBG and EBO proteins are shown in Fig. 3. The

t Fig. 1. The fragment of the MBG genome (cDNA plus-strand) with the predicted amino acid sequences of the VP35 and VP40 proteins (shown beneath the nucleotide sequence). The putative start signals for the RNA-dependent RNA-polytnerase are underlined and the stop signals are overlined. The striking hydrophobic region is boxed.

43 Volume 322, number 1 FEBS LETTERS May 1993

~TI;ATl;AAC;ATTAAAACCTTCATCATCCTTAL‘GTCMTTGMTTCTCTAGCACTCGMGC 60 TTATTi;'rl~TTL'MTGTAAAAi;AAAAGCTGGTCTMC~GATGACMCTAGMC~GGGC 120 MTTRTKG 7 AGGGGCCATACTGCGGCCACGACTCAAAACGACAGACAGMTGCCAGGCCCTGAGCTTTCGGGC 180 RGHTAATTQNDRaPGPELSG 27 TGGATCTCTGAGCAGCTMTGACCGGAAGAATTCCTGTMGCGACATCTTCTGTGATATT 240 w I SEQLNTGRIPVSDIFCDI 47 GAGMCMTCCAGGATTATGCTACGCATCCCAAATGCAA 300 E N N P G L C Y A[S_Q M 0 0 T K P N P K 67 ACr;CGCMCAGTC~CCCAAACGGACCCMTTTGCMTCATAGTTTTGAGGAGGTAGTA_;r_~-_~-_~__8-~_~-?;~ p 360 ___~___ ICNHSFEEVV 87 CAAACATTGGCTTCATTGGCTACTGTTGTGCMCMC~CCATCGCATCAGMTCATTA 420 QTLASLATVVQQQTIASESL 107 GMCMCGCATTACGAGTCTTGAGMTGGTCTAAAGCCAGTTTATGATATGGC~CA 480 E 0 R I TSLENGLKPVYDMAKT 127 ATCTCCTCATTGAACAGGGTTTGTGCTGAGATGGTTGC~TATGATCTTCTGGTGATG 540 I S S L N R V C AEMVAKYDLLVM 147 ACMCCGGTCGGGCMCAGCMCCGCTGCGGCMCTGAGGCTTATTGGGCCGMCATGGT 600 TTGRATATAAATEAYWAEHG 167 CMC(:ACCACZTGGACCATCACTTTATGMG~GTGCGATTCGGGGTMGATTGMTCT 660 Q P P P G P S L Y E E S A I RGKIES 187 AGAi;ATGAGACCGTCCCTCAAAGTGTGTTAGGGAGGCATTCMCMTCT~CAGTACCACT 720 RDETVPQSVREAFNNLNSTT 207 TCACTMCTGAGGAAAATTTTGGGAAACCTGACATTTCGGC~GGATTTGAG~CATT 780 SLTEENFGKPDISAKDLRNI 227 ATGTATGATCACTTGCCTGGTTTTGGMCTGCTTTCCACCMTTAGTACMGTGATTTGT 840 MYDHLPGFGTAFHQLVQVIC 247 AAATTGGGAAAAGATAGCMCTCATTGGACATCATTCATGCTGAGTTCCAGGCCAGCCTG 900 K L G KDSNSLDI IHAEFQASL 267 GCTi;MGGAGACTCTCCTCAATGTGTGCCCTMTTATCTTC 960 AEGDSPQCALI QITKRVPIF 287 (7MGATGCTGCTCCACCTGTCATCCACATCCGCTCTCGAGGTGACATTCCCCGAGCTTGC 1020 QDAAPPVIHIRSRGDIPRAC 307 CAGAAAAGCTTGCGTCCAGTCCCACCATCGCCCMGATTGATCGAGGTTGGGTATGTGTT 1080 QKSLRPVPPSPK IDRGWVCV 327 TTTCAGCTTCMGATGGTAACTTGGACTCAAAATTTTGAGCCMTCTCCCTTCCCTC 1140 F Q L Q D G K T L G L K I * 340 ~G~GAGGCGMTAATAGCAGAGGCTTCMCGGCTG~CTATAGGGTACGTTACATTM 1200 TGATACACTTGTGAGTATCAGCCCTGGATMTATMGTCMTT~CGACCMGAT~ 1260 TTGTTCATATCTCGCTAGCAGCTTAAAATATAT~TGTMTAGGAGCTATATCTCTGACAG 1320 TATTATMTCMTTGTTqTTAAGTMCCCAAACCMAAGTGATGMGATT!u4GAAMACC 1360 TACCTCGGCTGAGAGAGTGTTTTTTCATTMCCTTCATCTTGT~CGTTGAGC~TT 1440 (;TTAAAAATATGAGGCGGGTTATATTGCCTACTGCTCCTCCTGMTATATGGAGGCCATA 1500 MRRVILPTAPPEYMEAI 17 TACCCTGTCAGGTC~TTCAACAATTGCTAGAGGTGGCMCAGCMTACAGGCTTCCTG 1560 YPVRSNSTIARGGNSNTGFL 37 ACACCGGAGTCAGTCMTGGGGACACTCCATCGMTCCACTCAGGCCMTTGCCGATGAC 1620 TPESVNGDTPSNPLRP I AD D 57 ACCATCGACCATGCCAGCCACACACCAGGCAGTGTGTCATCAGCATTCATCCTTGMGCT 1680 TIDHASHTPGSVSSAFILEA 77 ATGc;TGMTGTCATATCGGGCCCCAAAGTGCTAATGATTGGCTTCCT 1740 MVNVISG PKVLMKQI P I w L P 97 I:TAGGTGTCGCTGATCAGACCTACAGCTTTGACTCMCTACGGCCGCCATCATGCTT 1800 LGVADQKTYS FDSTTAA I M L 117 GCTTCATACACTATCACCCATTTCGGCMGGCMCCMTCCACTTGTCAGAGTC~TCGG 1860 ASYTITHFGKATNPLVRVNR 137 CTGGGTCCTGGMTCCCGGATCATCCCCTCAGGCTCCTGCGMTTGG~CCAGGCTTTC 1920 LGPGIPDHPLRLLRIGNQAF 157 CTCCAGGAGTTCGTTCTTCCGCCAGTCCAACTACCCCCAGTATTTCACCTTTGA~T~CA 1980 LQEFVLPPVQLPQYFTFDLT 177 GCACTCAAACTGATCACCCACCACTGCCTGCTGCMCATGGACCGATGACACTCCMCA 2040 AL K L ITQPLPAATWTDDTPT 197 GGATCAAATGGAGCGTTGCGTCCAGGMTTTCATTTCATCC~CTTCGCCCCATTCTT 2100 G SNGALRPG ISFHPKLRPIL 217 TTACCCMCAAAAGTGGGP~GGGGMCAGTGCCGATCTMCATCTCCGGAG~TC 2160 LPNKSGKKGNSADLTSPEKI 237 CMGCMTMTGACTTCACTCCAGGACTTTMGATCGTTCCMTTGATCCMCC~T 2220 QAIMTSLQDFKIVPI D P T K N 257 ATCATGGGMTCGAAGTGCCAGAAACTCTCTGGTCCACMGCTGACCGGTMGMGGTGACT 2280 I M G IEVPETLVHKLTGKKVT 277 TCTAAAAATGGACMCCMTCATCCCTGTTCTTTTGCC~GTACATTGGG~GGACCCG 2340 SKNGQPI IPVLLPKYIGLDP 297 GTGGCTCCAGGAGACCTCACCATGGTMTCACACAGGATTGTGACACGTGTCATTCTCCT 2400 VAPGDLTMVITQDCDTCHSP 317 GCMGTCTTCCAGCTGTGATTGAGMGTMTTGCMTMTTGACTCAGATCCAGTTTTAT 2460 ASLPAVIEKK 326 AGMTCTTCTCAGGGATAGTGATMCATCTATTTAGTMTCCGTCCATTAGAGGAGACAC 2520 TTTTMTTGATCMTATACTAAAGGTGCTTTACACCATTGTCTTTTTTCTCTCCT~TG 2580 TAGMCTTMCAAAAGACTCATMTATACTTGTTTTT~GGATTGATTGATG~GATC 2640 ATMCTAATMCATTACATMTCCTACTATMTCMTACGGTGATTC~TGTTMTC 2700 TTTCTCATTGCACATACTTTTTGCCCTTATCCTCAAATTGCCTGCATGCTTACATCTGAG 2760 GATAGCCAGTGTGACTTGGATTGGAAATGTGGAGAAAAAATTCTAGGTT 2820 GTTCACMTCCAAGTACAGACATTGCCCTTCTMTTMGAAAAAA 2865 Volume 322, number 1 FEBSLETTERS May 1993

MBG ------HWDSSYH~VSEGLMTGKVPIDQVFGANPSEKLHKRRKPKG 41 EBO MTTRTKGRGHTAATTQNDR.PGPELSGWI..Q. ..RI VSDI.CDIENNPGLCYASQMQ 60

MBG TVGLQCSPCLMSKATSTDDIVWWLIVKKTLADLLIVKKTL~LLIPINRQISDIQSTLN~TTR~EIE101 EBO QTKPNPKTRNSQTQ.D-PICNHSFEE.VQ...S.ATWQQ TIASE.LEQRI.S------113

MBG RQLHEITPVLKMGRTLEAISKGHSEMLAKYDHLVISTGRTTAPAAAFDAYLNEHGVPPPQ 161 EBO -LENGLK..YD.AK. ISSLNRVCA..V....L..MT...A..T...TE..WA.. Q...G 172

RBG PAIFKDLGVA~ACSKGTMVKNETTDMDKMSKVLELSEETFSKPNLShKDLALLLFTHL 221 EBO .SLYEESAIRGKIE.RDET.PQSVRE.FNNLNSTTS.T..N G..DI.....RNIMYD.. 232

MBG PGNNTPFHILAQVLSKIAYKSGKSGAFLDAFHQI-LSEGENAQAALTRLSRTFDAFLGVV 280 Em FG.A .Q.V. IC.LG- DSN.LDIIH.EF.AS.A..DSP.C..IQITKRVPI.QDAA 291

MBG PPVIRVKNFQTVPRPCQKSLRAVPPNPTIDKGWVCVYSSEQGETRALKI 329 EBO HIRSRGDI..A... P...S.K..R.....FQLQD. K LG... 340 (A)

MBG PV\SS---SNYNTYMQYLNPPPYADHGA---NQLIPADQLSNQQGITPNYVGDLNLDD--- 51 EBO RRVILPTAPPE..FAIY.VRSNSTI.RGG SNTGFLTPESVN.D..SNPLRPIA..TID 60

MBG ---QFKGNVCHAFTLEAIIDISAYNEPTVKGVPAWLPLGIMSNFEYPLAHTVAALLTGSY 108 EBO HASHTP.S.SS.. I. .MVNVISGPKVLM.QI. I... .VADQKT.SFDS.T.. IMLA.. 120

MBG TITQFTHNGQKFVRVNRLGTGIPAHPLRMLREGNQ~IQNMVIPRNFSTNQFTYNLTNLV 168 Em H.GKATNPL...... P...D....L..I.....L.EF.L.PVQLPQY..FD..A.K 180

MBG LSVQKLPDDAWRPSKDKLIGNT~PAVSIHPNLPPIVLP--TVKKQAYRQHKNPNNGPLL 226 EBO IT.P..MT.TDDTPTGSNGALR.GI.F..K.R..L..NKSG..GNSADLTS.EK---- 236

MBG AISGILHQLRVEKVPEKTSLFRISLPADHFSVKEGMllKKRGENSPWYFQAPENFPLNGF 286 EBO -.QA.MTS.QDF. 1VPIDPTKN.MGIEVPETLVHKLTG.KVTSKNGQPIIPVLLPKYI.L 295

MBG NNRQWLAYANPTLSAV------303 Em DPVAPGDLTMVI.QDCDTCHSPASLPAVIEK 326 (B)

Fig. 3. Comparison of the amino acid sequences of (A) the MBG and EBO VP35 proteins (for EBO VP35 shown is the sequence deduced starting from the first methionine of the open reading frame), and (B) the MBG and EBO VP40 proteins. Dots indicate that the amino acid is the same as that in MBG. Gaps, included to maximize alignment, are indicated by dashes.

t Fig. 2. The fragment of the EBO genome @DNA plus-strand) with the predicted amino acid sequences of the VP35 and VP40 proteins. The marks are identical to those in Fig. 1; the second methionine in the VP35 open reading frame is boxed.

45 Volume 322, number 1 FEBS LETTERS May 1993

-38 ,,,,,,,, ,I,,(,,r, ,,,,,,,,, ,(, , , l’lr 1 68 ml II 248 388 B D Fig. 4. The hydropathicity plots of the deduced polypeptide sequences, computed by the method of Kyte and Doolittle [13] using an interval of 9 aa. The hydrophobic regions are shaded above the midline, and hydrophilic ones below it. (A) The VP35 of MBG; (B) the VP35 of EBO; (C) the VP40 of MBG; (D) the VP40 of EBO. In B the position of the second AUG codon is shown by the dotted line.

REFERENCES [9] Volchkov, V.E., Blinov, V.M. and Netesov, S.V. (1992) FEBS Lett. 305, 181-184. PI Brown, F. (1989) Intervirology 30, 181-186. [lo] Bukreyev, A.A., Kolichalov, A.A., Volchkov, V.E., Blinov, PI Martini, G. and Siegert, R. (1971) Marburg Virus Disease, Sprin- V.M., Netesov, S.V. and Sandakhchiev, L.S. (1991) Molekulyar- ger, New York. naya Genetika, Mikrobiologiya i Virusologiya 3,24-30 (in Rus- [31 Kiley, M.P., Cox, N.J., Elliott, L.H., Sanchez, A., DeFries, R., Sian). Buchmeier, M.J., Richman, D.D. and McCormick, J.B. (1988) J. [l l] Kozak, M. (1986) Cell 44, 283-292. Gen. Viral. 69, 1957-1967. [12] Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132. [41 Sanchez, A. and Kiley, M.P. (1987) Virology 157, 414420. [13] Elliott, L.H., Kiley, M.P. and McCormick, J.B. (1985) Virology PI Feldmann, H., Muhlberger, E., Randolf, A., Will, C., Kiley, 147, 169-176. M.P., Sanchez, A. and Klenk, H.-D. (1992) Virus Res. 24, 1-19. [14] Bukreyev, A.A., Shestopalov, A.M., Kolichalov, A.A., Brovkin, [6] Sanchez, A., Kiley, M.P., Klenk, H.-D. and Feldmann, H. (1992) AI., Blinov, V.M. and Netesov, S.V. (1991) International Con- J. Gen. Virol. 73, 347-357. ference on Medical Biotechnology, Immunization and AIDS, Ab- [7] Muhlberger, E., Sanchez, A., Randolf, A., Will, C., Kiley, M.P., stracts, R2-44, Leningrad, USSR, 1991. Klenk, H.-D. and Feldmann, H. (1992) Virology 187, 534547. [15] Volchkov, V.E., Chepumov, A.A. and Netesov, S.V. (1991) In- [8] Sanchez, A., Kiley, M.P., Holloway, B.P., McCormick, J.B. and ternational Conference on Medical Biotechnology, Immuniza- Auperin, D.D. (1989) Virology 170, 81-91. tion and AIDS, Abstracts, R2-45, Leningrad, USSR, 1991.