J. Mol. BioZ. (1991) 217, 603-610

Putative 65 kDa Protein of Beet Yellows Closterovirus Homologue of HSP70 Heat Shock Proteins

Is a

Alexey A. Agranovskylt, Vitaliy P. Boykol, Alexander V. Karasev2 Eugene V. Koonin2 and Valerian V. Doljal IA. N. Beloxersky Laboratory of Moscow State University 119899 Moscow, U.S.S.R. 21nstitute

of Microbiology, U.S.S.R. Academy Moscow 117811, U.S.S.R.

(Received 31 May

of Sciences

1990; accepted 23 October 1990)

A portion of the RNA genome of beet yellows closterovirus (BYV) has been sequenced encompassing a complete long open reading frame (ORF) potentially encoding a 65 kDa protein. The sequence of this putative protein was strikingly similar to those of HSP70-related heat shock proteins. The counterparts of all the eight segments strongly conserved in HSP70s could be confidently identified in the BYV 65 kDa protein. It is suggested that some of these segments might be the ATP-binding site(s) and that, similarly to the heat shock proteins, the 65 kDa is probably ATP-binding. Generally, however, the divergence between the 65 kDa sequence and the sequences of the HSP70s was much more pronounced than that between any two members of the latter family, allowing a clearer delineation of clusters of conserved residues that might be crucial for protein function. It is suggested that these observations will be helpful in functional dissection of the proteins of the HSP70 family. Analysis of the sequence of a portion of the ORF found upstream from the 65 kDa ORF showed that the C-terminal domain of the encoded protein could be an RNA-dependent RNA polymerase closely related to those of tricornaviruses, a family of RNA plant viruses with three component genomes.

The cell responds to heat shock and other environmental stresses by induction of specific proteins. Several families of heat shock proteins have been described, among which HSP70s are the most abundant. In Escherichia co&, a single HSP70related protein, the dnaK gene product, has been found, while in eukaryotes the HSP70$ family is represented by products of several genes (for a review, see Pelham, 1989; Craig, 1989). It has been shown that only some of the HSP70s are stressinducible; others are constitutive proteins abundant in major compartments of normally growing cells et al., 1987; Watowich & (Werner-Washburne Morimoto, 1988). All HSP70 proteins are thought to act as molecular chaperones. The stress-inducible species are apparently involved in post-stress cell recovery by acting on denatured cell proteins.

Constitutive HSP70 proteins appear to play an important role in protein transport within and between cell compartments, as well as in protein folding and assembly of multisubunit complexes (Rothman, 1989). All these HSP70 functions involve protein-protein interactions accompanied by ATP binding and hydrolysis (Pelham, 1986). The sequences of HSP70s are highly conserved from throughout a wide range of organisms, bacteria to mammals (Nicholson et al., 1990). These proteins have been detected in essentially all the living cells examined and have long been thought to be a prerogative of cellular organisms. We report here the first example of a virus gene encoding a putative HSP70-related protein. This gen.e was identified in the RNA genome of beet yellows virus (BYV). BYV is the type member of closteroviruses, a virus group composed of several filamentous plant viruses (Milne, 1988). BYV causes yellowing disease in many dicotyledonous plants and is transmitted by aphids semi-persistently. Its particles are composed of a single type of coat protein subunit

t Author to whom correspondence should be addressed. $ Abbreviations used: HSP70, heat shock 70 kDa proteins; BYV, beet yellows virus; kb, lo3 bases or base-pairs; ORF, open reading frame; nt, nucleotide(s). 603 0022-2836/91/040603%08

$03.00/O

0 1991 Academic Press Limited

A. A. Agranovsky et al.

604

and contain messenger-sense genomic RNA of about 145 kb (Bar-Joseph & Hull, 1974). The virus RNA is devoid of a 3’-poly(A) tail and bears a 5’-terminal cap structure. Cell-free translation of BYV RNA yields a major 250 kDa protein, presumably encoded by the 5’-terminal gene that covers about half of the virus genome (Karasev et al., 1989). It has been shown that BYV replication in plants is accompanied by formation of a set of subgenomic RNAs (Dolja et aE., 1998; and our unpublished results). In this work we have determined the sequence of 2677 nucleotides from the central part of the BYV genome, derived from the sequences of four overlapping cDNA clones (Fig. 1). The specificity of each of the cDNA clones was confirmed by dot,-blot hybridization with BYV-specific probe cDNA obtained by random-primed reverse transcription of purified virus RNA. A total of 52% of the sequence was deduced from both strands of the cloned cDNA, and 34% of it was read from independent overlapping clones (Fig. l(b)). The resulting sequence (deposited in the EMBL database under accession number X53462) was analysed using the GENBEE package of computer programs (Brodsky et al., 1991). The sequence contained only one complete long open reading frame (ORF), which was a good candidate for being an expressed gene when evaluated by the PROTMAKE/GENBEE program based on the

(a) I

, 1

0

PO1

KH I I

Trifonov algorithm (Trifonov, 1987). This QRF started at the AUG codon at’ nt 874 and terminated with the opal codon at nt 2668. The calculated molecular weight of the putative translation product was 65,156. Very recently we obtained the 65 kDa protein by the expression of the respective ORF in vitro using the phage T7 polymerase transcription/translation system (Karasev et al., unpublished results). Using the QUICK/GENBEE program, the amino acid sequence of the 65 kDa protein was compared to all prot,ein sequences available in the SWISSPROT database (release 12). To our surprise, the only group of proteins showing reliable similarity to the 65 kDa protein were HSP70-related heat shock proteins. Detailed dot matrix comparisons using the DOTHELIX/GENBEE program showed statistical significance of about 10 standard deviations above the random expectation for the regions of highest similarity between the 65 kDa sequence and different members of this family. Inspection of the alignment of the 65 kDa sequence with those of HSP70-related proteins revealed that all the eight sequence segments strongly conserved in the HSP70 family (Ting & Lee, 1988) had (partially) conserved counterparts in 65 kDa (Fig. 2). All these segments were located within the N-terminal 450 residues of HSP70s and the 65 kDa protein. This seems to be well compatible with the Xba I

Xho

M

Xhc I

I 2

65 kDa

I

(b)

a

a a

Figure 1. Map of cDNA clones and sequencing strategy

a

for the eentrai region of BYV genomic RNA. The cloning procedure was as follows. The full-length virion RNA purified in sucrose gradients (Karasev et al.; 1989) was used for the random-primed one-tube synthesis of cDNA (Gubler, 1988), followed by insertion into plasmids pUCl9, pTZ18 or pTZ19 E. coli XL-l cells (Maniatis et al., 1982). The virus-specific clones were selected by and transformation of competent colony hybridization followed by Southern and Northern blot hybridizations using 3ZP-labelled BYV cDNA and oligo-labelled clone r2 as respective probes (Maniatis et al.; 1982; Feinberg & Vogelstein, 1983). The clones were further characterized by restriction endonuclease mapping. (a) Schematic representation of the sequenced region. Numbers are distances in kb from the 5’ end of the sequence; the position of 0 could be roughly estimated at’ about 7 kb from the 5’ terminus of BYV RNA, assuming that the 250 kDa (putative polymerase) gene occupies the 5’-terminal half in the 145 kb genome. Letter symbols are the restriction sites used for Ml3 and pTZ subcloning: H, HindIII; K, KpnI; M, Mb&I; Xba, XbaI; Xho, XhoI. The incomplete open reading frame (ORB) for the putative polymerase and the ORF for the 65 kDa sequence are shown by open boxes. (b) The positions of overlapping clones ~43, 111-7, r2, and 36a are denoted by thick continuous lines. Thin continuous arrows indicate location, direction, and length of subcloned singlestranded DNA templates sequenced using a SequenaseTM kit (Pharmacia); the broken arrow shows the sequence determined by the chemical degradation method (Maxam $ Gilbert, 1980).

605

Communications +A

65K MVlVFGLDFGTTFSSVlCAYVGEELnFKQRDSAYIPTYVFVLSN DNAKE MGK IIGIDLGTTNSCV AIMDGTTPRVL-ENAEGDRTTPSIIAYTQDGETLVGQPAKRQA MTP70TC 22 SKVTGD VIGIDLGTTYSCV AVMEGDKPRVL-ENTEGFRATPSVVAFKGQ-EKLVGLAAKRQA GR78HUM 23 KEDVGT WGIDLGTTYSCV GVFKNGRVEII-ANDQGNRITPSYVAFTPEGERLIGDAAKNQL HS7OHUM MAK--AA AVGIDLGTTYSCVGVFQHGKVEII-ANDQGNRTTPSYVAFT-DTEBLIGDAAKNQV HS71MOU MSK--GP AVGIDLGTTYSCVGVFQHGKVEII-ANDQGNRTTPSYVAFT-DTERLIGDAAKNQV HS7CAEE MSK--I-IN AVGIDLGTTYSCVGVFMHGKVEII-ANDQGNRTTPSYVAFT-DTERLIGDAAKNQV HS71YEA MSK---- AVGIDLGTTYSCVAHFANDRVDII-ANDQGNRTTPSFVAFT-DTERLIGDAAKNQA HS70MAI MAKSEGPAIGIDLGTTYSCV GLWQHDRVEII-ANDQGNRTTPSYVGFT-DTERLIGDAAKNQV HS72DRO M-----P AIGIDLGTTYSCV GVYQHGKVEIN-AYDQGNRTTPSYVAFT-DSERLNGEPAKNQV consens *GID*GTTYScV a * Ii n qgnr Tps*Vaft D er*igD ak q L VI ed Iy E kvE F g L L Q Q 65K DNAKE MTP70TC GR78HUM HS70HUM HS71MOU HS7CAEE HS71YEA HS'IOMAI HS72DRO consens

DLSVRGGFYRDLKRWIGCDEENYRDYLEKLKPHYKTELLKLDCYSGTVPQNATLPGLI VTNPQNTLF-AIKRLIGRRFQD-EEVQRDVS-IMPFKIIAAPPQIS VTNPQSTFF-AVKRLIGRRFED-SNIQHDIK-NVPYKIGR TSNPENTVF-DAKRLIGRTWND-PSVQQDIK-FLPFKWE-EEIS ALNPQNTVF-DAWrLIGRKFGD-PVVQSDMK-HWPFQVIN--DGD~KVQVSY-KG-ETKAFYPEEIS AMNPTNTVF-DAKRLIGRRFDD-AVVQSDMK-HWPF-MVV-NDAGRPKVQVEY-KG-ETKSFYPEEVS AMNPHNTVF-DAKRLIGRKFDD-PAVQSDMK-HWPFKVIS-AEG~PKVQ~Y-KG-ENKIFTPEEIS AMNPSNTVF-DAKRLIGRNFND-PEVQADMK-HFPFKLID-VDG-~QIQVEF-KG-ETKNFTPEQIS AMNPTNTVF-DAKRLIGRRFSS-PAVQSSMK-LWPSRHLGAAEEIS AMNPRNTVF-D~IGRKMD-PKIAEDMK-HWPFKWS--DGG~KIG~Y-KG-ES~FAPEEIS *E G f p eIs np nt*F KR*IGr f D *q d*K *p D Y Y qv YN N L W

65K DNAKE MTP70TC GR78HUM HS7OHUM HS7lMOU HS7CAEE HS71YEA HS70MAI HS72DRO consens

ATFVKALISTASEAFKCQCTGVICSVPANYNCLQRSFTESCVNLSGYPCVYMVNEPSAAALSACSR[ AEVLKKMKKTAEDYLGEPVTEA VITVPAYFNDAQRQATKDAGRIAGLEVKRIINEPTAAALAYGLD AFVLEKMKETAENFLGRKVSNA WTCPAYFNGPQRQATKDAGTIAGLNVIRVVNGPTAAALAYGLD AMVLTKMKETAEAYLGKKVTHA WTVPAYFNDAQRQATKDAGTIAGLNVMRIINEPTAAAIAYGLD SMVLTKMKEIAEAYLGYPVTNA VITVPAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAIAYGLD SMVLTKMKEIAEAYLGKTVTNA VVTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIAYGLD SMVLLKMKKTAEAFLEPTVKDA VVTVPTYFNDSQRQATKDAGAIAGLNVLRIINEPTAAAIAYGLD SMVLGKMKETAESYLGAKVNDA WTVPAYFNDSQRQATKDAGTIAGLNVLRIINEPTAAAIAYGLD SMVLIKMKEIAEAYLGSTIKNA VVTVPAYFNDSQRQATKDAGVIAGLNVMRIINEPTAAAIAYGLD SMVLTKMKETAEAYLGESITDA VITVPAYFNDSQRQATKDAGHIAGLNVLRIINEPTAAALAYGLD *L kMk Ae y*g v a VvTVPAyFNd QRqaTkdag IaG*nv r*INEPTAAAIaygld v L f i IiS Y Le VSL

65K DNAKE MTP70TC GR78HUM HS70HUM HS71MOU HS7CAEE HS71YEA HS7OMAI HS72DRO consens

IK---GATSP VLVYDFGGGTFDVSVISALNN----T KG---TGNRT IAVYDLGGGTFDISII EIDEVDGMT KT---KDSM- IAVYDLGGGTFDISVLEIAGG----V KR---EGEKN ILVFDLGGGTFDVSLLTIDNG----V RTG--KGERN VLIFDLGGGTFDVSIL TIDDG----I KKV--GAERN VLIFDLGGGTFDVSIL TIEDG----I KKG--HGERN VLIFDLGGGTFDVSIL TIEDG----I KKG--KEEH- VLIFDLGGGTFDVSLLFIEDG----I KKATSSGJXN VLIFDLGGGTFDVSLLTIEEG----I KNL--KGERN VLIFDLGGGTFDVSIL TIDEGS---L k e V IFD*GGGTFDVS*L i Eg In IVY I I D N Fig. 2.

B

C

+

D --FVVRASGGDMNLGGRDIDKAFVEHL VEE FEVLATNGDTHLGGEDFDSRLINYL FEVKATNGDTHLaEDFDLCLSDYI LTE FEVVATNGDTHLGGEDFDQRVMEHF IKL VEE FEVKATAGDTHLGGEDFDNRLVNHF IAE FEVKSTAGDTHLGGEDFDNRMVNHF FEVKSTAGDTHLGGEDFDNRMVNHF CAE FEVKATAGDTHLGGEDFDNRLVNHF IQE FEVKATAGDTHLGGEDFDNRMVNHF VQE FEVRSTAGDTHLGGEDFDNRLVTHL AEE FeVK T GDthLGGeD*D r* * e RS

A. A. Agranovsky et al.

606

65K DNAKE MTP70TC GR78HUM HS70HUM HS71MOU HS7CAEE HS71YEA HS70MAI HS72DRO consens

YNKAQLPVNYKIDISFLKESLSKKVSFLNFPWSEQGVRVDVLVNVSE-- LAEVAAPFVERTI FKKDQGIDLRNDPLAMQRLKEAAEKAKIELSSAQQTDVNLPYITADATGPIKVTRAKLESLVE FKKSTGIDLSNERMALQRIREAAEKAKCELSTTMETEVNLPFITANQDGAQHVQMTVSRSKFESLAM. YKKKTGKDVRKDNRAVQKLRREVEKAK-ALSSQHQARIEIESFYEGED----FSETLTRAKFEEL FKRKHKKDISQNKRAVRRLRTACERFEGIDFYTSI~EEL~TL----SSSTQASLEIDSL FKRKHKKDISENKRAVRRLRTACERAKRTLSSSTQASIEIDSLYEGID----FYTSITR~FEELNAD FKRKHKKDLASNPRALRRLRTACERANETLSSSCaASIEIDSLFEGID----FYTNI~~FEELCA~ FKRKNKKDLSTNQRALRRLRTACERAKRTLSSSAQTSVEIDSLFEGID----FYTSITRPaRFEEL EKRKNKKDISGNPRALRRLRTACERAKRTLSSTAQTTIEIDSLFEGID----FTPRSSRARFEEL FKRKYKKDLRSNPRALRRLRTAAERAKRTLSSSTEATIEIDALFEGQD----FY~VSR~FEELCAN FkR dl n a* Rlr a era lss E e* * YK Kik k id tQ n

65K DNAKE MTP70TC GR78HUM HS70HUM HS71MOU HS7CAEE HS71YEA HS70MAI HS72DRO consens

IVKEVYEKYCSSMR---LEPNVKAKLLMVGGSSYLPGLLSRLSSIPFVDECLVLP LVNRSIEPLKVALQDAGLSVSDIDDVILVGGQTRMPMVQKKV AEFF-GKEPRKDV LVQRSLGPCKQCIKDAAVDLKEISE VVLVGGMTRMPKVIEAV KQFF-GRDPFRGV LFRSTMKPVQKVLEDSDWDIDE IVLVGGSTRIPKIQQLV KEFFNGKEPSRGI LFRSTLEPVEKALRDAKLDKAQIHD LVLVGGSTRIPKVQKLLQDFFNGRDLNKSI LFRGTLDPVEKALRDAKLDKSQIHD IVLVGGSTRIPKIQKLL QDFFNGKELNKSI LFRSTMDPVFKSLRDAKMDKSQVHD IVLVGGSTRIPKVQKLL SDLFSGKELNKSI LFRSTLDPVEKVLRDAKLDKSQVDE IVLVGGSTRIPKVQKLVTDYFNGKEPNRSI LFRKCMJZPVEKCLRDAKMDKSSVHD VVLVGGSTRIPKVQQ-LQDFFNGKELCKSI LFRNTLQPVEKALNDAKMDKGQIHD IVLVGGSTRIPKVQSLL QEFFHGKNLNLSI L* *p * da * id **L&G Tr*Pk'q i L I ve V M S v d t-D

E

G

F 65K

DNAKE MTP70TC GR78HUM HS7OHUM HS71MOU HS7CAEE HS71YEA HS70MAI HS72DRO consens

65K

DNAKE MTP70TC GR78HUM HS7OHUM HS71MOU HS7CAEE HS7lYEA HS7OMAI HS72DRO consens

sp----DARAAVAGGCALYSACLRND MLLVDCAAHNLSISSKYCESIVCVPAGSP NPDEAVAIGAAVQGGVLTGD VK---- D VLLLDVTPLSLGIETMGGVMTTLIAKNTT NPDEAVALGGATLGGVLRRD VK---- G LVLLDVTPLSLGVETLGGVFTRMIPKNTT NPDEAVAYGAAVQAGVLSGD QDTG--D LVLLHVCPLTLGIETVGGVMTKLIPSNTV NPDEAVGYGAAVQAAILMGD LIKRNST KSENVQDLLLLDVAPLSLGLETAGGVMTA NPDEAVAYGAAVQAAILSGD KSENVQDLLLLDVTPLSLGIETAGGVMTVLIKRNTT NPDEALAYGAAVQAAILSGD KSEAVQDLLLLDVAPLSLGIETAGGVMTALIKRNTT NPDEAVAYGAAVQAAILTGD ESSKTQDLLLLDVAPLSLGIETAGGVMTKLIPRNST NPDEAVAYGAAVQAAILSGE GNERS-D LLLLDVTPLSLGLETAGGVMTVLIPRNTT NPDEAVAYGAAVQAAILSGD QSGKIQD VLLVDVAPLSLGIETAGGVMTKLIERNCR NpdeAVA G A q iL D d *LLLDv plsLg*eT ggvmt 1I rnT D L v E vv t s VkS

IPFTGVRTVNMTGS-NASAVYSAALFEGDFVKCRLNKRIFVTG IPTKHSQVF-STAEDNQSAVTIH-VLQGE--RKRAADNKSLGQFNLDGINPAP IPTKKSQTFFSTAAFNQTQVGIK-VFQGE--REMAADNQMMGQFDLVGIPPAP VPTKNSQIF-STASDNQPTVTIK-VYEGE--RPLTKDNHLLGTFDLTGIPPAP IPTKQTQIF-TTYSDNQPGVLIQ-VYEGE--RAMTKDNNLLG~ELSGIPPAP IPTKQTQTL-TTYSDNQPGVLIQ-VYEGE--RAMTKDNNLLGKFELTGIPPAP IPTKTAQTF-TTYSDNQPGVLIQ-VYEGE--RAMTKDNNLLGKFELSGIPPAP ISTKKFEIF-STYADNQPGVLIQ-VFEGE--RAKTKDNNLLGKFELSGIPPAP IPTKKEQVF-STYSDNQPGVLIQ-VYEGE--RARTKDNNLLGKFELSGIPPAP IPCKQTKTF-STYSDNQPGVSIQ-VYEGE--RAMTKDNNALGTFDLSGIPPAP IPtk q * ST dNq V i V*EGE R dn *G *eL gIppap V e t LQD K d v n Fig. 2.

Communications

607

VSSVGTISFSLVGPTGVKKLIGGNAAYDFSSYQLGERVVAF ADGILHVSAKDKN-SGKEQKITIKASSG-LNEDEIQKMVRL-H MTP70TC PNGICHVTAKDKA-TGKTQNITITASGG-LSKEQIERMIRNNAETQA-N GR78HUM VNGILRVTAEDKG-TGNKNKITITNDQNRLTPEEIERMVNDAEKFAEEDKKLKERIDTRNELESYA-Y HS70HUM ANGILNVTATDKS-TGKANKITITNDKGRLSKEEIERMVQ-F HS71MOU ANGILNVSAVDKS-TGKENKITITNDKGRLSKEDIERMVQE~~AED~~DKVSSKNSLESYA-F HS7CAEE ANGILNVSATDKS-TGKAKQITITNDKDRFSKDD1ERMVNNGLESYA-F HS71YEA SNGILNVSAVMG-TGKSNKITITNDKGRLSKEDIMMVAEAQLESIA-Y HS70MAI VNNILNVSAEDKT-TGQKNKITITNDKGRLSKEEIMMVQE-Y HS72DRO ANGILNVSAKEMS-TGKAKNITIKNDKGRLSQAEIDRMVNALES~-F consens ngI1 VSa dk TG Iti 1s EeiERMV Eae D I m e dVITe S T Dd DKVI D Vk d 65K DNAKE

Qs Q 65K

DNAKE

MTP70TC GR78HUM HS7OHUM HS71MOU HS7CAEE HS71YEA HS70MAI HS72DRO consens

65K DNAKE

KLTDGDKALFLKRLTADYRR-EARKFSSYDDAVLNSSEL--LLGRIIPKILR ---AGDKLPADDKTAIESALTALETALKGEDKAAIEAKMQA STRKQVEE TAERQLTE---W-KYVSDAEKENVRTLLRACRKSME--NPV~ELSAATD~QLA~ECGRTEYSLKNQIGDKMLGGKLSSEDKET~AVEMIEWLESHQDADIEDFKAKKKELEEIVQPIISKLYG NMKSAVED-EGLKGKISEADKKKVLDKCQEVISWLDANTLAMDEFEHKRKELEQVCNPIISGLYQ NMKATvED-EKLQGKINDEDKQKILDKCNEIISWLDKNQTAMEEFEHQQKELMVCNPIITKLYQ NLKQTIED-EKLKDKISPEDKKKIEDKCDEILKWLDSNQT~EFESQ~LEGL~DLS~YQ SLKNTISE-A--GDKLEQADKDTVTKKAEETISWLDSNTTASKEEFDD~~LQDIANPIMSKLYQ NMRNTIKD-DKIASKLPAEDKKKIEDAVDGAISWLDSNQLAEVEEFED~LEGICNPII~YX NVKQSVEQ-AP-AGKLDEADKNSVLDKCNETIRWLDSNTTA~EFD~EEL~HCSPIMT~HQ a EE EL d K* DK * 1D DV e E mE DD Q 9

Q-M----------

---------------------------GSRVmLDV

MTP70TC GR78HUM HS70HUM HS71MOU HS7CAEE

TAGADASANNAKDDDV--VDAEFEEVKDKK QQA---- AAGNSSSSSGNTDSSQ--GEQQQQGDQQKQ SAG-- --------------PPPT--GEEDT~DEL GAG---GPG---PGGFGAQGPKG--GSGSGPTIEEVD SAG---GMPGGMPGGFPGGGAPPSGGASSGPTIEEVD SAG---GAP ---PGAAPGGAA----GGAGGPTIEEVD

HS71YEA HS7OMAI HS72DRO

AGGAPGGAAGGAPGGFPGGAPPA--PEAEGPTVEEVD GEG----AGMGAAAGMDEDAPSG--GSGAGPKIEEVD QGA---GAAGG-PGANCGQQAGGF-GGYSGPTVEEVD

QQQ----

consens

eE dD sQ

Figure 2. Alignment of the amino acid sequences of BYV 65 kDa (65 K) and selected HSP’IO-related proteins. The alignment was generated by the MULTALIN program (Corpet, 1988) using the Dayhoff log-odds matrix for amino acid residue comparison with the gap penalty of 8, and slightly modified by hand to maximize the number of invariant amino acid residues. The sequences are shown in their entirety, except those of MTP’IOTC and GRP78HUM, where the lengths of the N-terminal extensions are indicated. The consensus pattern was derived allowing for 1 exception in the HSP70s’ sequences. Upper case: residues conserved in 65 kDa and HSP70s; lower case: residues conserved in HSP70s but not in 65 kDa. Asterisks denote bulky hydrophobic residues (I, L, V, M; F, Y, W). The highly conserved segments of HSP70s (A to H), defined after Ting & Lee (1988), are boxed. Plus signs show the residues implicated in ATP binding and/or hydrolysis by X-ray diffraction analysis (Flaherty et al., 1990). DNAKE, E. coZi dnaK protein; MTP70Tf.2, Trypanosoma cruai mitochondrial HSP70-related protein; GR78HUM, human glucose-regulated protein; HS70HUM, human HSP70; HS71MOI.7, mouse HSP71; HS7CAEE, Caenorhabditis elegans HSP70; HS’llYEA, yeast HSP71; HS70MA1, maize HSP70; HS72DR0, Drosophila melanoguster HSP70. The sequences were from the SWISSPROT database (release 121, except that for MTP’IOTC which was from Engman et al. (1989). results of Chappell et aE. (1986), indicating that the ATPase activity of bovine brain uncoating ATPase, a member of the HSP70 family, was associated with this portion of the protein. Moreover, the X-ray

for

diffraction

data

with

demonstrated

ADP

tertiary

structure

showed

that

the complex

with

residues

overall those

of this

fragment

similarity

of hexokinases, Asp10 and Glu175

in its and were

A. A. Agranovsky et al

608

located in close proximity to the b-phosphate of ADP, implicating them in ATP hydrolysis (Flaherty in et al., 1990). Both these residues, residing segments A and B, respectively, are conserved in the 65 kDa sequence (Fig. 2; however, it can be noted parenthetically that the importance of the latter residue seems somewhat questionable because of its replacement in MTP’IOTC). Of further interest is segment C containing the longest stretch of residues conserved between the 65 kDa protein and HSP70s (Fig. 2). The organization of this segment, i.e. a stretch of hydrophobic residues followed by a tentative flexible loop constituted by glycine resembles that of various purine residues, NTP-binding sites (Moller & Ammons, 1985; Hanks et al., 1988; Gorbalenya & Koonin, 1989). It has been suggested that this region of HSP70s is related to the ATP-binding motif conserved in protein kinases (Nover et aZ., 1989). Although our analysis failed to confirm the statistical validity of this assertion (not shown), it remains a plausible possibility that segment C might constitut’e an ATP-binding motif of a new type. Together, these observations constitute strong evidence for an evolutionary relatedness between the 65 kDa protein and the HSP70 family and also suggest a functional analogy. It seems quite likely that the 65 kDa protein may share ATPase activity Concerning with the HSP70-related proteins. specific functions of the 65 kDa protein in BYV reproduction, it can be speculated that, like

HSP70s, it might exert chaperone activity, perhaps involved in assembly and/or transport of virusspecific ribonucleoproteins. Inspection of the alignment in Figure 2 shows that the 65 kDa sequence has diverged from the putative common ancestor with the HSP70s much further than any of the orthodox members of this highly conserved family. This alignment will thus be helpful in delineating more precisely the highly conserved segments that may be appropriate targets for site-directed mutagenesis aimed at functional dissection of the proteins of this family. Two alternatives for the origin of the 65 kDa gene, both implying the capture from the cell genome, can be envisaged: (1) the 65 kDa gene is a close relative of an as yet unidentified cell HSP70-related gene; and (2) the relatively low sequence similarity of the 65 kDa protein to the known members of the HSP70 family is due to the high rate of evolution of the gene within the virus RNA genome. The protein sequence encoded by the incomplete long ORF (nt 1 to 656) found upstream from that for the 65 kDa sequence (Fig. 1) encompassed three conserved segments typical of RNA polymerases of positive-strand RNA viruses (Kamer & Argos, 1984; Koonin et al., 1989). This result suggested that the C-terminal domain of the 250 kDa protein encoded by the 5’-terminal gene of BYV might be the polymerase. Judging by the alignments with the C-terminal conserved domains of other viral

II BMV CMV AlMV

518 567 583

BYV

?

III

SFQRRTGDAFTYFGNTLVTNAMIAYASDL--SDCDCAIFSGDDSLI-ISKVKPVLDTDMF SFQRRn;DAFTYFGNTIVTMAEFAWCYDT--DQFDRLLFS DFQRRn;DALTYLGNTIVTLACLCHVYDLMDPNVKFVVASEFLF * * * *** DNQRKSGASN~IGNSIETGILSMFYYT--NRFE(ALFV~DDSL~-FSESPI~SAD IV

BMV CMV AlMV

TSLFNMEIKVNDPSVPYVCSKFLVETEMGN---LVSVPDPLREIQRLAKRKIL--RDEQ TTLFNMEAKVNEPAVPYICSKFYSLMSLVT ----RFQSPT-IREIQRLGTKKIPYSDNND TTLFNLEAKF-PliNQPFICSKFLITMPTTSGGKWLPIPNPLKLLIRLGSKKV----NA

BYV

CTELGFETKFLTPSVPYFCSKFFVMTGHDV----FFV-PDP~L~GAS~E--VDDE

BMV CMV AlMV

MLRARFVSFCDRMK-FINQLDEKNITTLCNFVYLKYGKE FLFARFMSFVDRLK-FMDRNSQSCIDQLSIFFELKYKKS IFDEWYQSUIDIIGGFNDHHVIRVIRCVAANTARRYLRRPSL

BYV

FLFEVFTSFRDLTK---

DLVDERVIELLTNLVRSKYGYE

155 122 53 72

Figure 3. Alignment of a region of the putative BYV RNA-dependent RNA polymerase with the homologous regions of the tricornavirus polymerases. The conserved segments of positive-stra,nd RNA virus RNA polymerases are designated

II

to IV after

Koonin

et al. (1989).

The portion

of the BYV

genome

probably

encoding

the N-terminal

segment I is not yet sequenced. Asterisks denote amino acid residues conserved in (nearly) all positive-strand RNA virus polymerases. Other designations are as for Fig. 2. BMV, brome mosaic virus; CMV, cucumber mosaic virus; AlMV, alfalfa

mosaic

virus.

The tricornavirus

sequences

were from

SWISSPROT.

Communications

RNA-dependent RNA polymerases, the putative BYV polymerase is most similar to the polymerases of tricornaviruses, a family of tripartite RNA plant viruses; this similarity extends beyond the regions highly conserved in all positive-strand RNA virus polymerases (Fig. 3). As concerns the expression strategy of BYV, it seems most likely that the 250 kDa protein (the polymerase) is translated directly from the genomic RNA (Karasev et al., 1989), whereas the 65 kDa protein is from the largest subgenomic RNA. In accord with this, the clone r2 (Fig. 1) has been found to hybridize with the latter RNA species on Northern blots (not shown). Taken together, our observations suggest the following gene arrangement for the part of BYV genome studied in this work: 5’. . polymeraseputative ATPase.. .3’. This arrangement has a striking resemblance to the genomic organization of potexviruses (Skryabin et al., 1988) and carlaviruses (Mackenzie et al., 1989). An important distinction, however, is that in the latter two groups the putative NTPases are of a quite different type, i.e. they belong to a superfamily of (putative) helicases unrelated to HSP70s (Gorbalenya et aZ., 1988). It seems remarkable that in different positive-strand RNA viruses identical positions in the genome are occupied by genes encoding putative NTPases of two distinct types. An attractive hypothesis is that the respective gene products might be involved in phenomenologically related aspects of virus reproduction.

Addendum While the manuscript was being processed, we completed the sequence of 6746 nucleotides representing the 3’ half of the BYV genome (Agranovsky et al., 1991). Analysis of the sequence showed that the 65 kDa protein gene is separated from the 3’ end of the virus genome by five more genes, including the one encoding the capsid protein. We are greatly indebted to Drs S. Markovna Dracheva and N. Lunina for their help in cDNA cloning, and to V. Rogov for providing BYV-infected plant material. We also thank Professor J. G. Atabekov and Dr S. Morozov for helpful discussions.

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by J. Karn