VIROLOGY

188, 938-947 (1992)

A Gene Homologous to Topoisomerase II in African Swine Fever Virus R. GARCIA-BEATO,* J. M. P. FREIIE,t C. L&z-OTiN,t

R. BLASCO,+ E. VIFKJELA,*~' AND M. L. SALAS*

*Centro de Biologia Molecular, (CSIC-UAM), Facultad de Ciencias, Universidad AuQnoma, Cantoblanco, 28049 Madrid, Spain; tDepartamento de Biologia Funcional, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain; and +Laboratory of viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 Received January 2 1) 1332; accepted

March

12, 1332

A putative topoisomerase II gene of African swine fever virus was mapped using a degenerate oligonucleotide probe derived from a region highly conserved in type II topoisomerases. The gene is located within EcoRl fragments P and H of the African swine fever virus genome. Sequencing of this region has revealed a long open reading frame, designated Pi 192R, encoding a protein of 1192 amino acids, with a predicted molecular weight of 135,543. Open reading frame P1192R is transcribed late after infection into a 4.6-kb RNA. The deduced amino acid sequence of this open reading frame shares significant similarity with topoisomerase II sequences from different sources, with percentages of identity between 23 and 29%. The evolutionary relationships among the topoisomerase II sequences of ASF virus, eukaryotes and prokaryotes were analyzed and a phylogenetic tree was established. The tree indicates that the ASF virus topoisomerase II gene was present in the virus genome before protozoa, yeasts, and metazoa diverged. o 1992 Academic

Press,

Inc.

Here we report the mapping and sequence of an ASF virus gene homologous to topoisomerase II. To establish evolutionary relationships among prokaryotic, eukaryotic, and the ASF virus type II topoisomerases, the amino acid sequences of various of these enzymes have been compared and a phylogenetic tree has been constructed. Comparison of the amino acid sequences of type II topoisomerases shows a high degree of sequence similarity between the enzymes of both eukaryotic and prokaryotic origin (17). A highly conserved sequence of six amino acids, located in the amino-terminal half of the topoisomerases, was used to prepare a degenerate probe (Fig. 1A). The 32P-labeled probe was hybridized to cloned ASF virus DNA restriction fragments covering the complete length of the viral genome. As shown in Fig. 1 B, the probe hybridized specifically with fragment EcoRl P, indicating that the putative topoisomerase II gene was located, at least partially, in this fragment. The nucleotide sequence of a 5378-bp segment that includes the 2071 -bp EcoRl P fragment and part of the contiguous EcoRI S and EcoRl H fragments was determined (Fig. 2). A long open reading frame (ORF) encoding a polypeptide of 1192 amino acids with a predicted molecular mass of 135,543 Da was identified between nucleotides 1098 and 4673. Following the nomenclature used in a previous study for the ASF virus ORFs (78), this ORF was designated Pl 192R (letter of the &oRI fragment in which translation is started, number

African swine fever (ASF) virus, the causative agent of an important disease of domestic pigs (1, Z), is an icosahedral deoxyvirus that resembles the poxviruses in the DNA structure and in the strategy followed for the synthesis of early mRNA. Thus, the ASF virus genome is a double-stranded DNA of about 170 kbp (3,4) that contains terminal inverted repetitions and hairpin loops (5, 6), as does the poxvirus DNA (7, 8). Furthermore, both the ASFvirus and poxvirus particles contain all the enzymatic activities necessary for the synthesis and processing of early mRNA, such as RNA polymerase, poly A polymerase, and capping and methylating enzymes (9- 12). On the other hand, some results suggest the presence of a DNA topoisomerase II in the ASF virus particle. First, coumermycin Al, a specific inhibitor of this enzyme (13), inhibits in vitro transcription by permeabilized ASF virus particles (14). Secondly, a virion protein of about 155 kDa is able to form a covalent complex with DNA (unpublished results) under conditions similar to those used for the formation of covalent DNA-topoisomerase II intermediates (16). Furthermore, the virion protein involved in the covalent bond with DNA is similar in size to the eukaryotic type II topoisomerases, which have molecular weights between approximately 130,000 and 180,000 (16).

Sequence data from this article have been deposited with the EMBUGenBank Data Libraries under Accession No. M&38699. ’ To whom reprint requests should be addressed.

0042-6822192 $5.00 Copyright 0 1992 by Academtc Press. Inc. All rights of reproduction in any form reserved.

938

939

SHORT COMMUNICATIONS

a. Amino acid sequence: b. Oligonucleotide

probe:

A

Thr-Glu-Gly-Asp-Ser-Ala 5'-ACXGA:GGXGA:!$GC-3'

RK'$1 i RJ RAlSC 51' s0 - ,' RE'- i RM_ ;:;$ RC -,*t, --p 0

10 20

II

11

30

40

50

60

70

80

90

1

I

I

I

II

100 110 120 130 140 150 160 170 kbp I

I

I

IIll

I

UX’VYZU’X K’CIJ I ,';d l ' HHHC

F K E'M I

I

51'

I

II

II I

C N

B

I I

SB

G

C' OTN' 0 SPHR0.Q' E I I

I

I

SD

0' I

IEco RI

HJ

FIG. 1. Mapping of ASF virus topoisomerase II gene. (A) Amino acid sequence conserved between type II topoisomerases (a) (17) and mixed oligonucleotide probe used in the hybridization experiment(b). (B) Cloned ASF virus DNA fragments EcoRl (R), HindIll (H), SalI (S), and the double restriction fragment EcoRl A&r/l C (RA/SC) (38), covering the complete length of the viral genome. were spotted on nitrocellulose paper at a concentration equivalent to 10 rg of ASF virus DNA and the dot-blot was hybridized with 3 X 10” cpm/ml of 3*P-labeled oligonucleotide probe. Hybridization was carried out at 45” for 18 hr in a 50 mM sodium phosphate, pH 7.2, buffer containing 6X SSC (1 X SSC is 0.15 M NaCI, 0.015 M sodium citrate), 5X Denhardt solution (1 X Denhardt solution is 0.02% bovine serum albumin, 0.02% polyvinyl pyrrolidone, and 0.02% Ficoll), 0.1% SDS, and 50 pg/ml of yeast tRNA (Boehringer). After hybridization, the filter was washed twice for 30 min at 43” in 2X SSC and 0.1% SDS, and finally exposed to AGFA curix RP-2 films at -70”. The restriction maps of EcoRI, SalI, and HindIll ASF virus DNA (4) are shown below.

of amino acids of the encoded protein and rightward reading). The first AUG of this ORF is found in a favored context for initiation of translation according to Kozak’s rules (19). Upstream of this putative initiation codon an in-frame stop codon is present at nucleotides 885-887.

Two additional ORFs, extending for more than 60 amino acids, were identified in the sequenced fragment. One of them precedes ORF P1192R on the same strand and extends from nucleotide 242 to 1060, encoding a polypeptide of 273 amino acids. This ORF is designated S273R. Downstream of ORF P1192R and on the opposite strand, an incomplete ORF that terminates at nucleotide 4710 was found. ORF S273R was not significantly similar to any protein sequence of the databases. The characteristics of the amino acid sequence of ORF P1192R are described below. To determine whether the putative ASF virus topoisomerase II gene, ORF P1192R, was expressed during the viral infection, early and late poly(A)+ RNA isolated from Vero cells infected with ASF virus was analyzed by Northern blot hybridization using as probe a

synthetic oligonucleotide specific for ORF P1192R, as indicated in Fig. 26. An RNA of 4.6 kb was detected at late times after infection (Fig. 3). The size of this RNA is consistent with the length of the polypeptide encoded by ORF P1192R. With early CHM and CAR RNAs very weak bands were observed. These bands were more clearly seen with longer exposure times. ORF P1192R is, therefore, expressed mainly late in infection, which is consistent with its localization in a late transcriptional region (see Fig. 2A). A database search with ORF P1192R revealed a significant homology of the protein to type II topoisomerases. The FASTA scores obtained for the comparison of the P1192R protein with the eukaryotic topoisomerases II oscillated between 545 (the lowest for the enzyme of Tryoanosoma brucel) and 717 (the highest for Saccharomyces cerevisiae topoisomerase II). Using the BESTFIT program of the UWGCG, the following percentages of identity were obtained by pairwise comparison of ORF P1192R and different topoisomerases: S. cerevisiae,

28.6%;

Schizosaccharomyces pombe, Drosophila melanogaster,

28.7%; human, 26.5%;

940

SHORT COMMUNICATIONS

A

50

0

I

H

R

S273R

hi

s

100

150

I

I

RH

170 lb

H

S

P1192R (TOP0 II)

t

B AAGCTTCAAGAAAGGGGCAGTTGGGCTTTGACCC~~TGCTGACGAC~TATC~C~GCATGATGTATTC~AGTGAGGATAGTAGTTTACGGA~CT~TTCAGC~CCCGCCGTT TTATTTCGTATCTTGCCCAGTTATTCAGAGAGAGTA~CCACGCCTCCGAC~C~CAGACATCCTATCTATT AAAAAATAACAATAAAAACCTTATGAAATCTATGTATAGTGGCCGCTZUt AATGTCTATATTAGAAAAAATTACGTCAAGTCCCTCTCTG~TGCGCAGAG~C~TACA~C~GATAGCTGTTT~GT AAAAAAATACA&UAGAGC'CCACCTCTTTTTTGGAAAAAAA MSILEKITSSPSECAEHLTNKDSCL SKKIQKELT SFLEKK S2738> AGAGACACPCGGTTGCGATTCGGAGTCCTGCGTAATTACCCACCCCGCCGTG~GGCCTAT~GCAAC~~GGGA~GGAC~TTCC~G~~GGAGACTCGGTTT~~GCCAGG ETLGCDS ESCVITHPAVKAYAQQKGL DLSKELETRFKAPG

120 240 360

480

ACCCAGAAACAACACGGGTCT~TACAAACTTCAATATCGATG~CG~GCAGAGGTGGGCCAT~GTACACC~GTTTTTC~CTGTCCTTTTTCCATGATGGACTTTGAGAGGGT PRNNTGLLTNFNI DETLQRWAI KYTKFFNCPFSMMDFERV

600

CCATTATAAATTTRATCAAGTGGATATGGTAAAGGTATAT~GGGGG~GAG~A~ATATGTAG~GGC~GTGGTC~GCGTCCTTGT~CACCTTCGGATGCGTTTT~CACGGA HYKFNQVDMV KVYKGEELQYVEGKVVKRPCNT FGCVLNTD

720

CTTTTCAACGGGCACTGGAACACTGGGTAGCCATCTTTGTG~TATGCGG~CGACTGCTGGAGCATCG~TATTTT~TTCG~G~~TTCCCCCCCAGGTCCCGTTATT~CTG FSTGTGKHWVA IFVDMRGDCWSIEYFNSAGNSPPGPVIRW

840

GATGGAACGGGTCAAACAGCAGCTATTAAAAATACACCACACCGTG~CG~TGCGGTTACC~CATTCGTCACCAGCGGTCG~GACCGAGTGCGGCCCCTACAGC~GTTTTACAT MERVKQQLLK IHHTVKTLAVTNI RHQRSQT EC G PYSLFYI

960

CAGGGCACGC~CGACAACGTGTCATACGCCCATTTTATATCCGCTAGGATTACCGACG~GACATGTAT~GTTTAG~CC~T~GTTTCGCATCGCAT~CT~T~GTTTG~T RARLDNVSYAHFISARITDEDMYKFRTHLF R I A *

1080

TCTTTATAGGAATAAAAATGGAAGCGTTTGAAATCAGCGATTTC~GA~ATGCG~G~~GCATGTGGGCTGGCGCCCTC~C~GTCACTATTTCGGGTCTTATGGGGGT~ MEAFEISDFKEHAKKKSMWAGALNKVTI SGLMGVF P1192R> TTACCGAAGATGAGGACCTTATGGCGTTACCCATTCACAGAGACCA~GTCCCGCTTTGTT~~TTTTTGACGA~T~TCGT~TGCCACGGATCATG~GAGCTTGCCATAGCA TEDEDLMALPIHRDHC PA L L K IFDELIVNATDHERACHSK

1200

AAACAAAAAAGGTRACCTACATCAARATTTTCGTTTGATA~GGCGTGTTTTCTTGCG~CGATGGCCCGGG~TCCCCATTGC~AGCATGA~AGGCCAGTCTTATCGCC~GCGCG TKKVTYIKISFDKGVFSCENDG PGIPIAKHEQASLIAKRD

1440

ATGTGTATGTTCCCGAGGTGGCTTCATGCTTCTTTCTAGCCGG~CG~CATCAAT~GGCC~GGA~GTATC~GGGGGG~CC~CGGCGTCGGGCTG~GCTCGC~T~T~ATT v Y v PEVASCFFLAGTNINKAKDCIKGGT NGVGLKLAMVHS

1560

CGCAGTGGGCCATTCTTACCACCGCCGACGGCGCGC~GTATGTTC~CA~TC~CCAGCGCCTAGATATCATTGAGCCTCCTACCATTACACC~CCAGGG~TGTTTACACGTA QWAILTTADGAQ KYVQQINQRLDIIEPPT ITPSREMFTRI

1680

TCGAGCTWGCCCGT E L M P V

1800

Y

Q

AACTAGGGTACGCGGAGCCTCTGT~G~CGGAGCAAG ELGYAEPLS ETEQADLSAW

1320

IYLRACQCAAYVGK

AAGGCACCACCATTTATTACAATGATAAGCCTTGCCGCACGGG~CTGTGATGGCGCTGGC~A~TGTACACCCTGTTGAGCGCGCCT~TAGCACGATACATACGGCGACCATT~~ GTTIYYNDKPCRTGSVMALAKMYTLLSA PNSTIHTATIKA

1920

FIG. 2. Sequence of the ASF virus topoisomerase II gene. (A) EcoRl map of ASF virus DNA and organization of the ORFs within a 5378.bp fragment located within EcoRl restriction fragments S, P, and H. The restriction sites used for subcloning in M 13 phage vectors are indicated as follows: H, HindIll; R, EcoRI; M, Smal; S, Sall. The ORFs are indicated by arrows with their designation above each arrow. A transcriptional map of ASF virus DNA indicating the early (E, open boxes), early and late (EL, striped boxes), and late (L, filled boxes) is also shown (7 I). (B) DNA sequence of the 5378-bp fragment. Selected DNA fragments were cloned in M 13mpl8 and M t3mpl9 and the single-stranded templates were dATP (Amersham). sequenced by the dideoxynucleotide chain termination method (39) using T7 DNA polymerase (Pharmacia) and ?Yabeled

941

SHORT COMMUNICATIONS CCGACGCAAAACC~ATAGCCTGCACCCTCTCTGCAGGTTGCGGCGGTCGTGTC~CC~GTTT AAAAAATTTGAACACGTGTCCATTATCAACGGGGTAAATTGTGTAAAAGGAGAACATG IINGVNCVKGEHV PKFKKFEHVS PLQVAAVVS DAKPYSLH TTACCTTTTTGAAAAAGACCATTAATGAAATGGTCATTAAAAAA TFLKKTINEMVIKKFQQT

TTTCAACAGACGATTAAAGATAAAAACCGCAAAACAACATTACGTGA~G~GTTC~CAT~TTGTCGTTATAG F V IKDKNRKTTLRDSCSNI

2040

2160 V

IV

TGGGTTCCATTCCAGGCATAGAATGGACCGGACCGGCCA~GG~GGATG~CTTAGCAT~CAG~TGTTTTT~C~ATTA~CCATCCCTTCTAGTTTTTT~C~G~TGAC~GGT IAENVFKTHYSIPSSFLTSMTRS PGIEWTGQRKDELS G S I

2280

GATAACCATAAACAGGTCGACGTAGACAAATATACGCGTGTGCTAC CTATCGTGGATATTCTTCTGCAATCCATTTCTAAAAAA DKYTRARNAGGKRAQDCMLL I V D I L L Q S ISKKDNHKQVDV

2400

TCGCGGCGGAAGGGGATAGCGCACTTTCCCTGTTGCGCACGGGACTGACCCT~G~GTC~~C~GCGGGCC~C~TTGA~T~GCGGCATGAT~CCCTGGGAGG~TCATCA SLGGVIM PSFDFCGMI KSNPSG SLLRTGLTLG AAEGDSAL ==r==========:===

2520

TGAATGC~GCAAAAAGGTGACAAACATTAC~C~A~CTGGAG~CCATCATGGT~GC~CG~CAGCTTAC~T~T~GTGTTGCAGGG~TTGTGCAGGTATT~GTCTAG IVQVLGLD NACKKVTNITTDSGETIMVRNEQLTNNKVLQG

2640

A~TCRA~GCCATTACAARACGCAGGAAGAGCGAGCGAGC~GCTGAGATACGG~GCATTGTTGCGTG~TTGATC~GATCT~ATGGGTGTGG~T~TTGGACTGCT~T~C~ DQDLDGCGKILGLLLAY FNCHYKTQEERAKLRYGCIVACV

2760

ACTTTCACCTGTTTTGGCCTCAGCTTATTATCCATGGTTT~T~CGACTGCTTACCCC~TGATACGTGTGTACG~GGGC~GACTATGCCCGTAGAATTTTA~ATG~CAGG IIHGFVKRLLTPLIRVYEKGKTMPVEFYYEQE FHLFWPQL

2880

AGTTTGATGC~GGGCAAAGCAGACCAG~TAGT~TCATACTGT~~TATTAC~GGGATTGGC~C~ATGA~CCCATG~GT~G~TGTTC~ACATTTTGAC~~ SLVNHTVKYYKGLAAHDTHEVKSMFKH FDAWAKKQT

3000 FDNM

TGGTGTACACGTTTACCCTGATGA~CGGCAAAGGAGGAGTTGTTTCATATTTATTTTGG~GGGAGTC~AGTTGCG~GAGA~TTTGCACCGGCGTGGT~C~T~CTG~CCC ELRKRELCTGVVPLTETQ VYTFTLDDSAKELFHIYFGGES

3120

AGACGCAGTCCATTCATAGTGTCCGACGAATTCCTTCCTTGCAGCCTGCATCTGCAGGTAGATAC~GGCTTAC~GCT~ATGCCATCGA~G~AGATTCCC~~T~TAGA~G~TGA PNFLDGMT I E R Q I PCSLHLQVDTKAYKLDA T Q S IHSVRRI

3240

CGCGGGCGCGGCGCAAAATTTTAGCCGGGGGGGTG~ATG~TCGCTTCC~C~CCGTG~CG~~TTTTTCAGTTCGGGGG~ACGTTGC~ATCA~TGTTTTATCACCATGG~ RARRKILAGGVKCFASNNRERKVFQFGGYVADHMFYHHGD

3360

ACATGTCGTTAAACACRAGTATTATAAAAGCCGCCCAGTATTACCCGGG~C~C~ACCT~ATCCAGTATTCATAGGCATAGG~G~TCGG~CCAG~ACCTGGGAGG~AGGATG MSLNTSIIKAAQYYPGSSHLYPVFIGIGSFGSRHLGGKDA

3480

CAGGATCCCCAAGATACATCAGTGTGCAGCTTGCGTCTGAATTTATT~C~TGTTCCCCGCGGAGGA~CATGGCTTCT~C~ACGT~TTGA~ACGGCCAGCG~CGG~CCAG G S P R Y I SVQLASEFIKTMFPAEDSWLLPYVFEDGQRAEPE

3600

AGTACTACGTGCCTGTATTGCCGCTTGCTATTATGGAGTACGGCGCC~CCCATC~A~G~GG~GTACACCACTTG~C~GGC~CTGG~GA~TTTT~C~T~TGAGGGC~ ILALVRAY YYVPVLPLAIMEYGXNPSEGWKYTTWARQLED

3720

ACGTCGACAAAGACAACCCAACGAGCTACT~A~ATGC~T~CATAAGATTACTATACT~CGCT~G~C~C~TTAC~TTTC~GGGCCATTT~~GGTTTGGCC VDKDNPKHELLHYAIKHKITILPLRPSNYNFKGHLKRFGQ

3840

AATA~A~ACAGCTACGGCACGTACGACATCTCAGAGAGCAGCG~TAT~TTACTATTAC~AGCTTCCTCTGCGTGTTCCTACGGTTGCATATAT~~GTAT ITITELPLRVPTVAYIESIKKSSN YYYSYGTYDI SEQRNI

AAAAAAATCGAGTA

3960

ACCG04TGACATTTATTGAAGAAATCATCGA~A~GTAGTTCAG~CCATTG~TTCT~TG~CT~AGCC~TAGTCT~CCGTATCGT~~G~TTT~GGAGACTG~G RMTFIEEI I D Y S S S ET I EILVKLKPNSLNRIVEEFKETEE

4080

AGCAAGATTCCATAGAAAATTTTCTGCGCCTGCCTGCGC~TTGTTTACATTCGCATCT~A~TTGT~CCT~GGTGGTATTATCGAGTTT~~CATATTATG~TTTTATATGCGT QDSIENFLRLRNCLHSHLNFVKPKGGIIEFNSYYEILYAW

4200

GGCTACCTTACAGGCGTGAGCTTTACCAAAAGCGTCTTAT~GTGAGCACGCGGT~TT~GCTGCGCATTAT~TGG~CTGCTATTGTACG~ACAT~TGAGTCTGCAGAGCT~ LPYRRELYQKRLMREHAVLKLRIIMETAIVRYINESAELN

4320

AAAAAGCGCTGCAGGGCPGTTATACCTATATATACTTTT KALQGCYTYILSLQARELLIAAKTRRVEKIKKMQARLDKV

4560

AAAAAAAATGCAAGCTCGTCTTGATAAGG

TTGAGCAGCTTTTGCAGGAGTCTCCCTTTCCCCGG~CCAGCGTATGG~GGA~~TTGATGC~T~~~CTATTAT~GG~G~TACTCAGTGG~TTTCATT~CGC EQLLQES PFPGASVWLEEIDAVEKAI IKGRNTQWKFH"

4680

The M 13 sequencing primer and, when necessary, additional oligonucieotide primers corresponding to internal sequences, were used. The nucleotide sequence was determined in both strands at least once. The sequence is shown in the 5’. to 3’. direction and from left to right according to the restriction map. The protein sequences are shown below the DNA sequence in single-letter code. The designation for each ORF is given below the initiating ATG. The in-frame stop codon that precedes the initiating ATG of ORF Pl 192R is overlined. An oligonucleotide complementary to the underlined sequence was used as hybridization probe in the Northern blot analysis. Marked with a dashed double underline is a stretch of five amino acids that corresponds to the sequence encoded by the olrgonucleotrde probe used in the mapping of the topoisomerase II gene. The asterisks indicate the end of the ORFs.

942

SHORT COMMUNICATIONS

-7.4 - 5.3 -2.8 q-i -1:o - ..

8t

FIG. 3. Northern blot hybridization. Vero cells were mock-infected or infected with the Vero-adapted BA7 1V strain of ASF virus (38) at a multiplicity of 10 PFU per cell. To obtain early RNA, the cells were infected for 4 hr in the presence of 200 pg of cycloheximide (CHM) (Sigma) per ml or for 18 hr in the presence of 40 pg of cytosine arabinoside (CAR) (Sigma) per ml. Late RNA was isolated from cells infected for 18 hr in the absence of inhibitors. Whole cell RNA was prepared by the guanidine hydrochloride extraction procedure (40) and poly(A)+ RNAs were selected by oligo (dT)-cellulose (Invitrogen) chromatography as described by Maniatis et al. (41). The poly(A)’ RNAs (4 pg) were fractionated on a formaldehyde-agarose gel, transferred to a nitrocellulose membrane and hybridized with a % labeled oligonucleotide probe specific for ORF Pl 192R (see Fig. 2B) at 45”, as described in the legend of Fig. 1. The sizes (in kb) of RNA molecular weight markers are indicated on the right.

25.49/o; T. brucei, 25.4%; Crithidia fasciculata, 24.1010; Escherichia coligyrase B, 23% with the first 679 amino acids of ORF P1192R; E. co/i gyrase A, 21% between

the first 552 amino acids of gyrase A and amino acids 680 to 1192 of P1192R protein; phage T4 gene 39 protein, 22.49/owith the first 565 amino acids of ORF Pl 192R; and phage T4 gene 52 protein, 22.6% between the first 441 amino acids of the gene 52 protein and amino acids 669 to 1 175 of the Pl 192R protein. A multiple alignment of ORF Pl 192R and topoisomerases II from different origins is shown in Fig. 4. This alignment is similar to the one previously published (17) except that it includes the human, T. brucei and C. fasciculata topoisomerases II and omits the Bacillus subtilis gyrase. The Pl 192R sequence is essentially colinear with the topoisomerase II sequences. The ASF virus protein is close in size to the enzymes from the protozoa T. brucei and C. fasciculata and ends at about the same position in the multiple alignment than the phage T4 gene 52 protein. All these proteins lack a carboxy terminal segment of about 200-300 amino acids that is present in the other topoisomerases. This region is not well conserved among the topoisomerases, and in the case of the human, yeast, and Drosophila melanogaster enzymes is characterized by the

presence of stretches of positively and negatively charged amino acids. In the multiple alignment it can be noticed that a number of amino acid positions are invariant. Conservation of these residues in all the sequences compared suggests that they may be critical for the function of the enzymes. These invariant residues include the amino acids Arg-Tyr at positions 884-885, respectively, in the multiple alignment, which are involved in the formation of the transient covalent bond with DNA during the strand passage reaction (20, 21). Another highly conserved region is a stretch of five amino acids (EGDSA) that forms part of the signature pattern used in the PROSITE library to detect proteins belonging to the topoisomerase II family (22). As indicated before, a mixed oligonucleotide probe encoding this amino acid sequence was used to map the ASF virus topoisomerase II gene. Immediately preceding this invariant region there is a sequence that closely matches a modified consensus B structure found in ATP-binding proteins (23) as has been discussed by Tamura and Gellert (24) and Bugg et a/. (25). The consensus motif is as follows: [H, K, R] X,-, h X h, [D, E], where the amino acids enclosed in brackets signify alternatives for that position, h is a hydrophobic residue, Xs are nonconserved amino acids and the subscript indicates the number of intervening residues. The corresponding pattern in the topoisomerase II sequences, surrounding the invariant Leu residue at position 500 in the multiple alignment of Fig. 4, is [K, R] X,-, L [Y, I, V, F, L] h [v, T, A] E. Recently, the crystal structure of an N-terminal fragment of the E. co/i gyrase B protein, which contains the ATP hydrolyzing activity, has been determined and the amino acids involved in ATP binding have been identified (26). Some of these amino acids, overlined in the multiple alignment of Fig. 4, are conserved in all the sequences aligned. The invariant residues include the conserved GXXGXG motif, at positions 174-l 79 in the multiple alignment, that has been shown independently by affinity-labeling studies to form part of the ATP-binding site (24). The characteristics of the amino acid sequence of ORF P1192R, described above, strongly suggest that it encodes a topoisomerase II. This is the first example of a topoisomerase II encoded by an eukaryotic virus. It is known that vacciniavirus encodes a type I topoisomerase, which is encapsidated within the virion (27-29) while other viruses, such as adenovirus, cytomegalovirus, or simian virus 40, increase the levels of cellular type I or type II topoisomerases (30-32). The association of topoisomerase I activity with particles of Rous sarcoma virus, herpes simplexvirus, and human immunodeficiency virus has also been reported (33-35).

SHORT COMMUNICATIONS

943 100

1 EcoliB T4-39 Human Drome Schpo Schce Trybr Critfas ASFV

MEVSPLQPVNENMQVNKIKK MTASEQIPLV

MSNS&SSIKVLKGL MIKNEIKILSDI NEDAKKRLSVERIYQKKTQL MENGNKALSIEQMYQKKSQL TNNGNGNSNVSTQYQRLTPR MSTEPVSASDKYQKISQL MAEAHKYKKLTPI MTDASKYQKLTPI MEAFEISDF

.................... HERFMFGK . . . . ..WE.SVQ QQMWVYDEDVG.INYR.BVT ELMWVYDNSQNRMVQK.EIS SEX.lWVFDSEKNKLDYK.AVT QLQWIYDEETlXMIEK.NVT TPMFIYDEQKGHMVWE.TVK IPMFVFDPAKGKMVWE.SMQ GLMGVFTEDEDLMALPIHRD

. ..GLHHMVFEWD&IDEA YVFGLVKLIDEIIDNSVD.. FVPGLYKIFDEILVNAAD.. FVPGLYKIFDEILVNAAD.. YVPGLYKIFDEIIVNAAD.. IVFGLFKIFDEILVNAADN. LNHGLLKIVDEILLNASDNI VNQGLLKIVDEILLNAADNI HCPALLKIFDELIVNATDH.

eh.lkrpdmyiGsve..ttt

qlw.de.kg.mvw...vt

.vpgL.kifdEilvNaaD..

GIHPEEGVSAAEVIMTVLHA T...GEEIPGPVAAWTIPKA K...VEKMWPALIFGQLLT K... EQKMYVPTMIFGHLLT D...KEKIYIPELIFGNLLT N...KENIYIPEMIFGHLLT R...EHKLYIPEMVFGHLLT K...EHKMYIPEXVFGHLLT SLIAKRDVYVPBVASCFFLA

200 -GG;FDDNS&SG&HGVGV SWNALSQKL..ELVIQREG GGNFGDDKERVTGGMNGVGS SLTNIFSVMFVGETGDGQNN SSNYDDDEKKVTGGRNGYGA KLCNIFSTKFTVETASREYK SSNYNDDEKKVTGGRNGYGA KLCNIFSTSFTVETATREYK SSNYDDNQKKVTGGRNGYGA KLCNIFSTEFWETADKERM SSNYDDDEKKVTGGRNGYGA KLCNIFSTEFILETADLNVG SSNYDDDNQNAVAGRHGYGA KLTNILSLSFSV..CCRTNG SSNYNNDASSTTAGRHGYGA KLTNILSTKFSV..VCRTAG GTNINKAKDCIKGGTNGVGL KLAMVHSQWAILTTA..IX;A

DAVRKRPGMYIGDTDDGT.. EHIKKRSGMYIGSSA..N!ZT EHILLRPDTYIGSVE..LVT EHILLRPDSYIGSVE..FTK EHVLRRPDTYIGSIE..WT EHILKRPCTYIGSVE..TQE EHVLTRPBMYIGSLD..TTA DHVLLRPEMYVGSIE..TQS KEHAKKKSMWAGALNKVTIS

t

l

CONS

EcoliB T4-39 HUlll.%l Drome Schpo Schce Trybr Critfas ASFV

. . . . . . . . . . . . . . . . . . . .

. . . . . . . .

s.sskyqkl..l

EcoliB T4-39 Human Dr0me Schpo Schce Trybr Critfas ASFV

NSVSVQ;DGRGIP . . . . ..T NQVTVEDNGRGIPQAMVKTP VVEH QLISIWNNGKGIP... NMVSVWNNGQGIP...VTMH NVISIYNNGKGIP...IEIH IEIH HTIEXKNDGKGIP... IVRS GEITIENDGAGIP... GEIMVENDGAGLP...IVKS GVFSCENDGPGIPIAKHEQA

.nkvrd..m..ikvti..dk

n.isvendGkGiP...i.ih

k...eek.yipemifghllt

6snydddekkvtgGrnGyGa

k1cnifStkf.vetadre.g

201 KIHRQIYEHGVFQAPLAVTG IWRCS..NGMENKSWEDIP KMFKQTWMDNMGRAGEMELK KSFKQTWGNNMGKASDVQIK KKYKQTWYDNMSRKSEPVIT QKYVQKWENNMSICHPPKIT REFHMSWQDHMRKATAPRVS REFHMSWTDHMRMATTPRVS QKYVQQINQRLDIIEPPTIT

ETEKTG... TMVRFWPSLET GKWKG....TRVTFIPDFMS PF..NGEDYTCITFQPDLSK DF..NGTDYTRITFSPDLAK SL.KKPDEYTKITFKPDLAK SY.KKGPSYl'KVTFKPDLTR NVGTKEKNVTRVKFLPDYER NVDPKEKNVTRVTFMPDYAH P...SREMWRIELMPVYQE

FTNVTEFE............ FE..T...NELSQVLSLTLH FKM. . . ..QSLDKDIVAU.lV FKM.....DRLDEDIVALMS DKIDDDMVSIIK FGM..... FGM.... .KELDNDILGVMR FGMKE...KKISNDMKRVLY FGFPT . ..AAISLDMKRVLH LGYAEPLSEZTEQADLSAWIY

. . .YEILAKRLRELSFLNSG WIWQTLAWYPDIHL.... RRAYD.IAGSTKDVKVFLNG RRAYD.VAASSKGVSVFLNG RRIYD.MAGTVRETKVYLNN RRVYD.INGSVRDINVYLNG KRIMD.LSAMFPNIQITLNG KRIMD.LAAMFSKIBVRLNN LRACQCAAYVGKGTTIYYND

300 VSIRLRDKRLGKEDHFHYEG .PLMVKRFRAILR....... NKLPVKGFRSYVDMYLK... NKLGVRNFKDYIDLHIK... ERISISGFKKYVEMYLAS.. KSLKIRNFKNYVELYLKSLE SSFGFKSFKDYATLY..... VPFGFQTFNDYARLY..... KPCRTGSVMALAKMYTLLSA

kkfkq.wednm..as.p.it

. ..kkg..yTrvtf.Pdlak

fgm.......ldnd.vav..

rr.yd.la.s..di.vylng

..lgv..fkdy.ely..

301 GIKAFVEYLNKNKTPIHPNI . . . . . . . ..NMHGSTDEHAI . . . . . . ..DKLDETGNSLKV .... . NTDDDSGPPIKI . . . . . . . ..WTKPDEEPPRV KKRQLMJGEDGAAKSDIPPI . . . . ..SAMTPKGEKPPPPY . . . . ..SLPGADSAMPPEPF PNSTIHTATIKADAKP....

FYFSTEKDXGVEVALQWND VQEQENCS..IAVGRSP..D IHEQVNHRWEVCLTMSE..K VHEVANERWEVACCPSD..R IYMVNDRWDVAFAVSD..G LYERINNRWEVAFAVSD..I VYESKSGCVAFIPSWP..G VHTGPNGSIAFVPQLTQ..S . ..YSLHPLQVAAVVSPKFK

HLAGFRAAMI'RTLNAYMDKE HI...DCAMDDICEDLIPQI HV...DYVADQIVTKLVDW HV. ..DiiWDNLIKQLLEVL HV...NYVANKIVDAIDEW HV...NYITDQIVKKISEIL HC...NAAQDILTGCLDGVE HC...TSAMEILETGLDSLS VTFLKKTINEMVIKKFQQTI

400 GYSKKAKVSATGDDAREGLI KRKFKIDVTKRRCQRMFDYV KKKNKGGVAVKAHQVKNHMW KKKNKGGINLKPFQVRNHLW KKENKKA.PVKAFQIKNYVQ KKKKKK..SVKSFQIKNNMF RELKKENKVMIYPNRVLRHFT RSLKKDGKVIDTNRVARHFT KDKNRKTTLRDSC...SNIF

. . . . . . . . ..kdg..ppp.i

vye.vn.rweva..vs....

GFQENIYCFTNNIPQREGGT GFRQ..LTWNNIHTKNGGH GFQQ..ISFVNSIATSKGGR GFQQ..VSFVNSIATYKGGR QFKQ..VSFVNNISTIRGGT SFQQ..ISFVNSIATTMGGT VRRM..FGWNGWTYNGGT PKRI..VGWNGWTYNGGT KFBH..VSIINGVNCVKGEH t t gfqq..vsfvN.iaty.Ggt

hv....yamd.ivkkldev.

kkk.kkgv.vk.fqvknh..

VSSEXKSAVEQQMNELLAEY TSPFGEIRSHIQLDAKKISR TLQPKSFGSTCQLSEKFIKA TLGQKGFGSKCTLSEKFINN TTKVSAFGSQCTLSDKFLKA TTRVKDFGSRCEIPLEYINK VSTPTMPRVPRQDVMKYLLR VSTVTMPRVPRTALCQYLAA SIAENVFKTHYSIPSSFLTS

LLENPTDAKIWGKIIDAAR DILNN.EAILMPIIEAALAR AIGCGIVESILNWVKFKAQV MSKSGIVESVLAWAKFKAQN IKKSSWEEVLKFATAKAW IMKTDLATRMFEIADANEEN M...PFLEAHVSTITGQLAQ M...PFLEAHMNSMDDQLAA MTRS.IVDILLQSISKKDNH

ts.vkmfgs.cqlsekf..a

m.kspive..ln...akaa.

500 AREAARRAREMTRRKGALDL AGLPGKLAKCQERDPALSEL KLAAEKAAmKiUKKASKAK VHKHIKANLCGKDADT..TL QLNKKCSAVKH....NRIKG IPKLDDANDAGGRNSTECTL DIA.KTGGRKS....SKIKG IPKLEDANEAGGKNSIKCPL QL.SKGM;GLR....SRITG LTKLEDANKAGTKESHKCVL AL.KKSffiTRK....SRITN YPKLEDANKAGTKEGYKCTL ELNKEIGTGRRMSSKTLLTS ITKLVDATSTRRDPKHTRTL ELNKEIGTGKRLSSRSLISS ITKLVDATSSRSDXNIRTL . . . . . . . . . . . . . . ..KQVD VDKYTPARNAGGKRAQDCML t l.kkig.gkr....sritg i.kledan.aggk.s.kctL

Q..........AILPLKGKI K.......ELHGGYPLRGKV R.......DKYGVFPLRGKI R.......DLYGVFPLRGKL R.......DYYGVFPLRGKL R....... DYYGCYPLRGKM K.......RYTGVFPLRGKL K.......KFCGVFPLRGKL SNPSGPSFDFCGMISLGGVI

LNVEKA.............. LNSWGM.............. LNVREA.............. LNVREA.............. LNVREA.............. LNVREA.............. LNVRNK.............. LNVRNK.............. MNACKKVTNITTDSGETINV

RFDKMLSSQEVATLITALGC SYADMLKNKELFDICAITGL SHKQIMENAEINNIIKIVGL NFKQLSENAEINNLCKIIGL SHSQILNNKEIQAIKKIMGF SADQILKNAEIQAIKKIMGL NLKRLRNCKELQELFCALGL NLKRLKTCKELQDLFLALGL RNEQLTNNKVLQGIVQVLX;L

t

t

t

l

CONS

EcoliB T4-39 HUmaIl DlXXlle Schpo Schce Trybr Critfas ASFV CONS

EcoliB T4-39 Human DrOme Schpo Schce Trybr Critfas ASFV CONS

EcoliB T4-39 Human Drome Schpo Schce Trybr Critfas ASFV CONS

**

101 LAGHCKE....IIVTIHAD. .EGIRTKFKFANKINVTIKN .NKQRDPKMSCIRVTMIR.K .NKQRDKSMNTIKIDIDPER .NKVRDPNMNTLKVTLDPEA .NKVRDPSMKRIDVNIHAEE SN..RSARmYIRVTI.TL?T NNSVRGARMTYISIKI.SDS EPACHSKTKKVTYIKISFDK

l

CONS

l

401 AWSVKVPDPKFSSQTKDKL IWRDM.KNNRLIRQTKERL IFVNALIENPTFDSQTKENM VFVNCLIENPTFDSQTKENM VFVNCQIENPSFDSQTKmL IFINCLIENPAFTSQTKEQL ILVFLVQVQPKFDSQNKARL VLVFLIQSQPKFDSQSKARL VVIVGSIPGIEWTGQRKDEL t * .fvnclienpkfdsQtKerl 501 YLVEGDSAGGSAKQGRNRKN FLTEGDSAIGYLI...DVRD ILTEGDSAKTLAVSGLGWG ILTEGDSAKSLAVSGLGVIG ILTEGDSAKSLAVSGLSWG VLTEGDSALSLAVAGLAWG IVTEGDSAKALAQNSLSSCQ IVTEGDSAKALALNSLSSEQ LAAEGDSALSLLRTGLTLGK ****t iltEGDSAkslavsglswg

t

t

l

l

r.......dyygvfpLrGkl

l

*

l

lNvrea..............

600 GIGRDEY....NPDKLRYH. VLGEKAFEEKEDGEWFTFEL QY.KKNYEDEDSLKTLRY.. QY.KKKYLTEDDLKTLRY.. TH.KKTY...TDVKGLRY.. QH.RKKY...EDTKSLRY.. EL.DKDYTDADE...LRY.. EL.GKTYKSPAE...LRY.. DF.NCHYKTQEEFAKLRY..

*

s.kqllnnke.q.i.kilGl

ql.kk.y....d.k.lry..

FIG. 4. Comparison of the amino acid sequences of ORF Pl 192R and type II topoisomerases. The multiple alignment was obtained using the PILEUP and LINEUP programs of the UWGCG. The alignments were adjusted by hand. The arrowhead in the f. coligyrase B protein indicates a region of 172 amino acids that has been omitted, since this region is not homologous to other topoisomerase II sequences. Amino acids conserved in all sequences are indicated with an asterisk. The last line shows the consensus sequence. The arrow indicates the conserved

944

SHORT COMMUNICATIONS

EcoliB T4-39 Human Drome Schpo Schce Trybr Critfas ASFV CONS

EcoliB T4-52 Human Drome Schpo Schce Trybr Critfas ASFV

601 . SIIIMTDADVDGS.HIRT NGDTIIVNENDEVQ.INGKW .GKIMIMTDQDQDGS.HIKG .GKVMIMTDQDQDGS.HIKG .GHLMIMTDQDHDGS.HIKG .GHLMIMTDQDHDGS.HIKG .QRILIMTlX)DADcS.HIKG .QRLLVMTIMDACGS.HIKG .GCIVACVD;)DLDGCGKILG t .g.imimtdqD.dgs.hikg

LLLTFFYRQMPEIVERGHVY ITVGELRKKSIMT/ LLINF1HHNWPSLL.RHR.F LLINF1HTNWPELL.RLP.F LIINYLESSYPSLL.QIFGF LIINFLESSFLGLL.DIQGF LVINAFESLWPSLLVRNPGF LVINAFESLWPSLLQHNFGY LLLAYFHLFWPQLI..IHGF

IAQ..PPLYKVKKGKQEQYI

v KDDEAMWYQISI......R

700 GLSIQRYKGLGEMNPEQLWE

LEEFITPIVKVS...KNKQE KKNEE LEEFITPIVKAT... LIQFITPIIKCT...RGNQV LLEFITPIIKVSITKPTKNT ISIFSTPIVKARL..RDKSV ISLFSTPIVKIKVNGKAKEV VKRLLTPLIRVYEKGKTMP.

MAFYSLPEFEBWKSSTPNHK LSFYSLPEFEEWKNDTANHH QAFYTLPEYEYWKEANNNGR IAFYNMPDYEKWREEESHKF VSFFSMKEFHKWQRSNANTP VAFHSFRDFHRWQRANPNAR VEFYYEQEFDAWAKKQTSLV

KWKVKYYKGLGTSTSKEAKE TYNIKWKGLGTSTSKEAKE GWKIKYYKGLGTSDHDDMKS TWKQKYYKGLGTSLAQBVRE .YTCKYYKGLGTSTTAEGKE .YSAKYYKGLGTSTTAEGKE NHTVKYYKGLAAHDTHEVKS

llinffes.wpsll.ripgf

l.efitPivkv....ktk.v

vafys.pefe.wk..n.n.r

..kikyYKGLgtstt.e.ke

*et.

l

701 TTMDPESRRMLRVTVKDAIA

BOO YAMSVIVGRALPDVRKGLKP YAMYTVENRAIPNMIDGFKP FSN.SDNERSIPSMVlXLKP FSN.ADNERSIPSLVM;LKP FSM.ADNIRSIPSWDGLKP FSL.ADNIRSIPNVLDGFKP FAL.VGNARALAHSVCGLKP FAL.VGNARAIPHCVDGLKP YKLDAIE.RQIPNFLDGWl'R

ADQLFTTLMGDAVEPRRAFI

EENALKAANIDU

YFADMK.RHRIQFKYSGPED YFQDMD.RHRILFKYDGSVD YFSDLD.RHMKYFHAMQEKD YFSNLD.RHLKIFHSLQGND YFKDME.KHTMRL.LVDRSD YFADME.RNVMRL.WEPKD MFKHFD..NMVYTFTLDDSA

MSD LAREITPVNIEEELKSSYLD MQLNNRDLKSIIDNEALA DAAISLAFSKKQIDDRKEWL TNFMEDRRQRKLLGLPEDYL YGOTTTYLTYNDFINKELIL DESIVMAFSKKHIESRKVWL TNHMDBVKRRKELGLPERYL YTKGTKSITYADFINLELVL AELIEMAFAKKKADVRKBWL RTYRPGI...... . . . . ..Y MDYTQPQIPIDDFINRELIQ KDYIDLAFSKKKADDRKEXWL RQYEPGT.... . . . . . . ..V LDPTLKEIPISDFINKELIL HKLLDNVFDSQEVEWRKDWM T......... KANAWGEVD IDRSKKMLTVTDFVHKEMVH HRLLDSVFDSAEVEWRKBWM S......... KANAFQGEVD IDRSKKLLTIGDFVHKEMVH . . . ..ETQTQ SIHSVRRIPCSLHLQVGTKA KELFHIYFGGESELRKRELC TGWPLT...

yf.dmd.rhm.rf...dp.d

.elid.afskk.ve.rkewl

t...............

801 VHRRVLYAMNVLGNMNKAY VQRFVIARALDLARGNKDKF GQRKVLFTCF...KRNDKRE GQRKVMFTCF...KRNDKRE GQRKWYYCF...KRNLVHE GQRKVLYGCF...KKNLKSE SQRKIIWALM...RRSGNEA SQRKILWAML...KRHSSEA ARRKILAGGVKCFASN.NRE t gqRkvlyacf...km.kre

KKSARWGDVIG.KYHPHGD HKLASIAGGVADL.GYHHGE VKVAQLAGSVABMSSYHHGE VKVAQLSGSVAEMSAYHHGE TKVSRLAGWASETAYHHGE LKVAQLAPYVSECTAYHHGE AKVAQLSGYISEASAFHHGE AKVAQLSGYISEVSSFHHGE RKVFQFGGWADHMFYHHGD * It .KvaqlagyvaemsayhHGe

SAVYDTIVRMAQPF...SLR T.LHKSQCU.U+NTW..NNNF MSL&fMTIINLAQNFVGSNNL VSLQMTIVNLAQNFVGANNI VSMEQTIVNLAQNFVGSNNI QSLAQTIIGLAQNFVGSNNI TSLQETMIKMAQSFTGGNNV ASLQETIVKMAQNFTGGNNI MSLNTSIIKAAQYYPGSSHL .slq.ti...Aqnfvgsnni

.nll.p.GqFGsr.qggkda

aapRYift.l.sklarllfp

901 DLEKETVDFVDNYIXTEKIP VYKDTEYAPVHQ.DKEHIPP PKDDHTLKFLYD.DNQRVEP PLDDPLLDYQVD.DGQKIEP SNDDQLLNYQND.EGQWIEP PADDPLYKYIQE.DEKTVEP SEDDPLLDYIVE.EGQQVEP EDDDPLLDYIDE.EGTMVEP AEDSWLLPYVFE.lXQRAEP

DVMPTKIPNLLVNGSSGIAV AFYLPIIPTVLLNGVSGIAT EXYIPIIPMVLINGAEGIGT LWYLPIIPMVLVNGAEGIGT EYWPILPMVLVNGAEGIGT EWYLPILPMILVNGAEGIGT NHYVPILPLLLCNGSVGIGF NHYVPILPLLLCNGAVGIGF EYYVPVLPLAIMEYGANPSE

GMATNIPPHNLTEVINGCLA GYATYILPHSVSSVKKAVLQ GWSCKIPNFDVREIVNNIRR GWSTKISNYNPREIMKNLRK GWSTFIPNYNPKDITANLRH GRSTYIPPFNPLEIIKNIRH GFSSNIPPFHRLWSAAVRA GFATNIPSFHPLDVSAAVRA GWKYTTWARQLEDILALVRA

YIDDEDISIEGLMEHIPGPD ALQGKKV............. LMIZEEP............. MINGQEP............. MLNGEPL............. LMNDEEL............. MISGERA............. MIXESA............. YVDKDNPKHELLHYAI....

1000 FPTAAIINGRRGIEEAYRTG . ..TKPKVE....FPEFRGE . ..LP....MLPSYKNFKGT . ..SV....MHPWYKNFLGR . ..EI....MTPWYRGFRGS . ..EQ....MHPWFRGWTGT . ..KSWRRLVPWAVGFCGE . ..KQWRNLVFWAVGFQGT .KHKITILPLRPSNYNFKGH

mingeep.............

. ..k.....m.pwy.gfrgt

DESDKffiMRIVIEVKRDAVG GFGFKVKFRKEYSLSDNE.. EYHTGTTVKFWKMTEEKLA EYHTDTTVRFVISFAPGEFE SHHGEGIVHFNVTLTEAGMK EQHDDNI.KFIITLSPEEMA DYSGANHVDIDVEVAQGAVN DYSGANHIDIDLIVREGSMT DYSSSWIEILVKLKPNSLN

1100 . . . . . . ..EXVLNNLYSQTQ . . . . . . EERHAKIMKDFG . . . . . . ..EAERVGLHKVFK RI......HAEEGGFYRVFK . . . . . . ..EALNESLEVKFK ... . . KTRKIGFYERFK TY......AECESEL....G TW......AECFPDL....A RIVEEFKETEEQDSIENFLR

l

CONS

EcoliA T4-52 Human Drome Schpo Schce Trybr Critfas ASFV CONS

EcoliA T4-52 Human Drome Schpo Schce Trybr Critfas ASFV

l

CONS

EcoliA T4-52 Human Drome Schpo Schce Trybr Critfas ASFV

EcoliA T4-52 Human Drome Schpo Schce Trybr Critfas ASFV CONS

l

l *

.dr..k.itisdfinkel.l

f.l.adn.R.ip.wCGlkp

YMLVLXQGNFGS..IDGDSA .PLLDcQGNFGSR..TVQKA .NLLQPIGQFGTRLHGGKDS .NLLEPRGQFGTRLSGGKDC .NLLMPNGQFGTRSffiGKNA .YLLLPNGAFGTRATGGKDA .NLLVPEGQFGSRQQLGNDH .NLLVPEGQFGSRQQLGNDH YPVFIGIGSFGSRHLGGKDA

900 1 AAMRYTEIRL.AKIAHELMA AASRYIFARV.S...KNFYN ASPRYIFTML.SSLARLLFP ASARYIFTIM.SPLTRLIYH SASRYLNTAL.SPLARVLFN AAARYIYTEL.NKLTRKIFH AAPRYIFTKL.SKVARLLFP AAPRYIFTKL.SRFARLLFP GSPRYISVQLASEFIKTMFP

t

tt

t*

l

l

p.ddplldy..e.dgqrveP

ewyvpilPmvlvngaegigt

1001 RGKVYIRARAEVEVDAKTGR VVEI.DGQYEIRGTYKFTSR IEELAPNQWISGEVAILNS MEYVSDGRYIQTGNIQILSG ITKVAPDRYKISGIINQIGE IEEIEPLRYRMYGRIEQIGD IRRGPEGEFIAVGTYTYCKG VRRGPEKEYIAVGKYTAHRN LKRFGQYYYSYGTYDISWR

ETIIVHEIPYQVNKARLIEK IAELV. ..KEKRVEGISALR TQMHITEIPYKYDREX'YVSK ILDPLENKGFITWDDACGEH TTIEISELPVRTWTQTYKEQ VLEPMLNGTEKTPPLITDYR NRLEISELPVGVWTQNYKEN VLEPLSNGTEKVKGIISEYR NKVEITELPIRFWTQDMKEY LEAGLV.GTEKIRKFIVDYE NVLEITELPARTWTSTIKEY LLI&LS.GNDKIKPWIKCME GRVHVTELFWTCSVEAFREH I......SYLATKDIVNRIA GRLHVSELPWMTSIEAFRSH I......SSLASSDWQRIA NIITITELPLRVPTVAYIES I..... KKSSNRMTFIEEII

ie.v.pgryiivgt....gr

nr.eitElP.r.wt..yke.

il..l..gtektkd.isd.r

dy.gdntvr.w.l..gem.

. . . . . . ..eeee.gly..fk

NLKDIIAAFVRHRREVWRR VYDNVVDLIKDF.VEV..RK KYDTVLDILRDL.FEL..RL RFPTAIDILKEY.YKL..RR KYDSVEDILTEF.YEV..RL KYNSVNEILSEF.YW..RL PLESDLTPVLQWHYDR..RL PIDADLSPVLQWHYDR..RL EFNSYYEILYAW.LPY..RR

TIFELRKARDRAHILEALAV TYVQKRIDNKIKETESAFRL KYYGLRKEWLLGMLGAESAK EYYARRRDFLVGQLTAQADR RTYQRRKEHMVNELEKRFDR EYYQKRKDHMSERLQWEVEK DLYKKRRQRNLTLLEQELAR DLYKRRRTRQIGLLEMDLAR ELYQKRLMREHAVLKLRIIM t eyyqkRkdr..g.leaelar

ALANIDPIIELIR.HAPTPA AFAKAHFIKKVISGEIWQG LNNQARFILEKIDGKIIIEN LSIX)ARFILEKCEKKLWEN FSNQARFIHMIIEGELWSK YSFQVKFIKMIIEKELTVTN EKSTLKFVQHFGAGHIDFAN LQSTRKFVEHFRQGHIDFLA ETAIVRYINESAELNLS.HY

1200 EAKTALVANPWQLGNVAAML KTRKELTEELSKIDMYSSYV KPKKELIKVLIQRGYDSDPV KQRKAMCDELLKRGYRPDPV KKKKDLIVELKEKKPQP... KPRNAIIQELENLGFPR... ATEATLEKVCSKLGLVR... ATDmLTKTCVKLGLVR... EDEKEASRILSEHGFPP...

lsaqarfi.e.ieg..wen

kt.k.likelsklgfv....

l

CONS

. ..d

1101 LQVSFGINMVALHHGQPKIM LIERRSQNITV.INEKGKLQ LQTSLTCNSMVLFDHVGCLK LTTTLSTNQMHAFDQNNCLR LSRTQATSNMIAFDASGRIK LISPISLMNMVAFDPHGKIK LTQRIHINGTV.FSPNGTLS LSQRIYINGTV.FSPDGTLS LRNCLH.SHLNFVKPKGGII * L..r.sin.mv.fdp.gklk

Gwstnippfnpl.i.anvra

*

t kydsvldil.e..yev..Rl

active site tyrosine residue (20,27). Amino acids that have been shown to be involved in ATP binding, in the case off. coligyrase B (24,26) are overlined. The sequences are designated as follows: EcoliB and EcoliA, E. coligyrase B (42,43) and E. co/i gyrase A (44) respectively; T4-39 and T4-52, phage T4 gene 39 protein (46) and phage T4 gene 52 protein (46) respectively; Human (47); Drome, Drosophila melanogasfer (77); Schpo, S. pombe (48); Schce, S. cerevisiae (49); Trybr, T. brucei(50); Critfas, C. fasciculafa (S. G. Pasion, J. C. Hines, R. Aebersold, and D. S. Ray, unpublished data; EMBL accession number X59623); ASFV, ASF virus.

945

SHORT COMMUNICATIONS

EcoliA T4-52 Human Drome

Schpo Schce Trybr Critfas ASFV CONS

EcoliA T4-52 Human DrOme Schpo Schce Trybr Critfas ASFV CONS

EcoliA Human Drome Schpo Schce Trybr Critfas CONS

EcoliA Human Drome Schpo Schce

1201 ERAGDDAARPEWLEPEFGVR .................... KAWK......EAQQKVPDEE KEWQRRIKMEDAEQADEEDE ISKPKKGHLVDLEVENALAE FNKEGKPYYGSPNDEIAEQI .................... . .. .... ...............

CGLYYLTECQAQAILDLRLQ .................... ENEES..........DNEKE EEEEAAPSVSSKAKKEKEVD EEQSGDVSQDEDSD...... NDVKGATSDEEDEESSHEDT .................... ............ . LNHTLIISPEFA

KLTGLEHEKLLDEYKELLW . . . . . . . . . . . . . . DKLVGM TEKSDSVTDSGPPFNYLLDM PEKAFKKLTDVKKFDYLLGM . . . . . . . . . . ..AYNYLLSM ENVINGPEELYGTYEYLLGM . VDDSFDYILRK . . . . . . . ..VDDGYDYILKK SIEELNQKALQGCYTYILSL

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . l.dtydyllgm 1301 ..QGYVKYQP SADINLEDLITQEDWVTLS H......... .VTEYTKDLEEIK/ .SDLWKEDLATFIEELEAVE AKEKQDEQV.......GLPG .EMLWLDDLDALESKLNEVE EKERAEEQGINLKTAKALKG MLEEEQSSRD .KELWLHDLDAFEHAWNKVM DDIQRE.... .KDIWNTDLKAFEVGY.... . . ..QE....FLQRDAEARG .VQLWLGELDQFDRFFQDHE . . . . . . . . . . . . . . . . ..KK .VKMWLTDLDKFDKTFQEYE . . . . . . . . . . . . . . . . ..RV GASVWLEEIDAVEKAIIKGR NTQWKFH/

CONS

piw.l...tk.er..ellkk

1300 ELELVREQFGDKRRTEITAN AKAKKEEN..EYWKTTDV.. RNEKEQEL..lXLKRKSP.. RlYl'KLSEL..ESLRKKTP.. KDEXMAEL..DALIKKTP.. KQEKE?l'EL..ENLLKLSA.. IAETERRI..EALKKTTP.. IKKTQDSI..AVLKQTTP.. MQARLDKV..EQLLQESPFP . eke.el..ealkk.tp

LSEYEAQRRGGKGKSAARIK

EEDFIDRLLVANTHDHILCF

1400 SSRGRVYSMKVYQLPEATRG

.KGGKAKGKK.......TQM QKSASAKGRKVKSMGGGAGA FVNRTKKKPRGKSTGTRKPR .GNVPNKGSKTKGKGKRKLV MVEAILKERRQRS.PPSDLL LIHSIQKEQRPASITGGEEV

AEVLPSPRGQRVIPRITIEM GDVFPDPDGEPVEFKITEEI AIAGSSSSTAVKKEASSESK DDEDYDPSKKNKKSTARKGK PGLQQPR............. PALRQPP.............

KAEAEKKNKKKIKNENTEGS . . . . . . . . . ..IKKMAAAAK PSTTNRK..QQTLLEFAASK KIKLEDKNFERILLE...QK . . . . . . . . . ..LEVEE.AKG . . . . . . . . . . LMLEAPAKG

dp.............

.

. i.le..a..

.vdlwledldafeka..."e

. . . . . . . . . . . . . . . . ..rg

. . . ..akgr..ks...

1401 ARGRPIVNLLPLEQDERITA PQEDGVELEGLKQRLEKKQK VAQAAKEPKKPKEPKEPKVK EPEKSSDINIVKTEDNSHGL LVTKSKAPTKIKKEKTP... GKKFEMRVQVRKY.VPPPTK AASSSYRVHICRYEEPPASK

ILPVTEFEEGVKVFMATANG REFGTKTKKQTTLAFKPIKK KEPKGKQIKAEPDA..SGDE SVEENRISKSPGLDSSDSGK SVSETKTEEEENAPSSTSSS RGAGGRSDGD...GGATAAG RKPEDTYGGALSSGGSTRNV

TVKKTVLTEFNRLRTAGKVA GKKRNPWPDSESDRSSDESN VDEFDAMVEGGSKTSPKAKK SRKRSQSVDSEDAGSKKPVK SIFDIKKEDKDEGELSKISN AAAAVGGRGEKKGPGRAGGV GKRLTGARGAKKKKWRRTR

IKLV........DGDELIGV FDVPPRETEPRRAATKTKFT AWKKEPGEKKPRQKKENGG .KIAASASGRGRKTNKPVAT .KFKKISTIFDKMGSTSATS RP.MVL........DALAKRV TKMSLGTRVAEFAGAQLGRL

. . ..s......k...ep..k

r.petk..k.......t...

..k.....d.....s.k...

.k...........g.k....

1501 FKESSVRAMGCNTTGVRGIR FVPSDASPPKTKTSPKLSNK V..EEVTPRAERPGRRQASK LPSSLKPSTITSTKAS.AKN TKKNQTTAKKTAVKPKLAKK

LGEGDKVVSLIVPRGDGAIL ELKPQKSWSDLEADDVKGS KID.YSSLFSDEEE!XGNVG KGKKASSVKKQSPEDDDDDF PVRKQQKVVELSGESDLEIL

TATQNGYGKRTAVAEXPTKS VPLSSSPPATHFPDETEITN SDDDGNASDDDSP....... IIPGSSSTP........... . . ..DSYTD...........

RATKGVISIKVTERNGLWG PVPKKNVTVKKTAAKSQSST . . . . . KRPAKRGREDE . . . . . . KASSTNAEPPE . . . . . . . ..REDSNKDEDDA

1600 AVQVDDCDQIMMITDAGTLV STTGAKKRAAPKGTKRDPAL SSGGAKKKAPPK........ DSDSPIRKRPTRRAAATVKT IPQRSRRQRSSR..AASVPK

. ... .... ... .. .......

. .... ... .... .......

CONS

EcoliA Human DroIlle Schpo Schce

IAELLRILGSADRLMEVIRE NIFHM...TS.DEAKKLAEE PLWYL...TK.EKKDELCRL SMWML...TE.EKKNELLKQ PLWSL...TY.ERYVELLKK RIWSL...TK.ERYQKLLKQ PITFY...TK.TSFENLLKK PITFY...TK.TSTEKLQAD QARELLIAAK.TRRVEKIKK

1601 RTRVSEISIVGRNTQGVILI NSGVSQKPDPAKTKNRRKRK . . . . . KRRA PIYVDPSFDSMDEPSMQDDS KSYVET.......LELSDDS

RTAEDENVVGLQRVAEPVDE PSTSDDSDSNFEKIVSKAVT VIESDDDDIEID........ FIVDNDEDVD.........D FIEDDEEENQ.........G

EDLIYTIDGSAAEGDDEIAPE VDVDDEPEEE/ SKKSKGESDDFHMDFDSAVA PRAKSVRAKKPIKYLEESDE . . . . ..EDDDDDSDFNC/ . . ..YDESD/ SDVSFNEED/

....................

....................

....................

1500 DLTSGEDEVMLFSAEGKWR MDLDSDEDFSDFDEKTDDED GLKQSKIDFSKAKAKKSDDD TIFSSDDE.........DDL KENTPEQD.........DVA TRLLPRLLF/ LPQLPRLLF/ d............

1685 DDLF/

................

Fig. 4-Continued

As mentioned before, previous results suggested the presence of a topoisomerase II in ASF virions. The finding that coumermycin Al inhibited in vitro transcription by the virion-associated RNA polymerase also suggested an involvement of the topoisomerase activity in RNA synthesis. The predicted molecular weight of the protein encoded by ORF Pl 192R (135,543) agrees rather well with that of the virion protein that binds covalently to DNA (about 155,000) which supports the idea that this protein might be the product of ORF Pl 192R. To investigate this possibility and to initiate biochemical and genetics studies, attempts will be made to express the topoisomerase II gene in bacterial or eukaryotic systems. Since topoisomerase II sequences are highly conserved in different organisms, it may prove to be a use-

ful tool in establishing distant evolutionary relationships. We have reconstructed a possible history of the ASF virus gene by generating a phylogenetic tree relating the topoisomerase II genes from nine organisms (Fig. 5). The tree confirms the expected grouping of the species in metazoa (human, Drosophila), yeast (Saccharomyces, Schizosaccharomyces), protozoa (Trypanosoma, Crithidia), and prokaryotes (E. co/i, T4). The most striking feature in the tree is the branching of the ASF virus sequence before the divergence of the different groups of eukaryotes. This indicates that the ASF virus gene was diverged from an ancestral cellular gene before the divergence of protozoa and metazoa. Although we cannot absolutely rule out the possibility of a more recent gene transfer from other organisms (such as archaebacteria) to an ASF virus ancestor, we

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946 13.8

future, a virus similar to ASF virus will be isolated from unicellular organisms.

Human Drosophila

18.6

ACKNOWLEDGMENTS 4.5

122

T. brucei

44A

- 12.2 C. fasciculata

We thank J. Dopazo and A. von Haeseler for performing the split decomposition analysis of the phylogenetic trees and L. Yuste for expert technical assistance. This work was supported by grants from the Comisi6n Interministerial de Ciencia y Tecnologia. the Consejeria de Agricultura de la Junta de Extremadura, and by an Institutional grant from Fundacidn Ramdn Areces.

REFERENCES 1. VI~UELA, E., Curr. 2.

a.2

I I

E. coli B

3.

11.8

q

I

4. 67.0

T4 -39

5. Genealogic tree relating different topoisomerase II sequences. Topoisomerase II sequences were aligned by the progressive method of Feng and Doolittle (51). The tree in the figure was derived from a distance matrix (52) obtained from the multiple alignment of the amino terminal region of the proteins. The regions aligned were as follows: human, residues 81 to 488; Drosophila melanogaster, residues 62 to 469; S. pombe, residues 72 to 479; S. cerevisiae, residues 60 to 478; T. brucei, residues 55 to 466; C. fasciculata, residues 55 to 468; ASF virus, residues 54 to 480; E. co/i gyrase B, residues 36 to 449; T4 gene 39 protein, residues 48 to 441. Trees obtained using an alignment of the central region of the eukaryotic enzymes and the amino end of the E. co/i gyrase A and T4 gene 52 subunits, or of the amino plus central regions of the eukaryotic topoisomerases, were consistent with that shown in the figure. The trees obtained by the distance matrix method were corroborated using a parsimony method (53). The reliability of the phylqgenetic trees was checked by means of the recently developed split decomposition analysis (54, 55). It demonstrated that no other alternative groupings of the taxa than that described by the tree are significantly supported. The numbers in the figure indicate the genetic distance for each branch. Horizontal lines are proportional to the genetic distance. Vertical lines are of arbitrary length and are introduced to separate the different branches of the tree.

5.

FIG.

favor the interpretation that the branching of ASF virus in the tree reflects the “host-to-virus” gene transfer event. That would imply the existence of an ancestral virus at least as old as the appearance of lower eukaryotes. This conclusion is compatible with the results of previous phylogenetic analysis carried out with thymidine kinase sequences (36,37). Our data support the hypothesis that an ancestor of ASF virus was infecting a unicellular organism, possibly a lower eukaryote, and that the virus acquired the topoisomerase II gene at that time. It is tempting to postulate that, in the

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