VIROLOGY

190, 654-665

Genetic

(1992)

Relationships

MICHEL BUBLOT,* JOHN NICHOLAS,+*

between Bovine Herpesvirus 4 and the Gammaherpesviruses Epstein-Barr Virus and Herpesvirus Saimiri

PATRICK LOMONTE,” ANNE-SOPHIE LEQUARRE,* JENS-CHRISTIAN ALBRECHT,1BERNHARD FLECKENSTEIN,t PAUL-PIERRE PASTORET,” AND ETIENNE THIRY*v’

*Virology Department, Faculty of Veterinary Medicine, Molekulare Virologie der Friedrich-Alexander-Universita’t of Virology, National Institute for Medical Received

University of Lisge, B6, Sart Tilman, B-4000 Lisge, Belgium; tlnstitut fur Klinische und Erlangen-NUrnberg, 7, Loschgestrasse, D-8520 Erlangen, Germany; and *Division Research, The Ridgeway, Mill Hill, London NW7 IAA, United Kingdom January

3 1, 1992;

accepted

June 3, 1992

The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA sequences. Twenty-three genes conserved in at least one of the completely sequenced herpesviruses have been identified and localized. All of these genes encoded amino acid sequences with higher similarity to proteins of the gammaherpesviruses Epstein-Barr virus (EBV) and herpesvirus saimiri (HVS) than to the homologous products of the alphaherpesviruses varicella-zoster virus and herpes simplex virus type 1 or the betaherpesvirus human cytomegalovirus. The genome organization of BHV-4 had also an overall collinearity with that of the gammaherpesviruses EBV and HVS. Furthermore, the BHV-4 genes content and arrangement were more similar to those of HVS than to those of EBV, suggesting that BHV-4 and HVS are evolutionarily more closely related to each other than either are to EBV. BHV-4 DNA sequences were generally deficient in CpG dinucleotide. This CpG deficiency is characteristic of gammaherpesvirus genomes and suggests that the BHV-4 latent genome is extensively methylated. Despite several biological features similar to those of betaherpesviruses, BHV-4 displays the molecular characteristics 0 1992 Academic PWSS. h. of the representative members of the gammaherpesvirinae subfamily.

served genes are clustered in blocks of genes and the arrangement of these blocks is identical for the members of a subfamily but differs from the other subfamilies (Davison and Taylor, 1987; Gompels et a/., 1988; McGeoch, 1989; Kouzarides et al., 1987). Another molecular criterion which can differentiate the three subfamilies is the CpG dinucleotide frequency: there is a global deficiency in CpG dinucleotide in the genomes of gammaherpesviruses, a local deficiency in the major immediate-early genes of betaherpesviruses, and no deficiency at all in alphaherpesviruses genomes. The CpG deficiency is probably the result of methylation of the latent genome (Honess et al., 1989). There is a correlation between these biological and molecular criteria for most herpesviruses. Nevertheless, Marek’s disease virus (MDV), its close relative being the herpesvirus of turkey (HVT), and human herpesvirus 6 (HHV-6), which present biological features of gammaherpesviruses, do not share the molecular characteristics of these viruses. MDV and HVT genames have the same gene content and gene organization as alphaherpesviruses (Buckmaster et a/., 1988) and HHV-6 is closely related to betaherpesviruses (Lawrence el a/., 1990; Josephs et al., 1991; Neipel et a/., 1991; Martin et al., 1991); furthermore, they are not globally CpG-deficient (Honess et a/., 1989; Lawrence et a/., 1990; Martin et a/., 1991). These results show that the classification on the basis of certain biological

INTRQDUCTION Members of the family Herpesviridae are classified, primarily on the basis of biological behavior, into three major subfamilies: the Alphaherpesvirinae, including the neurotropic herpes simplex virus 1 (HSV-1) and 2 (HSV-2) and varicella-zoster virus (VZV); the Betaherpesvirinae, including cytomegaloviruses; and the Gammaherpesvirinae, including the lymphotropic EpsteinBarr virus (EBV) and herpesvirus saimiri (HVS) (Honess and Watson, 1977; Roizman, 1982). Analysis of complete (Baer et a/., 1984; Davison and Scott, 1986; McGeoch et a/., 1988; Chee et al., 1990; J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein and R. W. Honess, submitted) or partial sequences (see for example, Gompels et al., 1988) of several mammalian herpesviruses has allowed the identification of genes common to all these viruses as well as the recognition of genes specific to either a particular virus or virus subfamily. The con-

Sequence data from this article have been deposited EMBUGenBank Data Libraries under Accession Nos. through M90800. ’ To whom reprint requests should be addressed. p Present address: Department of Oncology, Johns School of Medicine, 418 North Bond Street, Baltimore, MD 0042.6822/92

$5.00

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

with the M90768

Hopkins 21231. 654

GENE

ORGANIZATION

properties does not necessarily reflect a true evolutionary relationship. The group of bovine herpesvirus 4 (BHV-4) includes a collection of antigenically related isolates distinct from other bovine herpesviruses. These viruses are distributed worldwide and are isolated from a variety of clinical diseases and even healthy cattle (reviewed in Thiry et a/., 1990, 1992). It is mainly isolated from cattle and, in some African regions, from buffalo (Syncerus cakier) (Rossiter et al., 1989). It has been also occasionally isolated from American bison (Bison bison) (Todd and Storz, 1983) and sheep (Van Opdenbosh et a/., 1986). Feline cell-associated herpesvirus (FeCAHV) isolated from cat (Fabricant et a/., 1971) and herpesvirus aotus type 2 (HVA-2) isolated from owl monkey (Aotus trivirgatus) (Barahona et al., 1973) have been shown to be BHV-4 strains (Kit et al., 1986; Kruger et al., 1989; Bublot et al., 1991 b; Dubuisson et al., 1991). On the basis of some of its biological characteristics, BHV-4 was assigned to be a member of the betaherpesvirinae subfamily (bovine cytomegalovirus) (Storz et a/., 1984); however, recent molecular data have shown a clear relationship with the members of the gammaherpesvirinae subfamily (Honess, 1984; Kit et a/., 1986; Bublot et al., 1990, 1991 a; Van Santen, 1991; Thiry et a/., 1992). The genome structure of BHV-4 (V. Test strain) is similar to that of HVS and consists of a unique coding part (LiDNA) of approximately 108 kb flanked by (G + C)-rich tandem repeats of 2.65 kb called polyrepetitive DNA (prDNA or H-DNA) (Ehlers eta/., 1985; Bublot et a/., 1990). The H-DNA contains potential open reading frames (M. Goltz and H.-J. Buhk, manuscript in preparation) but no RNA derived from these repetitive sequences has been detected (V. L. Van Santen and L.-Y. Chang, unpublished results). In order to determine the genetic relationship of BHV-4 to well characterized representative members of the alpha-, beta-, and gamrnaherpesvirinae subfamilies, an analysis of short nucleotide sequences from cloned restriction fragments distributed across the coding region of the viral genome has been carried out. This analysis has clearly shown a genetic relationship of BHV-4 with gammaherpesviruses EBV and HVS; furthermore, BHV-4 is more closely related to HVS than to EBV. MATERIALS Recombinant

AND

METHODS

plasmids

Several cloned EcoRl fragments from BHV-4 (V. Test strain) DNA have been chosen (Bublot et al., 1990). Some of them have been subcloned (using HindIll or

OF

BHV-4

655

BarnHI) in the phagemid Bluescript pSK+ (Stratagene, La Jolla). Orientation of most of these clones was determined by restriction analysis (Bublot et al., 1990). Other ones were oriented using homology results of sequences from these clones and those from contiguous ones, with genes of well-characterized herpesviruses.

DNA sequencing Nucleotide sequences were determined on dsDNA by the dideoxynucleotide chain termination method (Sanger et al., 1977) using the T7-sequencing Kit (Pharmacia LKB, Uppsala, Sweden). Sequences were obtained from one or both ends of the cloned fragments using appropriate primers recognizing a plasmid sequence and were therefore derived from one strand of the DNA.

Computer

analysis

of sequence

data

The sequence data were compiled using the software package version 7.0-Unix of the University of Wisconsin Genetic Computer Group installed on Ultrix (Devereux et al., 1984). Nucleotide sequences were translated into all six reading frames and resulting amino acid sequences were compared to protein databases (SWISS-PROT, EMBL release 17.0) and to the herpesvirus saimiri amino acid sequences using the FASTA program (Ktuple of 2) (Pearson and Lipman, 1988). Comparisons based on amino acid similarities and identities have been done with the GAP program using a gap weight of 3.0 and a length weight of 0.1 (Devereux et al., 1984). Multiple alignments have been done using the program PILEUP (gap weight of 3.0 and length weight of 0.1) (Feng and Doolittle, 1987; Higgins and Sharp, 1989).

Dinucleotide

frequencies

analysis

Observed frequencies (0) of dinucleotides were counted and expected frequencies (e) of dinucleotides were calculated from observed mononucleotide frequencies. Deviations of the observed from the expected frequencies of dinucleotides were first displayed as o/e when o > e or as e/o when e > o (Fig. 4a). In order to examine the correlation between the deficiency of CpG dinucleotide and the excess of TpG and CpA dinucleotides, the absolute magnitudes of the differences o - e were computed (Fig. 4b) (Honess et al,, 1989).

656

BUBLOT

ET AL.

EOORI map:

O5

35

31

FIG. 1. Localization

of sequenced

regions

RESULTS 1. Localization

of sequenced

regions

Thirty-three sequences of the coding part (L-DNA) of BHV-4 (V. Test strain) genome were obtained; they are localized on the EcoRl restriction map in Fig. 1. The total length of the sequenced regions was 9569 bp (about 9% of the L-DNA) with a (G + C) content of

40.3%. 2. Identification

of conserved

kbp

genes

Homologues of 27 translated sequences were present in one of the completely sequenced mammalian herpesviruses (EBV, VZV, HSV-1, HCMV, and HVS). The similarity was always greater to EBV and HVS genes than to homologous products of alphaherpesvirusesVZVand HSV-1 or betaherpesvirus human cytomegalovirus (HCMV). Twenty-three HVS homologues were identified (Table 1). Nineteen of these HVS homologues belong to blocks of genes which are conserved among gamma- and alphaherpesviruses and/or betaherpesviruses. Five BHV-4 sequences (21,22,23,30, and 31) were found to be homologous to gammaherpesvirus-specific genes. The BHV-4 sequences 21,22, and 23 were homologous to the products of BRRF2, BRRFI, and BLRF2 of EBV (Baer et al., 1984) and those of HVS 48, 49, and 52 (J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, 8. Fleckenstein and R. W. Honess, submitted) (corresponding to EDLF5, EDLF4, and EDLFS, respectively, in Nicholas eta/., 199213) (Table 1). These genes are not conserved in the alphaand betaherpesviruses. The two other sequences (30 and 31; Table 1) were homologous to the EBV 140,000-M, membrane antigen encoded by BNRFl. This gene is conserved in the gammaherpesviruses HVS (gene 75 or ElLFl in Nicholas eta/., 199213) (Cameron et a/., 1987) and murine herpesvirus 68 (Efstathiou et a/., 1990b) but no related gene has been identified in the alpha- and betaherpesviruses. The BHV-4 sequences 30 and 3 1 were also homologous to the HVS gene 3, a second BNRFl homologue in the HVS genome (J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann,

on BHV4

(V. Test strain)

genome

(EcoRI

33

map).

M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess, submitted) (data not shown). The BHV-4 amino acid sequences were therefore more closely related to that of the gammaherpesviruses than that of the alpha- or betaherpesviruses.

3. Homology sequences

between

BHV-4, HVS, and EBV

Figure 2 shows alignments of BHV-4 amino acid sequences with those of homologous products of several herpesviruses. BHV-4 sequence 14a was homologous to the N-terminal part of spliced gene exon 2 which is highly conserved in all herpesviruses subfamilies (Fig. 2a); the DNA sequence of the predicted splice acceptor site was also very well conserved (data not shown) (Davison and Scott, 1986; Lawrence et al., 1990). BHV-4 sequence 6 was homologous to a part of the gene coding for the HSV-1 glycoprotein B which is also conserved in all herpesviruses subfamilies. This 59amino-acid BHV-4 sequence contains three potential IV-glycosylation sites; two sites are conserved only with HVS and the third one is conserved with the two gammaherpesviruses as well as with alphaherpesviruses HSV-I and VZV (Fig. 2b). Alignment of BHV-4 sequence 25, that is homologous to a part of the small subunit of ribonucleotide reductase, is shown in Fig. 2c; the betaherpesvirus HCMV genome does not encode this small subunit, although it encodes the large subunit of this enzyme (Chee et al., 1990). Figure 2d shows alignment of BHV4 sequence 14b which was found to be homologous to the gammaherpesviruses HVS gene 26 and EBV BDLF3.5 and the betaherpesvirus HCMV UL91 but not to any alphaherpesvirus gene: EBV BDLF3.5 is a new EBV open reading frame located between BDLF3 and BDLF4 and discovered by analysis of the complete sequence of HVS (J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess, submitted). Alignment of BHV-4 sequence 30 with that of the membrane antigen (HVS 75 and EBV BNRFl genes product), which is a gammaherpesvirusspecific gene, is also shown (Fig. 2e). Table 1 indicates the percentage of identical and similar amino acids between the homologous sequences of BHV-4, HVS, and EBV. The most con-

GENE

ORGANIZATION TABLE

OBSERVED HOMOLOGY

BHV4 sequence no.8

No. of amino acids of homology

(IDENTIIY AND SIMILARIT/)

Gammaherpesviruses homologous genes HVSb

EBV

2 3 4 5a 5b 6 7 10 11 12 13a 13b 14a 14b 15 16 17 18 19 20a 20b 21 22 23 24 25

108 61 103 47 12 59 77 87 90 114 156 66 126 59 58 97 86 33 139 34 140 116 63 29 43 82

6 6 6 6 7 8 9 19 19 25 25 26 29b 30 29a 37 40 40 44 46 47 48 49 52 56 60

BALF2 BALF2 BALF2 BALF2 BALF3 BALF4 BALFS BVRFl BVRFl BcLFl BcLFl BDLFl BDRFl BDLF3.5 BGRFl BGLF5 BBLF2 BBLF2 BBLF4 BKRF3 BKRF2 BRRF2 BRRFl BLRF2 BSLFl BaRFl

26

91

61

BORF2

27 28 30 31

73 80 120 94

64 64 75 75

BPLFl BPLFl BNRFl BNRFl

OF

657

BHV-4

1

OF BHV-4 (E?) PREDICTED AMINO ACID SEQUENCES AND THOSE OF THE GAMMAHERPESVIRUSES HVS (H) AND EBV (E) USING THE PROGRAM GAP

Subfamilies in which the gene is conservedC

Percentage identity Gene

function/commentsd

Major DNA-binding Major DNA-binding Major DNA-binding Major DNA-binding Transport protein HSV-1 glycoprotein DNA polymerase Virion protein Virion protein Major capsid protein Major capsid protein

protein protein protein protein

Spliced

2

gene exon

Percentage similarity

of

B-H

B-E

H-E

B-H

B-E

H-E

small

52.8 56.7 62.1 40.4 75.0 64.4 75.3 47.1 66.7 62.3 71.8 56.1 80.2 39.0 62.1 36.3 32.9 21.2 69.1 39.4 31.8 28.9 28.6 34.5 41.9 64.6

42.6 44.8 39.2 25.5 66.7 44.1 64.9 41.4 58.9 57.9 62.2 53.1 73.0 18.6 39.7 28.8 19.8 35.5 55.1 31.3 30.5 20.9 27.0 41.4 27.9 61.0

39.8 37.9 31.4 19.6 58.3 40.7 68.8 43.5 52.2 57.9 66.0 50.0 70.6 25.9 43.1 26.3 16.3 20.7 57.2 31.3 22.8 18.8 23.8 31.0 34.9 65.9

74.1 75.0 78.6 57.4 83.3 72.9 79.2 63.5 78.9 78.9 89.1 75.8 91.3 61.0 72.4 64.8 50.6 45.5 81.3 54.5 60.5 54.4 49.2 62.1 65.1 78.0

68.5 65.5 64.7 55.3 66.7 64.4 72.7 60.9 73.3 70.2 84.0 71.9 86.5 51.4 62.1 52.5 41.9 48.4 68.8 59.4 54.7 46.1 47.6 62.1 48.8 76.8

67.6 65.5 59.8 45.7 66.7 62.7 76.6 57.6 68.9 72.8 82.7 67.2 88.9 51.7 63.8 53.8 38.4 44.8 68.1 65.6 52.8 46.4 55.6 51.7 55.8 75.6

large

47.3

39.3

44.0

64.8

54.8

63.1

23.6 37.5 48.1 33.0

22.9 26.6 29.0 21.3

19.4 21.5 29.8 24.5

50.0 61.3 71.7 53.2

47.1 49.4 45.8 40.4

40.3 46.8 47.1 46.8

B

Spliced gene exon 1 Deoxyribonuclease (HSV-1 ori-binding protein) (HSV-1 ori-binding protein) Helicase Uracil-DNA glycosylase

DNA replication Ribonucleotide reductase subunit Ribonucleotide reductase subunit Large tegument protein Large tegument protein Membrane antigen Membrane antigen

of

Note. Gap weight, 3.00; length weight, 0.10. a A BHV-4 sequence number followed by “a” or “b” means that this sequence contains part of two adjacent open reading frames; sequences designated “a” are positioned on the left of the corresponding ones designated “b” in the genome oriented as in Fig. 1. ’ Nomenclature of HVS genes (1 to 75) is that used in J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess (submitted); alternative names of genes 48,49, 52, 56, 60, 61, 64, and 75 (EDLF5, EDLF4, EDLFB, EDRF4. EELFB, EELF2, EERF2, and ElLFl , respectively) have been used in Nicholas et al. (1992b). ’ Subfamily in parentheses means that the potential homologous gene in this subfamily is only positionally conserved. d Most of the gene functions and comments come from data concerning HSV-1 homologues.

served sequence found in this study was sequence 14a (126 aa) which was homologous (80.2% identity with HVS 29b) to the N-terminal part of exon 2 of the spliced gene (see also Fig. 2a). The homology of most BHV-4 sequences was greater to the homologous products of HVS than to the equivalent genes of EBV. The homology between HVS and EBV sequences was

generally similar to that measured between BHV-4 and EBV. These results indicate that BHV-4 sequences are more closely related to that of HVS than to that of EBV. 4. Localization

of conserved

genes

The analysis of the complete sequence of HVS has recently allowed the identification of genes which are

BUBLOT ET AL.

658

a BEIY-4

10 20 30 40 SIRGQTFNILWDEANFIFX.EALPAILGFMLQKDARIIFISSVNSADKSTSFLFN~NAR

EYS EBY EICMY E8Y-6 my-1 YZV

SIRGOTPNLLYIDEANFIKKDSLPAILGFMLQKD~LIFISSVNSGDRATSFLPNLKNAS SIR~TFELLFVDEANFIKKEALPAILGFMLQKDAKIIFISSVNSADQATSFLYKLKDAQ SIRGQNFBLLLVDEABFIKKEAFNTILGFLAQNTTKIIFISSTNTTSDSTCFL~NNAP SIRGQSFNLLIYDESEFIID~STILGFLPQASTRILLNNSP GIRGQDFNLLFVDEANFIRPDAYQTIHGFLNQANCKIIFVSSTNTGKASTSFLYNLRGAA GIRGQDFNLLRrDEANFIRPDAV~IYGFLNPTNCKII~SSTNT~STSFLYN~GSS

50

60

IlIIIIII:II:IIIIIIII::IIIIIIIIIIIIII:IIIIIIII:I::IlIIlII:Il:

l ***

l

l

l *

tt

t

**

*

l

l

*II

t



SW-4

70 80 90 100 EKMLNVVNYVCPEEKEDFNLQSTLTSCPCYRLBIPTYITIDESIKNTTNLFLDDVFT~L

EYS EBV EICMY KEY-6 ESY-1 Y&Y

EKMLNIVNYICPDEKDDFSLQDSLISCPCYLYIPTYITIDETIKNTTNLFLD~T~L ERLLNWSWCOEERODFDMODSMVSCPCPC~EIPSYITMDSNI~TTNLFLDGAFSTEL FDMLNWSWCEEELESFTEKGDATACPCYRLBKPTFISLNSQVRKTANMFMPGAFMDEI FEHLSWSWCEDEABMLNERGNATACSCYRLEKPKFISINAEVKKTANLFLEGAFIEEI DELLNWTYICDDEMPRWTETNATACSCYILKRPVFITnDGI DQLLNWTYVCDDEHPRVLABSDYTACSCYVLNKPYFITMDGAl4RRTADLFMADSFVQEI l

BEY-4

MGDIST

BYS EBY BCMY

NGDMSG MGDTSS IGGTNK MGGATC IGGQAR VGGRXQ *

RSY-1 VZY

b BBV-4

C BEV-4

**

IIl:l:

l

l

*

l

l

t

:I1 l::Il:I

I:I:

BEV-4 BYS EBY ECMY

l

l

t

50

:/IIIIIIIII:IIIIII:IIIIl:IIIlI::III:

10 20 VNGVCLANDYISRDELLBTRAAALL

:562-620) 1593-651) (615-673) [ 638-696) 1587-645) l

*

30 40 50 YNTMVSGADKPEIILWVBNLFKKAVEVKREFILVKS

I:::Il:IlIIlI

60

:/I

:I:

MricI~~biriARo~rae~S~~~~~SS~R~SED~I~~~~~~~Fi~G MPGICLANNYISRDELLETRAASLLYNSMTAKADRPRATWIQELFRTAVKVXTAFIEARG FWTCQFNDLISRDEAIBTSASCCIYNNYVP--EKPAITRIEQLFSEAVEIECAFLKSBA LRVTCQSNDLISRDEAYETTASCYIYNNYLGPPPDRYY~~~~IEIGFIRSQA t

t

.**

tt

t

70 SG----YSAVNVDDIRSFLCATADRI

BBY-4

d

l

ETFFTAK~YBFKNYVBVETLPVN~TLDTFLAL~FIENIDFFAVELYSSGERKL QYYFQSGNEIBVYNDYBBFKTIELDGIATLQTFISLNTS LKIFIAGNSAYEYVDYLFKRMIDLSSISTVDSMIALDIDPLENTDFRVIXLYSQKELRS RRYFTFGGGYVYFKEYAYSBQLSRADITTVSTFIDL~KLaEDEEE%'PLEYYTREEIKD KRYFLFGBBYVYYEDYRYVREIAVBDYGMISTYVDL~LLKDREFMPLQYYTRDELRD t t l *

l

EVS EBY YZY ESV-1

120

(304-429) 1326-4411 (‘297-422) (293-418) (344-469) (353-478)

:ll:illllll:IIl:lil:lllllllll::::::I EVS EBY YZV ESY-1

110

40 10 20 30 RSLFFVK~EYFKDYKFVKTMDTN~TLDTFLTL~FIDNIDFKTVKLYSETEP.KM

::I EYS EBV BCMV ESY-1 VZY

l

IIIII:III:II:II:II:II::I:IIIIl:I:IIIIIIIII:IIIIIIIIII::IIIII

l

HRV-6

l *

I

II lIl::II

l

*

l

t*

ttt

l

t

80

Il:llIIII

YG----VSLVNVWIRQFLQATADRI EG----YTLVDVRAIKQFLKATADRI PK----l'RLVNVDAITQYVKFSADRL PTDSEILSPAALAAIENYVRFSADRL *

(185-266) (174-255) (179-258) (207-292)

*tt

10 30 40 50 20 MDSGSLTEKDFSDCKBFFSQP~RLIDD~S~NDI-DL~~IEN~~SLLLDLVG~ I :: t:l:Il:Il: Il::II I::: l::l::l I: :I: :::::I:::lII:III kSKPCIS~R~~?rb~QAiP~~PI~Y~I~KS~i-DW KKRGTIGBREFGELLSWDPTDLPRTVARVYVAVGGLFKQEYSEYQRLKNICTLLDLAGVB MNSLLARLNRLGVABATTKDVFIFVDRLFQBFSFLFQAEESGPRRXXLYASVFEBLTVS

(l-59) (12-71) (l-59) l

l

e EVS BEY-4: EBV

EVS BBY-4: EBV

10 20 30 40 50 P~~~~~.~T.~TP~AS~~---RDTPTPDTPIQDTD~~~~~~~--N~TAS~YTS-IERITPA -..-- --_ ..-. - ----------. I:1 I:I(ItII:I:IIII II:) l:I(Il:I I::1 --I,-,~,,,, :: IIGLSFTSASDSIPMGE~SMDPTAMDLGIPTFINTPNF EFCRDL1mmm~

:

: ::I:::1

:I:I

I::I

:::

I

ASDY~GLcvKLTFGsAs--CPETGSSASNF~---------~~V~~~FSGPLITPV t * l

l:l:Il:

**

l

l *

t*

*

:

l t

70 90 100 80 LKXAENALYBVCLSKELTLSGSVnNSPThPSSHLPDLDTSKUtDMFYAVKBLISKNLY :::II::II:II:II: )I: : III:::I::I::II I(:::::l: LKMDGSSLICLSISKQVTLAGSTFKBIFTEQIE~PDYSSSQIRNLFYLVKKLMSENLI ):::II 11 :: :: : :I1 Il::l:: :: :: :t ::I11 LQRTGSLLIAYRCGDGRIQGGSLFBQLFSDVATTPRAPBALS~NLPaAVPQLVXSGIY l

60 ::l:Il:

:I\ l *

110 (768-875) Il:l:l:Il: I::I:::::: 1

t

(770-878)

l

FIG. 2. Amino acid comparisons between BHV-4 sequences and parts of homologous products of other herpesviruses: (a) BHV-4 sequence l4a and the N-terminal parts of the spliced genes exons 2 (conserved in alpha-, beta-, and gammaherpesviruses), (b) BHV-4 sequence 6 and genes coding for HSV-1 glycoprotein B homologues (conserved in alpha-, beta-, and gammaherpesviruses), (c) BHV-4 sequence 25 and

GENE

ORGANIZATION TABLE

COMPARISON

OF THE ESTIMATED LENGTH OF BHV-4 Gene

Genomic Left end Block 1 Block 1 Block 2 Block 2 Block 3 Block 3 Block 4 Block 4 Block 5 Block 5

regions to block

1

to block

2

to block

3

to block

4

to block

5

OF

BHV-4

659

2

GENE BLOCKS WITH THOSE OF GAMMAHERPESVIRUSES

Length

content

HVS*

EBV”

HVS

1 to 5 6 to 9 10 to 16 17to47

BNLFl a to BALFl BALF2 to BALF5 BlLFl to BdRFl BVRF2 to BKRF2 BKRFl BRRF2 to BRLFl BZLFl to BLLFla BLRF2 to BFLF2 BHLFl to BCRFl BNRFl

12.6 R, V 11.0 7.3 36.5 ':P,,.

>,,I ,,,, ',. 1.3

,I.

,.,,

I

I.. ., i:

:4

,

I I I I

m

El

T6

STP-A HSU-RNAS DHFR CCPH

GCR

cyclin

CD59

I33

IR4

EBV

I 0

I

I

I

10

20

30

I I 40

I I 50

I

I

I

60

70

60

I I 90

I 1 100

I I 110

I 120

I I 130

I I 140

I I 150

I I 160

I 170 kb

FIG. 3. Comparison of the order and spacing of gene blocks on the mature, linear genome of BHV-4 (V. Test strain), herpesvirus saimiri (HVS, strain No. 11) (Nicholas eta/., 1992b; J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess, submitted) and Epstein-Barr virus (EBV, 695-8 strain) (Baer et al., 1984). The 5 gene blocks, which are conserved within gammaherpesviruses, are indicated by large shaded rectangles. Gene content of each block is reported in Table 2. Genes conserved with beta- and alphaherpesviruses are located in blocks 1, 2, and 4; blocks 3 and 5 include only gammaherpesvirus-specific genes. Also shown as horizontal arrows are the positions of the major immediate-early gene of BHV-4 (IEl) (Van Santen, 1991) and HVS (IE-G) (Nicholas era/., 1990). Vertical arrows indicated important genes located outside of gene blocks and which are not conserved between EBV and HVS; most commonly investigated genes are marked by their abbreviations (STP-A, saimiri transformation-associated protein; HSU-RNAs, herpesvirus saimiri U-RNAs; DHFR, dihydrofolate reductase; CCPH, complement control protein homologue; CD 59, cluster designation 59 homologue; TS, thymidylate synthase; GCR, G protein-coupled receptor homologue; LMP, latent membrane protein; EBNA-2, -3A, B, C, -LP, EBV nuclear antigen 2, 3A, 3B, 3C, leader protein; bcl-2, bcl-2 proto-oncogene homologue; IL1 0, interleukin 10 homologue). EBV origins of replication are indicated by triangles termed “orilyt” (origin of replication for the lytic cycle) and “ori,,” (plasmid origin of replication); terminal repeats (TR) and large internal repeats (IR) are represented by open rectangles; small internal repeated sequences are indicated by vertical lines or black rectangles. Vl , V2, V3, and V4 are genomic regions which vary in size between BHV-4 isolates (Thiry et a/., 1992); the Vl region, at least, contains tandem repeats (M. Bublot, unpublished results). The orientation of the EBV genome is inverted relative to the conventional orientation (Baer et a/., 1984).

length of HVS genome from the region homologous to the rightmost BHV-4 sequence to the right end of the block. Lengths of BHV-4 blocks 3 and 5 have been also estimated by analysis of BHV-4 (DN 599 strain) sequences kindly provided by V. Van Santen and G. Keil. The results are reported in Table 2. Lengths of blocks 1 and 5 were similar for BHV-4, HVS, and EBV; EBV block 2 and especially block 4 were longer than those of BHV-4 and HVS; and HVS block 3 longer than those of BHV-4 and EBV. This bigger size of HVS block 3 is due to the presence of a repeat in gene 48 which causes an expansion of the acidic C-terminal domain of this BRRF2 homologue (J. C. Albrecht, J. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein, and R. W. Honess, submitted) (Fig. 3; Table 2). The space between, BHV-4 gene blocks was also more similar to that of HVS than that of EBV. It was very obvious for that between blocks 3 and 4 and that between blocks 4 and 5 which are rich in tandem repeats in EBV (Table 2; Fig. 3). The EBV genomic region located between blocks 2 and 3 contains the BKRFl

gene coding for EBNA-1 (Baer eta/,, 1984); homologue of this gene has not been found in HVS nor in BHV-4 genome (A.-S. Lequarre and M. Bublot, unpublished results). The intervals between ends of the unique portion of the genome and the leftmost and the rightmost block vary widely between these three viruses (Table 2). The gene arrangement of BHV-4 is therefore also more similar to that of HVS than to that of EBV. 5. Dinucleotide

frequencies

analysis

Deviations of observed from expected frequencies of dinucleotides in BHV-4 sequences are presented in Fig. 4a. In total, there was an observed sixfold decrease in CpG dinucleotide frequency. An increase in TpG and CpA dinucleotides was also detected. The relationship between deviations of observed from expected frequencies of CpG and the sum of deviations of observed from expected frequencies of TpG and CpA are presented in Fig. 4b. While the correlation between CpG deficit and TpG + CpA excess was rela-

GENE

a

oe

I

OF

o/e

AG ATCA ccCGCTGAGcaGTTA TCTG,

e-I 765432123456 Deviations

-6 of observed (0) from expected frequencies of dinucleotides

(e)

-7 Deviations

-6 -5 of observed

-4

-3 -2 from expected

-1 0 frequencies

1 of CpG

2

FIG. 4. (a) Deviations of observed (0) from expected (e) frequencies of dinucleotides in BHV-4 sequences. Filled bars indicate the magnitude of deviations of observed occurrence of each dinucleotide from occurrence expected in random DNA sequences with the observed mononucleotide compositions [for o c e, e/o = fold deficit (left side) and for o > e, o/e = fold excess (right side)]. Arrows indicate the excess of CpA and TpG dinucleotides. (b) Relationships between deviations of observed (0) from expected (e) frequencies of CpG [CpG(%), - CpG(%),] (abscissa) and the sum of deviations of observed from expected frequencies of TpG and CpA { [TpG(%), - TpG(%),] + [CpA(o/,& - CPA(%),]} (ordinate) in the 33 sequences (filled squares). Dots of the line correspond to an exact correlation between CpG deficiency and TpG + CpA excess. The open square is the mean value for the 33 sequences.

tively weak for individual sequences, it was strong on the average (open square in Fig. 4b). This weak correlation for individual sequences was probably due to their short length allowing greater variations between expected and observed values. This deficiency in CpG is likely to be the result of methylation of viral DNA (Bird, 1980). DISCUSSION This study has shown that the BHV-4 presents the molecular characteristics of the representative members of the gammaherpesvirinae subfamily. Indeed, the BHV-4 genes content and organization were similar to those of EBV and HVS and the BHV-4 coding sequence was globally deficient in CpG dinucleotide. The CpG deficiency suggests that BHV-4 latent genome is methylated and that it is present in dividing cells capable of de nova methylation (Honess et al., 1989). The latency site of BHV-4 has not been characterized yet; trigeminal ganglia (Homan and Easterday, 1981; Krogman and McAdaragh; 1982; Castrucci et a/., 1987) and spleen (Osorio and Reed, 1983; Osorio eta/., 1982, 1985) have been proposed to harbor latent BHV-4. The CpG deficit in the BHV-4 genome suggests that the neuronal cells of the trigeminal ganglia are not likely to be the main site of latency because these differentiated nondividing cells are unable to methylate DNA (Honess et a/., 1989). The spleen, especially dividing cells, could be a potential latency site of BHV-4 as could be cells of the basal layer of an epithelium or

stem cells of the bone marrow. Alternatively, it is also possible that latently infected cells are immortalized by BHV-4 but such immortalization has never been described in viva or in vitro. The CpG deficiency suggests also that the BHV-4 latent DNA is replicated (Honess et al., 1989); it could persist as an episome like EBV (Kieff et al., 1983) or HVS (Kaschka-Dierich et a/., 1982). BHV-4 is genetically related to the gammaherpesviruses HVS and EBV. Furthermore, several data indicate that relationship of BHV-4 with HVS is stronger than with EBV: (1) BHV-4 (Ehlers et a/., 1985; Bublot et a/., 1990; this study) and HVS (Fleckenstein et al., 1975; Bornkamm eta/., 1976) possess the same genomic structure and a similar (G + C) content differing from those of EBV (Baer et al., 1984); (2) BHV-4 amino acid sequences are more similar to those of HVS; (3) the length of conserved gene blocks and the space between these blocks are more similar between BHV-4 and HVS than between BHV-4 (or HVS) and EBV. Another finding that underlines the different degrees of relatedness between, on the one hand, BHV-4 and HVS and, on the other hand, EBV is the presence of a major immediate-early gene between block 1 and 2 in BHV-4 (Van Santen, 1991) and HVS (Nicholas et al., 1990) genome but not in EBV genome (Baer et a/., 1984). The two genes are transcribed from similar genomic regions and in identical directions, giving the most abundant immediate-early RNA (Fig. 3); these RNAs are spliced but the potential open reading frame (ORF) of HVS is located in one exon (Nicholas et a/., 1990; L. S. Coles and R. W. Honess, unpublished re-

662

BUBLOT

sults) and that of BHV-4 in four exons (Van Santen, 1991). These two potential ORFs are not homologous at the amino acid sequence level. Whether they play the same role in the biology of infection is not known. All these results suggest that, in the gammaherpesvirinse subfamily, HVS and BHV-4 are evolutionarily more closely related to each other than either are to EBV. Consistent with this division of thegammaherpesvirinae subfamily into two genetically distinct subgroups, is the observation of Albrecht and Fleckenstein (1990) who have shown that EBV (gamma, prototype) and HVS (gamma, prototype) (Honess, 1984) were more distinct from each other than VZV and HSVI, the prototypes of alpha, and alpha, subgroups, respectively. The genomic structure of HVS is also found in other herpesviruses: herpesvirus (h.) ateles (Fleckenstein et al., 1978) herpesvirus sylvilagus (Medveczky et al., 1989) murine herpesvirus 68 (MHV 68) (Efstathiou et al., 1990a) and alcelaphinae herpesvirus 1 (Bridgen et al., 1989). The genetic content has been partially studied for h. ateles (Richter et al., 1988) and MHV 68 (Efstathiou et a/., 1990b). These two viruses seem to be also more closely related to HVS than to EBV. All these viruses having the same genomic structure are able to grow in fibroblastoid cell lines unlike EBV. Further studies should be done on these viruses to confirm their closer genetic relationships to HVS and, therefore, their belonging to a gammaherpesvirus subgroup evolutionarily distinct from EBV. Six BHV-4 sequences were not found to be homologous to any HVS or EBV genes. These sequences could contain parts of BHV-4 specific genes but they could as well be parts of noncoding regions of the BHV-4 genome or parts of nonconserved domains of conserved proteins. All these sequences were located outside of conserved gene blocks. These nonconserved regions between HVS and EBV genomes contain genes coding for proteins playing an important role in the pathobiology of these viruses. EBV genes coding for the latently expressed proteins such as EBNAs and latent membrane proteins (LMP) as well as the BZLFl gene coding for EBV transactivator ZEBRA and BLLFl coding for the major glycoprotein complex (gp 350/ 220) recognizing EBV cellular receptor CD 21 have no counterpart in HVS (Baer et al., 1984; Kieff and Liebowitz, 1990; Nicholas et al., 199213; J. C. Albrecht, I. Nicholas, D. Biller, K. R. Cameron, B. Biesinger, C. Newman, S. Wittmann, M. A. Craxton, H. Coleman, B. Fleckenstein and R. W. Honess, submitted). Genes involved in lymphocyte immortalization by EBV (those coding for EBNA-2 and LMP and BARFl) (Kieff and Liebowitz, 1990; Wei and Ooka, 1989) or by HVS (HVS gene 1 coding for the saimiri transformation-asso-

ET AL

ciated protein) (Murthy et al., 1989) are also not conserved within these gammaherpesviruses. Furthermore, genes homologous to cellular genes are located in these nonconserved regions too; these genes are EBV BCRFl (IL-10 homologue; Moore et a/., 1990), EBV BHRFl (bcl-2 homologue; Cleary et al., 1986), HVS gene 2 (dihydrofolate reductase homologue; Trimble et al., 1988) HVS gene 4 (complement control protein homologue; J.-C. Albrecht and B. Fleckenstein, submitted), HVS gene 15 (Human CD59 homologue; J. C. Albrecht, J. Nicholas, K. R. Cameron, C. Newman, B. Fleckenstein, and R. W. Honess, submitted), HVS gene 70 (ECLF4; thymidylate synthase homologue; Bodemer et a/., 1986; Honess et al., 1986), HVS gene 72 (ECLF2; cyclin homologue; Nicholas et al., 1992a), and HVS gene 74 (ECRF3; protein G-coupled receptor; Nicholas et al., 1992a). HVS U-RNAs (Lee et al., 1988; Wassarman et al., 1989; Albrecht and Fleckenstein, 1992) and EBV EBER RNAs (Baer et al., 1984) are located between a conserved block and one end of the coding part of the genome and are not conserved (Fig. 3). Genes involved in important biological properties such as latency, immortalization, lytic-cycle transactivation, and other virus-host interactions are therefore not conserved between EBV and HVS. The available space for such genes in the BHV-4 genome is roughly similar to that of HVS, and therefore it is likely that these BHV-4 nonconserved regions contain genes involved in the specific biology of this virus. Further sequence analysis of these regions will assess if HVS genes having no counterpart in EBV are conserved in BHV-4 genome, if such regions contain ORFs homologous to cellular genes and therefore if there are preferential sites for insertion of cellular genes into such viral genomes. Gammaherpesviruses are lymphotropic (Honess and Watson, 1977; Roizman, 1982; Honess, 1984). Nevertheless, BHV-4 infection has some biological characteristics common to members of the Betaherpesvirinae subfamily (Storz et al., 1984) and the lymphotropic viruses MDV, HVT, and HHV-6 have a gene organization different from that of EBV and HVS. These results show that there is no relationship between lymphotropism and the overall gene organization of herpesviruses and therefore this biological property cannot be used to identify genetically related herpesviruses. Members of the gammaherpesvirinae subfamily are divided, on the basis of biological behavior, into two subgroups: the gamma,-herpesvirinae subgroup (including EBV and related viruses of Old World monkeys) and the gamma,-herpesvirinae subgroup (including viruses of the New World monkeys HVS and h. ateles). The gamma,-herpesviruses are typically associated with B-lymphocytes and the gamma,-herpesvi-

GENE

ORGANIZATION

ruses with T-lymphocytes (Honess, 1984). MHV-68 which has a genome more similar to that of HVS (Efstathiou eta/., 1990b) seems to persist in B-cells (N. P. Sun+Chandra, S. Efstathiou, and A. A. Nash, unpublished results) and BHV-4 in macrophages at least in the rabbit model of infection (Osorio et al., 1985). Therefore, neither T-cell or B-cell lymphotropism seem to be related to the general gene organization of herpesviruses. The genomes of EBV, HVS, MHV-68, and BHV-4 have similar gene organization and are all deficient in CpG dinucleotides (Baer et a/., 1984; Honess et a/., 1989; Efstathiou et a/., 1990b; Albrecht and Fleckenstein, 1990). They all possess tandem repeats at both ends (Fleckenstein eta/., 1975; Bornkamm eta/., 1976; Given et a/,, 1979; Ehlers et al., 1985; Efstathiou et a/., 1990a). Additional studies, especially on the molecular control of latency, will be necessary to assess the relationships between these common molecular characteristics and the biological properties of gammaherpesviruses.

ACKNOWLEDGMENTS We thank Marc Collet and Robert Herzog of the Free University of Brussels (Department of Molecular Biology) for their help in the computer analysis. We thank also Vicky van Santen and Gunther Keil for providing us their BHV-4 (DN 599 strain) sequences, Brigitte Biesinger and Doris Biller their HVS sequences, and Marie-FranCoise Van Bressem its BHV4 (V. Test strain) sequences. The text presents research results of the Belgian National incentive program on fundamental research in life sciences initiated by the Belgian State, the Prime Minister’s Office Science Policy Program and was in part supported by the Deutsche Forschungsgemeinschaft, Forschergruppe “DNA-Viren des haematopoetischen Systems.” Scientific responsibility is assumed by the authors. We also thank Marinette Muys for the patient application of her secretarial skills.

REFERENCES ALBRECHT, J. C., and FLECKENSTEIN, B. (1990). Structural organization of the conserved gene block of herpesvirus saimiri coding for DNA polymerase, glycoprotein B, and major DNA binding protein. Virology 174, 533-542. ALBRECHT, J. C., and FLECKENSTEIN, B. (1992). Nucleotide sequence of HSURG and HSUR7, two small RNAs of herpesvirus saimiri. Nucleic Acids Res. 20 (in press). BAER, R., BANKIER, A. T., BIGGIN, M. D., DEININGER, P. L., FARRELL, P. J.. GIBSON, T. J., HATFULL, G., HUDSON, G. S., SATCHWELL, S. C., SEGUIN, C., TUFFNELL, P. S., and BARRELL, B. G. (1984). DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 310, 207-211. BARAHONA, H. H., MELENDEZ, L. V., KING, N. W., DANIEL, M. D., FRASER, C. E. O., and PREVILLE, A. C. (1973). Herpesvirus aotus type 2: A new viral agent from owl monkeys (Aotus trivirgatus). J. Infect. Dis. 127, 171-178. BIRD, A. P. (1980). DNA methylation and the frequency of CpG in animal DNA. Nucleic Acids Res. 8, 1499-l 504. BODEMER, W., NILLER, H. H., NITSCHE, N., SCHOLT~, B., and FLECKEN-

OF

BHV-4

663

STEIN, B. (1986). Organization of the thymidylate synthase gene of herpesvirus saimiri. J. Viral. 60, 1 14-l 23. BORNKAMM, G. W., DELIUS, H., FLECKENSTEIN, B., WERNER, F.-J., and MULDER, C. (1976). Structure of herpesvirus saimiri genomes: arrangement of heavy and light sequences in the M genome. J. Viral. 19, 154-161. BRIDGEN, A., HERRING, A. J., INGLIS, N. F., and REID, H. W. (1989). Preliminary characterization of the alcelaphine herpesvirus 1 genome.1. Gen. Viral. 70, 1141-1 150. BUBLOT, M., VAN BRESSEM, M.-F., THIRY, E., DUBUISSON, J., and PASTORET, P.-P. (1990). Bovine herpesvirus 4 genome: Cloning, mapping and strain variation analysis. /. Gen. Viral. 71, 133-l 42. BUBLOT, M., WELLEMANS, G., VAN BRESSEM, M.-F., DUBUISSON. J., PASTORET, P.-P., and THIRY, E. (1991 a). Genomic diversity among bovine herpesvirus 4 field isolates. Arch. Viral. 116, l-l 8. BUBLOT, M., DUBUISSON, J., VAN BRESSEM, M.-F., DANYI, S., PASTORET, P.-P., and THIRY, E. (1991 b). Antigenic and genomic identity between simian herpesvirus aotus type 2 and bovine herpesvirus type 4. J. Gen. Viral. 72, 715-719. BUCKMASTER, A. E., Scorr, S. D., SANDERSON, M. J., BOURSNELL, M. E. G., Ross, N. L. J., and BINNS, M. M. (1988). Gene sequence and mapping data from Marek’s disease virus and herpesvirus of turkeys: Implications for herpesvirus classification. J. Gen. Viroi. 69, 2033-2042. CAMERON, K. R., STAMMINGER, T., CRAXTON, M., BODEMER. W., HoNESS, R. W., and FLECKENSTEIN, B. (1987). The 160,000-n/r, virion protein encoded at the right end of the herpesvirus saimiri genome is homologous to the 140,000-M, membrane antigen encoded at the left end of the Epstein-Barr virus genome. J. Vkol. 61, 20632070. CASTRUCCI, G., FRIGERI, F., FERRARI, M., PEDINI, B., ALDROVANDI, V., CILLI, V., RAMPICHINI, L., and GATTI, R. (1987). Reactivation in calves of latent infection by Bovid herpesvirus-4. Microbiologica 10,37-45. CHEE, M. S., BANKIER, A. T., BECK, S., BOHNI, R., BROWN, C. M., CERNY, R., HORSNELL, T., HUTCHISON, Ill C. A., KOUZARIDES, T., MARTIGNETTI, J. A., PREDDIE, E., SATCHWELL, S. C., TOMLINSON, P., WESTON, K. M., and BARRELL, B. G. (1990). Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD1 69. Curr. Top. Microbial. Immunol. 154, 125-l 69. CLEARY. M. L., SMITH, S. D., and SKLAR. J. (1986). Cloning and structural analysis of cDNAs for bcl-2 and hybrid bcl-2/immunoglobulin transcript resulting from the t(14; 18) translocation. Cell 47, 1928. DAVISON, A. J., and SCOTT, J. E. (1986). The complete DNA sequence of varicella-zoster virus. /. Gen. Viral. 68, 1759-l 816. DAVISON, A. J., and TAYLOR, P. (1987). Genetic relations between varicella-zoster virus and Epstein-Barr virus. f. Gen. Viral. 68, 1067-l 079. DEVEREUX, J., HAEBERLI, P., and SMITHIES, 0. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12, 387-395. DUBUISSON, J., DANYI, S., BUBLOT, M., PASTORET, P.-P., and THIRY, E. (1991). Comparison of proteins of simian herpesvirus aotus type 2 and bovine herpesvirus type 4. J. Gen. Viral. 72, 1 145-l 150. EFSTATHIOU, S., Ho, Y. M., and MINSON, A. C. (1990a). Cloning and molecular characterization of the murine herpesvirus 68 genome. J. Gen. Viral. 71, 1355-l 364. EFSTATHIOU, S., Ho, Y. M., HALL, S., STYLES, C. J., SCOTT, S. D., and GOMPELS, U. A. (1990b). Murine herpesvirus 68 is genetically related to the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri. /. Gen. Viral. 71, 1365-l 372. EHLERS, B., BUHK, H.-J., and LUDWIG, H. (1985). Analysis of bovine cytomegalovirus genome structure: Cloning and mapping of the

664

BUBLOT

monomeric polyrepetitive DNA unit, and comparison of European and American strains. J. Gen. Viral. 66, 55-68. FABRICANT, C. G., GILLEPSIE, J. H., and KROOK, L. (1971). Intracellular and extracellular mineral crystal formation induced by viral infection of cell cultures. Infect. Immun. 3, 416-419. FENG, D.-F., and DOOLITTLE, R. F. (1987). Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25, 351-360. FLECKENSTEIN, B., BORNKAMM, G. W., and LUDWIG, H. (1975). Repetitive sequences in complete and defective genomes of herpesvirus saimiri. 1. Viral. 15, 398-406. FLECKENSTEIN, B., BORNKAMM, G. W., MULDER, C., WERNER, F.-J., DANIEL, M. D., FALK, L. A., and DELIUS, H. (1978). Herpesvirus ateles DNA and its homology with herpesvirus saimiri nucleic acid. J. Viral. 25, 361-373. GIVEN, D., YEE, D., GRIEM, K., and KIEFF, E. (1979). DNA of EpsteinBarr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA. J. Viral. 30, 852-862. GOMPELS, U. A., CRAXTON, M. A., and HONESS, R. W. (1988). Conservation of gene organization in the lymphotropic herpesviruses, herpesvirus saimiri and Epstein-Barr virus. J. Viral. 62, 757-767. HIGGINS, D. G., and SHARP, P. M. (1989). Fast and sensitive multiple sequence alignments on microcomputer. C.A.5.I.O.S. 5, 151153. HOMAN, E. J., and EASTERDAY, B. C. (1981). Further studies of naturally occurring latent bovine herpesvirus infection. Am. J. Vet. Res. 42, 1811-1813. HONESS, R. W. (1984). Herpes simplex and “the herpes complex”: Diverse observations and a unifying hypothesis. J. Gen. Viral. 65, 2077-2107. HONESS, R. W., BODEMER, W., CAMERON, K. R., NILLER, H. H., FLECKENSTEIN, B., and RANDALL, R. E. (1986). The A + T-rich genome of herpesvirus saimiri contains a highly conserved gene for thymidylate synthase. Proc. Natl. Acad. Sci. USA 83, 3604-3608. HONESS, R. W., GOMPELS, U. A., BARRELL. B. G., CRAXTON, M., CAMERON, K. R., STADEN, R., CHANG, Y.-N., and HAYWARD, G. S. (1989). Deviations from expected frequencies of CpG dinucleotides in herpesvirus DNAs may be diagnostic of differences in the states of their latent genomes. J. Gen. Viral. 70, 837-855. HONES?,, R. W., and WATSON, D. H. (1977). Unity and diversity in the herpesviruses. J. Gen. Viral. 37, 15-37. JOSEPHS, S. F., ABLASHI, D. V., SALAHUDIN, S. Z., JACODZINSKI, L. L., WONG-STAAL, F., and GALLO, R. C. (1991). Identification of the human herpesvirus 6 glycoprotein H and putative large tegument protein genes. J. V/r-o/. 65, 5597-5604. KASCHKA-DIERICH, C., WERNER, F. J., BAUER, I., and FLECKENSTEIN, B. (1982). Structure of nonintegrated, circularherpesvirussaimiriand herpesvirus ate/es genomes in tumor cell lines and in vitro-transformed cells. J. Viral. 44, 295-310. KIEFF, E., DAMBAUGH, T., HUMMEL, M., and HELLER, M. (1983). Epstein-Barr virus transformation and replication. /n “Advances in Viral Oncology” (G. Klein, Ed.), Vol. 3, pp. 133-l 82. Raven Press, New York. KIEFF, E., and LIEBOWITZ, D. (1990). Epstein-Barrvirus and its replication. /n “Virology” (Fields et a/., Eds.), 2nd ed., Vol. 2, pp. 8891958. Raven Press, New York. KIT, S., KIT, M., ICHIMURA, H., CRANDELL, R., and MCCONNELL, S. (1986). Induction of thymidine kinase activity by viruses with group B DNA genomes: Bovine cytomegalovirus (bovine herpesvirus 4). Virus Res. 4, 197-212. KOUZARIDES, T., BANKIER, A. T., SATCHWELL, S. C., WESTON, K., TOMLINSON, P., and BARRELL, B. G. (1987). Large-scale rearrangement of homologous regions in the genomes of HCMV and EBV. Virology 157, 397-413.

ET AL. KROGMAN, L. A., and MCADARAGH, J. P. (1982). Recrudescence of bovine herpesvirusin experimentally infected calves. Am. J. Vet. Res. 43, 336-338. KRUGER, J. M., OSBORNE, C. A., WHETSTONE, C. A., GOYAL, S. M., and SEMLAK, R. A. (1989). Genetic and serologic analysis of feline cellassociated herpesvirus-induced infection of the urinary tract in conventionally reared cats. Am. J. Vet Res. 50, 2023-2027. LAWRENCE, G. L., CHEE, M., CRAXTON, M. A., GOMPELS, U. A., HONES& R. W., and BARRELL, B. G. (1990). Human herpesvirus 6 is closely related to human cytomegalovirus. J. Viral. 64, 287-299. LEE, S. I., MURTHY, S. C. S., TRIMBLE, J. J., DESROSIERS, R. C., and STEITZ, J. A. (1988). Four novel U RNAs are encoded by a herpesvirus. Cell 54, 599-607. MARTIN, M. E. D., NICHOLAS, J., THOMSON, B. J., NEWMAN, C., and HONES& R. W. (1991). Identification of a transactivating function mapping to the putative immediate-early locus of human herpesvirus 6.1. Viral. 65, 5381-5390. MCGEOCH, D. J., DALRYMPLE, M. A., DAVISON, A. J., DOLAN, A., FRAME, M. C., MCNAB, D., PERRY, L. J., Scorr, J. E., and TAYLOR, P. (1988). The complete DNA sequence of the long unique region in the genome of herpes simplex virus type 1. J. Gen. Viral. 69, 15311574. MCGEOCH, D. J. (1989). The genomes of the human herpesviruses: Contents, relationships and evolution. Annu. Rev. Microbial. 43, 235-265. MEDVECZKY, M., GECK, P., CLARKE, C., BYRNE% J., SULLIVAN, J. L., and MEDVECZKY, P. G. (1989). Arrangement of repetitive sequences in the genome of herpesvirus sylvilagus. 1. Viral. 63, 101 O-l 014. MOORE, K. W., VIEIRA, P., FIORENTINO, D. F., TROUNSTINE, M. L., KHAN, T. A., and MOSMANN, T. R. (1990). Homology of cytokine synthesis inhibitoty factor (IL-l 0) to the Epstein-Barr virus gene BCRFl Science 248, 1230-l 234. MURTHY, S. C. S., TRIMBLE, J. J., and DESROSIERS, R. C. (1989). Deletion mutants of herpesvirus saimiri define an open reading frame necessary for transformation. J. Viral. 63, 3307-3314. NEIPEL, F., ELLINGER, K., and FLECKENSTEIN, B. (1991). The unique region of the human herpesvirus 6 genome is essentially collinear with the U, segment of human cytomegalovirus. 1. Gen. Viral. 72, 2293-2297. NICHOLAS, J., GOMPELS, U. A., CRAXTON, M. A., and HONES% R. W. (1988). Conservation of sequence and function between the product of the 52-kilodalton immediate-early gene of herpesvirus saimiri and the BMLFl-encoded transcriptional effector (EB2) of Epstein-Barr virus. 1. Viral. 62, 3250-3257. NICHOLAS, J., SMITH, E. P., COLES, L., and HONES% R. (1990). Gene expression in cells infected with gammaherpesvirus saimiri: Properties of transcripts from two immediate-early genes. Virology 179, 189-200. NICHOLAS, J., COLES, L. S., NEWMAN, C., and HONES% R. W. (1991). Regulation of the herpesvirus saimiri (HVS) delayed-early 1 1 O-kilodalton promoter by HVS immediate-early gene products and a homolog of the Epstein-Barr virus R fransactivator. J. Viral. 65, 2457-2466. NICHOLAS, J., CAMERON, K. R., and HONES& R. W. (1992a). Herpesvirus saimiri encodes homologues of G protein-coupled receptors and cyclins. Nature 355, 362-365. NICHOLAS, J., CAMERON, K. R., COLEMAN, H., NEWMAN, C., and HoNESS, R. W. (1992b). Analysis of nucleotide sequence of the rightmost 43kbp of herpesvirus saimiri (HVS) L-DNA: General conservation of genetic organization between HVS and Epstein-Barr virus. Virology 186, 296-310. OSORIO, F. A., REED, D. E., and ROCK, D. L. (1982). Experimental infection of rabbits with bovine herpesvirus-4: Acute and persistent infection. Vet Microbial. 7, 503-513.

GENE

ORGANIZATION

OSORIO, F. A., and REED, D. E. (1983). Experimental inoculation of cattle with bovine herpesvirus-4: Evidence for a lymphoid-associated persistent infection. Am. J. Vet. Res. 44, 975-980. OSORIO, F. A.. ROCK, D. L., and REED, D. E. (1985). Studies on the pathogenesis of a bovine cytomegalo-like virus in an experimental host. J. Gen. Viral. 66, 1941-l 951. PEARSON, W. R., and LIPMAN, D. J. (1988). Improved tools for biological sequence comparisons. Proc. Nat/. Acad. Sci. USA 85, 24442448. RICHTER, J., PUCHTLER, I., and FLECKENSTEIN, B. (1988). Thymidylate synthase gene for herpesvirus ateles. J. Viral. 62, 3530-3535. ROIZMAN, B. (1982). The family Herpesviridae: General description, taxonomy and classification. In “The Herpesviruses” (B. Roizman, Ed.), Vol. 1, pp. l-23, Plenum, New York/London. ROSSITER, P. B., GUMM, I. D., STAGG, D.A., CONRAD, P. A., MUKOLWE, S., DAVIES, F. G., and WHITE, H. (1989). Isolation of bovine herpesvirus-3 from African buffaloes (Syncerus caffer). Res. Vet. Sci. 46, 337-343. SANGER, F., NICKLEN, S., and COULSON, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Nat/. Acad. Sci. USA 74, 5463-5468. STORZ. J., EHLERS, B., TODD, W. J., and LUDWIG, H. (1984). Bovine cytomegaloviruses: Identification and differential properties. J. Gen. Viral. 65, 697-706.

OF

BHV-4

665

THIRY, E., DUBUISSON, J., BUBLOT, M., VAN BRESSEM, M.-F., and PASTORET, P.-P. (1990). The biology of bovine herpesvirus 4 infection of cattle. Dtsch. Tier%ztl. Wochenschr. 97, 72-77. THIRY, E., BUBLOT, M., DUBUISSON, J., VAN BRESSEM, M.-F., LEQUARRE, A.-S., LOMONTE, P., VANDERPLASSCHEN, A., and PASTORET, P.-P. (1992). Molecular biology of bovine herpesvirus type 4. Adv. Vet. Viral., in press. TODD, W. J., and STORZ, J. (1983). Morphogenesis of a cytomegalovirus from an American bison affected with malignant catarrhal fever. J. Gen. Viral. 64, 1025-l 030. TRIMBLE, J. J., MURTHY, S. C. S., BAKKER, A., GRASSMANN, R., and DESROSIERS, R. C. (1988). A gene for dihydrofolate reductase in a herpesvirus. Science 239, 1 145-l 147. VAN OPDENBOSCH, E., WELLEMANS, G., and OUDEWATER, J. (1986). Toevallige isolatie van het boviene herpesvirus 4 uit de long van een schaap. Vlaams Diergeneesk. Tijdschr. 55, 432-433. VAN SANTEN, V. L. (1991). Characterization of bovine herpesvirus 4 major immediate-early transcript. 1. Viral. 65, 521 1-5224. WASSARMAN, D. A., LEE, S. I., and STEITZ, J. A. (1989). The nucleotide sequence of HSUR 5 RNA from herpesvirus saimiri. Nucleic Acids Res. 17, 1258. WEI, X. M., and OOKA, T. (1989). A transforming function of the BARFl gene encoded by Epstein-Barr virus. EMBO 1. 8, 28972903.

Genetic relationships between bovine herpesvirus 4 and the gammaherpesviruses Epstein-Barr virus and herpesvirus saimiri.

The overall arrangement of genes in the unique central part of the bovine herpesvirus type 4 (BHV-4) genome has been deduced by analysis of short DNA ...
1MB Sizes 0 Downloads 0 Views