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VIROLOGY 184, 404-410 (1991)
Nucleotide Sequence of the Gene Encoding Infectious Laryngotracheitis Virus Glycoprotein B’
KRITAYA KONGSUWAN,’ C. T. PRIDEAUX, M. A. JOHNSON, M. SHEPPARD, AND K. J. FAHEY
CSIRO Division of Animal Health, Animal Research Laboratory, Private Bag No. 1, Parkville, V/C 3052, Australia
Received February 19, 199 1; accepted May IS, 199 1
The nucleotide sequence of the infectious laryngotracheitis virus (ILTV) gene encoding the 205K complex glycopro- tein (gp205) was determined. The gene is contained within a 3-kb EcoRl restriction fragment mapping at approximately map coordinates 0.23 to 0.25 in the U, region of the ILTV genome and is transcribed from right to left. Nucleotide sequence analysis of the DNA fragment identified a single, long open reading frame capable of encoding 873 amino acids. The predicted precursor polypeptide derived from this open reading frame would have a calculated M, of 98,895 Da and contains nine potential glycosylation sites. Hydropathic analysis indicates the presence of an amino terminal hydrophobic sequence and hydrophobic carboxyl terminal domain which may function as a signal peptide and a membrane anchor sequence, respectively. Comparison of the predicted ILTV gp205 protein sequence with those of other herpesviruses revealed a significant sequence similarity with gB-like glycoproteins. Extensive homology was observed throughout the molecule except for the amino and carboxyl termini. The high homology in predicted primary and secondary structures is consistent with the essential role of the gB family of proteins for viral infectivity and pathogenesis. 0 1991 Academic Press, Inc.
Infectious latyngotracheitis virus (ILlV) is an impor- tant pathogen of chickens, causing an acute upper re- spiratory tract infection. The disease is found world- wide and may result in severe productions losses due to mortality and reduced egg production (1). The ge- nome of ILlV, an alphaherpesvirus, is linear double- stranded DNA approximately 155 kb in size and has a structure similar to that of pseudorabies virus (PRV). The genome has two covalently linked components, U, and U,, of which only the Us sequence is bounded by inverted repeats (2, 3). From random DNA sequencing of ILTV DNA, Griffin (4) was able to identify 21 ILTV genes. Of these genes 20 were identified by compari- son to varicella-zoster virus (VZV) and 19 by compari- son to herpes simplex virus type 1 (HSV-1); only 12 genes were found by comparison with the gammaher- pesvirus Epstein-Barr virus (EBV). Sequence data of the thymidine kinase gene and the upstream overlap- ping genes of ILTV also provide evidence that homol- ogy exists at the DNA level between ILTV and other alphaherpesviruses (5).
As part of a research program to characterize ILTV antigens responsible for stimulating protective immu- nity in chickens, we have focused on the glycoproteins since these proteins are known to be among the pri- mary targets for both humoral and cell-mediated im- mune responses (8). The characterization of ILTV gly-
’ The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database.
’ To whom requests for reprints should be addressed.
coproteins is still at an early stage with the exact num- ber of glycoproteins expressed still unknown. York et al. (7) showed that chicken antisera to the ILTVvaccine strain and to a virulent isolate immunoprecipitated five major viral glycoproteins of 205, 160, 115, 90, and 60K. Additional glycoprotein bands were recognized by immune chicken and rabbit sera in Western blotting using a glycoprotein fraction purified from extracts of virus-infected cells (7). Further work using monoclonal antibodies defined the five major glycoproteins into two groups; the 205K complex (205, 160, 115, and 90K glycoproteins) and the 60K glycoproteins (8). To identify ILTV glycoprotein genes, we used the monoclo- nal antibodies 12-1, 22-7, 23-1, and 131-6 which reacted with the glycoprotein 205K complex in West- ern blot (kindly provided by Dr. Jenny York) to screen the library of Agtl 1 -ILTV recombinant phages. Recom- binant phages encoding the desired epitope were identified with the epitope localized to the 3-kb EcoRl fragment at 0.23-0.25 map unit in the U, on the ILTV genome with reference to the previously published map (3). The nucleotide sequence of this EcoRl frag- ment revealed a single, long open reading frame en- coding a protein which shares significant amino acid sequence homology and many predicted structural features with the glycoprotein B (gB) family of proteins.
The strain used in the present study is the SA-2 vac- cine strain of ILTV used in Australia. ILTV was propa- gated in monolayer cultures of primary chicken kidney cells grown in Eagle’s basal medium (Commonwealth Serum Laboratories) supplemented with 5% newborn
0042-6822191 $3.00 404
Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.
406 SHORT COMMUNICATIONS
FIG. 2. Expression of ILW “gB” gene. Total cytoplasmtc RNAs from ILTV that were infected (lane 1) and mock infected (lane 2) were fractionated on an agarose/formaldehyde gel and transferred onto a Hybond-N membrane. The membrane was hybridized to the 8g/ll- Pstl fragment (Fig. 1, region between horizontal arrows). Arrow- heads indicate the location of chicken ribosomal RNAs 28 S and 18 S which were used as size standards and were estimated to be 4.2 and 1.6 kb, respectively.
calf serum (Flow Laboratories Australasia Pty Ltd). Cell-associated viral DNA was prepared using the slightly modified method of Whalley et al. (9) from in- fected cell cultures at 24 hr postinfection. Approxi- mately 2 pg of ILTV DNA was sheared by sonication, made flush-ended with T4 DNA polymerase, methyl- ated by EcoRl methyltransferase, ligated to EcoRl linkers (Biolabs), and digested with EcoRl endonucle- ase. Fragments of 0.5-2 kb were isolated by agarose
gel electrophoresis and ligated to Xgtl 1 arms previ- ously digested with fcoRl and dephosphorylated with calf intestine alkaline phosphatase (Amersham). After ligation the DNA was packaged using Packagene ex- tract (Promega), transfected, and resulted in a library of 100,000 phages. The library was screened using the Super immunoscreening system of Amersham accord- ing to the instructions of the supplier. High titer stocks of each positive phage and phage DNA were prepared by making a plate lysate (IO) on Escherichia co/i Y 1090. All the subcloning into plasmid and M 13 vec- tors was performed using standard methods (10).
DNA sequencing was carried out by the dideoxynu- cleotide chain termination method using the Sequen- ase sequencing kit (United States Biochemical). The DNA synthesis reactions were primed with either a 17- mer oligonucleotide residue that hybridized to pUC or M 13 sequences adjacent to the insert DNA or with custom 20-mer oligonucleotides complementary to a specific region of the insert DNA. Specific oligonucleo- tides were synthesized using a Pharmacia LKB Gene Assembler Plus. DNA sequence reading and analyses were done using a HIBIO DNAsis software package (Hitachi America, Ltd.) and with the programs available through the Australian National Sequence Analysis Fa- cility (ANSAF). Searches of protein databases and com- parison of homologous sequences were performed with the FASTNiP program of Lipman and Pearson (1 I).
The complete nucleotide sequence of the region
I i 1 110 219 328 437 546 655 764 074
FIG. 3. Hydropathy plot of the predicted ILTV “gB” amino acid sequence. The plot was based on the algorithm of Kyte and Doolittle (38) using a 1 1 -amino acid window. The two most hydrophobic regions in the N- and C-termini are predicted to represent the signal sequence and the transmembrane anchor region, respectively, of the glycoprotein molecule.
EcoRI GMTTCGACCTCGACGGCCCGATTTTGGAAAACGGG~TTMCATTTGMGAGG --_-_
MCATCCATTAGTTGCAGTGTEG_G_GFGTCGATGA~~G~~~~~CTG~TCCAGCGTCTACTATCATAGTAG [HASLKN
AGAAAGATTTATACGCGGTCCTGTATGCAATCCTACATCCTACATCGCCGTGMCATTGACAT~CTAGCTTG~TG LICVCVAILIIPSTLSQDSHGIGW~
CTGATCTGCGTGTGCGTGGCMTCCTGATCCCATCTACCCTATCTCMGATTCACAC~MTTGGCTGGMT NSPHDTASMDVGKISFSEAIGSGA
MTAGCCCTCATGATACAGCCAGCATGGATGTTGGAAAAAA PKEPQIRNRIFACSSPTGASVARL
CCG~GMCCCCAGATTAGAAACAGAATTTTTGCGTGCGCTT AQPRHCHRHADST NMTEGIAVVFK
GCCCAGCCACGACATTGTCACCGACATGCCGATTCGATTCGACTMCATGACTGM~MTT~CGTAGTCTTCMG QNIAPYVFNVT L Y Y K H ITTVTTWA
CRAAACATTGCCCCGTACGTCTTTMTGTGACTCTATACTAT~CATATMCCACAGTTACTACGTGGGCA LFSRPQ ITNEYVTRVP IDYHEIVR
TTATTCTCMGACCCCAAATMC~TGAGTACGTGACCAG~TTCCMTAG~CTATCATG~TTGTCA~G IDRSGECSSKATYHKNFHFFEAYD
ATTGATCGATCGGGAGMTGCTCATCCAAAGCAACGTATCAT~TTTCATGTTTTTTGM~TTACGAC NDEAEKKLPLVPSLLRSTVSKAFH
MTGATGMGCAG AAAAAAAATTGCCCCTGGTTCCATCACTGTTMGATCMCTGTCTCCAAGGCGTTTCAT TTNFTKRHQTLGYRTSTSVDCVVE
ACMCTMCTTTACTMGCGACATCRAACCCCT~ATACCGMCGTCTACATC~TCGACTGTGTTGTGGM YLQAEl-SVYPYDYFGMATGDTVEIS
TATCTACAGGCTAGATCTGTATACCCGTATGATTACTTTGTTTCT P F YT K NTTGPRRHSVYRDYRFLEI
CCTTTTTATACCAAAAACACGACCGGACCAAGGCGTCACAGTGTCTACAGAGACTATAGATTTCTCG~TC ANYQVRD LETGQIRP PKKRNFLTD
GCAAATTATCMGTCAGGGATTTGGAAACCGGACAAATAAGACCCCCT MAAMAGAAACTTTCTMCAGAT E Q F T IGWDAMEEKESVCTLSKWIE
GMCMTTCACTATAGGCTGGGATGCMT~MG~~MTCTGTATGTACTCTCAGT~TGGATTGM v P EAVRVSYKNSYHFSLKDMTMTF
GTCCCGGMGCAGTTCGTGTTTCGTACAAAAACAGTTACCGTTC SSGKQPF NTSR LH LAE CVP T I AS E
TCGTCCGGAARACMCCTTTTMCATCA~A~TTCATTT~TGMT~GTTCCTACCATAGCCTCGGAG A IDGIFARKYSSTHVRSGDIEYYL
GCCATAGATGGCATCTTTGCCAGRAAGTATAGTATAGTTCGACTCATGTCCGTTCT~ACATCGMTACTATCTC GSGGFLIAFQKLHSHGLAEMYLEE
GGTAGTGGCGGATTTCTGATCGCATTTTCAG~CTCATGA~CAT~TT~TG~TGTACCTAGMGAG AQRQNHLPRGRERRQAAGRRTASL
GCACAAAGACAAAATCATCTCCCGAGAGGGAGAGAGCGTCTG QSGPQGDRI TTHSSATFANLQFAY
CAGTCTGGACCTCAGGGTGATAGMTTACTACCCACAGTTCT~MCATTT~CATGTTACMTTT~ATAC ~KIQAHvNELIGNLLEAw~ELQNR GACAAAATCCMGCCCATGTTMCGA~TTATCGGAAATTCGC
QLIVWHEMKKLNPNSLMTSLFGQP CMCTGATTGTATGGCACGAGATGMG~CTAAhCCCCGMCTCACTGATGACATCTTTGTTC~ACMCCT
VSARLLGDIVAVSKCIEIP I EN I R GTMGCGCCAGGCTATTGGGAGACATCGTAGCGGTATCAATATTAGG
HQDSHRVPGDPTMCYTRPVLIFRY ATGCAGGATTCCATGCGCGTGCCAGGGGACCCAACCATGTTAT
SSSPESQFSANST ENHNLGILGQL TCGTCCTCCCCTGAGTCACAGTTTTCTGCtAACTCAACAGCTC
GEHNEILQGRNLIEPCMINHRRYF GGAGMCATMTGRAATTTTACAAGGCCtGAATTTGATAGMC~T~ATGATCMTCACAGAC~TACTTT
LLGENYLLYEDYTFVRQVNASEIE CTGTTGGGAGAAAACTACCTTCTTTACGMGACAT
EVSTFINLNATI LEDLDFVPVEVY GMGTGAGCACATTCATCMCTTGMCGCCACTATACTAG~GATTTGGACTTTGTGCCCGTCG~GTATAC
T R E ELRDTGTLNYDDVVRYQNIYN ACTCGCGAGGMCTCAGAGATACTGGGACTTTAAACTATGC
K R F RD I D TV I R[GDRGDAIFRA I A D AAAAGGTTCAGAGACATTGACACTGTMTACGTGGAGATAGGGGAGATGC~TCTTTAGAGCMTAGCAGAT
FFGNTLGEVGKALGTVVMTAAAAV TTTTTTGGCAACACTCTTGGAGMGTAGGCATTGGCATTG~MCTGTAGTGATGACAGCCGCGGCAGCAGTA
IS TVSGIASFLSNPFAALAIGIAV ATTTCTACAGTATCTGGCATCGCCTCATTTCTTTCTAACCTTGGGATAGCGGTG
VVSIILGLLAF]KYVMNLKSNPvQV GTGGTGAGCATTATTTTAGGACTGCTGGCGTTCAAATATGTMTG~CCTG~TC~CCCAGTTCAGGTT
LFPGAVP PAGTPP RPSRRYYKDEE CTGTTCCCAGGCGCAGTTCCCCCGGCCGGAACTCCTCCACGACCCTCTAGACGTTACTAC~GGATGAGGAG
EVEEDSDEDDRI LATRVLKGLELL GAGGTTGAGGAGGATAGTGATGAGGACGACAGGATACTTGCCACCAGAGTTCTG~GGCCTTGAGCTTCTA
HKDEQKARRQKARFSAFAKNMRNL CACAAGGATGMCAGRAAGCTCGMGACAG~GCGCGGTTTTCTGCTTTTGCT~TATGAG~CCTA
FRRKPRTKEDDYP LLEYPSWAEES TTTCGCAGAAAACCCCGMCCRAGGAAGATGACTACCCCCTGCTCG~TACCCTTCGTGGGCAG~G~GC
ED E * GAAGACGMTMGTTTAAATGCAGTTTATTTAUAUA TGACATTACTATTCACATGACTCAGTCTGCCATC . . . . . . . ATTTGCGCAAATGCGGCTGCTTCTTTCTTTCTTTTCAATTGCATCTTTCAGTCGTTTTGGCATA GAAGCATCGACTGTCTCCCGAGCAGACTCTTGATTACTATTTTCTAGTTCCTCTTTTCTCTCTGMGACG~ TCGGCATTGGMGCTGATTTMGAATTC
EcoRI
59 130
6 202 30
274 54
346 78 418 102 490 126 562 150 636 174 706 198 778 222 850 246 922 270 994 294
1066 318
1138 342
1210 366
1282 390
1354 414
1426 430
1498 462
1570 406
1642 510
1714 534
1786 558
1858 582
1930 606
2002 630
2074 654
2146 678
2218 702
2290 726
2362 750
2434 744
2506 798
2578 822
2650 846
2722 870
2794 873
2866 2936 3010 3038
FIG. 1. The nucleotide sequence of the EcoRl fragment encoding the “gB” homologue of ILTV and the deduced amino acid sequence. The putative TATA box of the promoter is boxed. The polyadenylation site, AATAAA, is dotted underlined. Broken lines indicate GC-rich regions. The presumed signal sequence at the N-terminus and the membrane-spanning region at the C-terminus are indicated by italics and are bracketed. The putative N-linked glycosylation sites of the consensus N-X-S/T are underlined. Numbers at right indicate positions of nucleotides and the predicted amino acids.
405
SHORT COMMUNICATIONS 407
containing the putative gp205K coding sequence is shown in Fig. 1. There is a single large open reading frame within this region extending from the ATG codon beginning 185 bp 3’ of the EcoRl site (Fig. 1) to a TAA termination codon, starting at nucleotide 2804 (Fig. 1). Translation of this 2619 bp would produce a polypep- tide of 873 amino acids. There are two other possible initiation codons in the vicinity of the assigned ATG, one at position 155 to 157 and the other at nucleotides 200 to 202 (see Fig. 1). Both of these codons are in frame with the above-mentioned open reading frame. However, the assigned initiation codon ATG at 185 bp resides within the sequence GACATGG which con- forms well to the consensus sequence (A/G)CCATGG (12). It has a purine (G) at position -3, a C at -1, and a G at +4 which are considered to be the most strongly conserved features of the flanking sequence of the initi- ation codon of eukaryotic mRNAs.
Searching for the upstream cis-regulator-y sequence, two potential TATA box homologues were found. They are located 45 and 148 bp upstream of the putative start codon (at 140 and 38 bp in Fig. 1). We suggest that the TATA box position at bp 38-41 (Fig. 1, in box) is the functional TATA box of this gene for two reasons: (i) its local sequence TATAllT has some features pro- posed for the consensus TATA box sequence TATA(A/ T)A(A/T) (13) and (ii) Sl nuclease mapping indicated that the potential RNA polymerase initiation site of this gene mapped at about 144 nucleotides upstream of the ATG (C. T. Prideaux, unpublished data). Other pu- tative cis-regulatory elements found are the GC-rich regions (Fig. 1, indicated by broken lines) which are potential binding sites for the promoter-specific tran- scription factor Spl (14). A potential polyadenylation signal, AATAAA, was found 20 bp downstream from the termination codon (Fig. 1, dotted underlined). The G + C content of the sequence is 44.4%, which is close to the estimate of 45% for total ILTV DNA as determined from buoyant density measurements (15).
Northern blot analysis (Fig. 2) detected a single tran- script 2.9-3 kb in size from cells infected with ILTV. The probe used for hybridization was derived from the 632-bp Bglll-Pstl fragment (region defined by horizon- tal arrows in Fig. 1). Using the same probe, a similar result (not shown) was obtained with the poly(A)+ RNA indicating that the RNA is polyadenylated. Allowing 100 bp for polyadenylation, this transcript size is con- sistent with the predicted 5’ and 3’ ends of the mRNA (see above).
The deduced amino acid sequence for the polypep- tide encoded by the 2619-bp open reading frame is shown above the DNA sequence in Fig. 1. The molecu- lar mass of the 873 amino acids primary translation product is 98,895 Da. The predicted protein has fea- tures common to other membrane-spanning glycopro-
teins. A hydrophobicity plot (Fig. 3) identified a se- quence of 16 hydrophobic amino acids at the extreme NH2 end (Figs. 1 and 3) which may function as the signal peptide. Applying the weight matrices criteria of von Heijne (16) for the prediction of the cleavage site, the cleavage might occur at the serine residue 21. A broad hydrophobic domain at amino acids 690 to 761 near the C-terminus (Figs. 1 and 3) represents a mem- brane anchor sequence. A large extracellular domain (amino acids 17 to 689) contains nine potential N- linked glycosylation sites (underlined in Fig. 1). C-ter- minal amino acids 762 to 873 have a net positive charge and may function as the cytoplasmic domain.
To obtain the identity of the gp205 predicted trans- lated product, we searched the Swiss, NBRF, and GenBank protein databases for homologous se- quences. The results indicated high homology with the gB family of proteins in herpesviruses. Identities are scattered in the central portion of the proteins with little or no identities at the N- and C-termini. To date the amino acid sequences of the gB-type glycoproteins of 13 distinct herpesviruses have been published: HSV-1 (17, 18), HSV-2 (19) PRV (20), VZV (21), bovine herpes- virus type 1 (BHV-1) (22, 23) BHV-2 (24) EHV-1 (25), EHV-4 (26) EBV (27) human cytomegalovirus (HCMV) (28), herpesvirus saimiri (29), and MDV (30). Multiple alignments of 10 herpesvirus gBs (Fig. 4) have high- lighted several characteristics of a conserved se- quence. The common structural features of the gB-like proteins shown in Fig. 4 are (i) the conservation of 10 cysteine (C) residues which were perfectly aligned in gB of all 10 viruses. This accounts for all cysteines of the ILTV protein except for the two which occur in sig- nal sequence. This observation indicates that the pro- teins are conserved in their secondary and tertiary structures since C-C disulfide bonds are important de- terminants of the tertiary structure of the protein. (ii) Six sites of prolines occur at conserved positions (Fig. 4). (iii) The triple hydrophobic transmembrane regions were found in similar positions and consisted of three distinct peaks of hydrophobicity (positions 851 to 918 in Fig. 4). This structure is believed to enable the pro- tein to traverse the membrane three times (27). (iv) Some of the putative N-linked glycosylation sites exist in similar positions but are not strictly conserved (at positions 184, 298, 457, 486, 652, and 793 in Fig. 4). The motif CYSRP at positions 702-706 which was noted by Ross et al. (30) to be conserved in mammalian herpesviruses and in MDV has the sequence CYTRS in ILTV.
Taken togetherthe data confirmed the overall similar- ity in the primary, secondary, and tertiary structures of the proteins. These data are consistent with the notion that gB is multifunctional and essential for viral replica- tion. It is also likely that these proteins perform similar
408 SHORT COMMUNICATIONS
I LTV
HSV BHV-2
PRV
vzv
EHV- 1
HVS
HDV
HCHV
EBV
I LTV
HSV BHV-2
PRV
vzv
EHV- 1
HVS
MIS4
HCMV
EBV
ILTV
HSV
BHV-2 PRV
vzv
EHV- 1
HVS
MDV
HCHV
EBV
I LTV
HSV BHV-2
PRV
vzv
EHV- 1
HVS
HDV
HCMV
EBV
ILTV
HSV BHV-2
PRV
vzv
EHV- 1 HVS
MDV
HCMV
EBV
1 120 ~-------------------LKMLICVCVAILIPSTLS-----QDSHGIGUNN~HDTASMOVGKISF--------------------- ---------------------------SE
MRQCAPARGRRUFWV--------ALLCLTLGVLVASAAP---SSPGTPGVA--------------------MTaMNGGPA-----TPAPP-AP~PPT~PKPKKNRKPKPPKPPRP
H-----AISRRSLHA-----------IILTVLLLAATAAP---SPSGSRSRSRRKSERPSTNRGRDNNSIRGGVA~TPESSPLPALDLTP~PPHEKEEPDTLAPRASRDAPGTPKVP~P
MPAGG--------GLURGPRGHRPGHHG--------------GAGLGR---LUPAPHH~RGAVALALLLLALAAAPPCGAAAV-TRMSA----SPTPGTGATPNDVSAEASLEEIE ~‘-------------------------------------------------------------------FVTAVVSVS~SSF~ESL~VE~T~S--------------EO,TRSAHLGOGD
MSSGCRSVGGSTUGNWRGDGGDLRPRRVLSPVCSAPMGSUIGSQLGNVGNLLATPHPLGKPASSRVGTIVLACLLLFGSCVVRAVPTTPSPP----TSTPTSMSTHSHGT~PTLL--- H---------------------------------------------------------------Vp”KHLLL,,LSFSTAC----------- --------------------------GQ HH------------YFR-----RNC,FFL,V,LyGT”S~--------------------------------------------------------------------------------
“---------------------------------------------------------------EsR,UCLWCV”LC,VCLGAAVSSSSTSHATSSTH”-GSHTSRTTSA~TRSVyS~H
M---------------------------------------------------------------TRRRVLSWVLLAALAC----------- ------RL-GAQTP-----------EQP *
121 . . 240 AIGSCAPKEPPI-------------------RNRIFACSSPT~SVARLA9PRHCHRHADST-~lEGIAWFK~NlAPYVF~LVYKHITTVTT---UALFSRP~ITNEYVTRVPIDY A-----CDYATVMGHATLREHLRDIKAEIITDANFYVCPPPT~TVV9FEQPRRCPTRPEG~-NYTEGIAWFKENIAPYKF~TMYYKOVTVS~V---UFGHRYSOFNGIFEDRAPVPF
GVTPEPSGMSEPADPAELRADLRGLKGSSDDPNFYVCPPPT~TVVRLEEPRPCPELPKGL-NFTEGIAVTFKENLAPYKFKATMYYKAVTVASV---USGYSYNPFNNIFEDRAPIPF
AFSPGPSEAPDGEYGOLOARTAVRAAA--TERDRFYVCPPPSGSTVVRLEPEPACPEYSQGR-NFTEGIAVLFKENIAPHKF~HlYYKNVIVTTV---USGSTYMITNRFTDRVPVPV
-----------------EIREAIHKSaDAETKPTFYVCPPPTGSTIVRLEPTRTCPDYHLGK-NFTEGIAWYKENIMYKF~TVYYKDVIVSTA---UAGSSYT~ITNRYADRVPIPV
-----PTETPDP------LRLAVRESGILAEDGDFYTCPPPTGSTVVRIEPPRTCPKFDLGR-NFTEGIAVIFKENIAPYKFRANWYKOIWTRV---UKGYSHTSLSDRYNDRVPVSV
TTPTTAVEKNWTQAIYPEY-------------FKYRVCSASTTGELFRFDLDRTCPSTED-KV-HKEGILLVYKKNIVPYIFKVRRYKKITTSVRIFNGUTREG-VAITNKUELSRAVPK
------------STQNYTSREWSSVQLSEEESTFYLCPPPVGSTVIRLEPPRKCPEPR~T-EUGEGIAILFKENI~YKFKVTLYYKNII~TTT---UTGTTYRQITNRYTDRTPVSI
VTSSEAVSHRANETIYNTTLKYGDWGVYTTKYPYRVCSMA~GTDLIRFERNIICTSMKPINEDLDEGIMWYKRNIVAHTFKVRVYPKVLTFRRSYA--YIYT-TYLLGSNTEYVAPPM
APPATTVPPTATRP--PTS-------------FPFRVCELSSHGDLFRFSSDI~CPSFGT-RENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNG~ADS-V--TNRHEEKFS~S
* . * . . . . . . **. . .* *. . . *.. * * .
241 . . f 360 HEIV-RIDRSGECSSKATYHKNFMFFEAYDNDE-AEKKLPLVPSLLRSTVSKAFHTTN--FTKRHaTLG----YR-TSTSVDCWEYLQARSVYPYDYFG~T~TVEISPFYT-KNTTG
EEVIDKINAKGVCRSTAKYVRNNLETTAFHRDD-HETDMELKPANMTRTSRGUHTTDLKYNP~VEAF----HRY-GTTVNCIVEEVDARSVYPYDEFVLATGDFVYNSPFYGYREG-S EEIVDRIHGRWlCLSTAKYVRNNLETTAFHNDA-DEHEMKLVPAESAPGLHRGUHTTRLKNNPTGSAUI----HRH-GTTVDCIVDEVEAKSSYPYNEFVLATWFVYASPFFGYRDG-S
QEITDVIDRRGKCVSKAEYVRNNHKVTAFDRDE-NPVEVTYTKIGAAGF----YH-TGTSVNCIVEEVEARSVYPYDSFALSTGDIVYMSPFYGLREG-A
SEITDTIDKFGKCSSKATYVRNNHKVEAFNEDK-NPPDGTPGT----YR-TGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGLRDG-A
EEIFGLIDSKGKCSSKAEYLRDNIMHHAYHDOE-DEVELDLVPSKFATPGARA~TT~TTSYVG~PU----RHYTSTSVNCIVEEVEARSVYPYOSFALSTGDIVYASPFYGLRM-A
YEI-DIMDKTYQCHNCMQIEVNGMLNSYYDRDG-NNKTVDLKP~GLTGAITRYIS~P~FADPG--UL-UGTYR-TRTTVNCEI~MFARSADPYTYFVTALGDTVEVSPFC--D~NS
EEITDLIDGKGRCSSKARYLRNNVYVEAFDRDA-GEKQVLLKPSKFNTPESRAUHTTNETYTVWGSPUI----YR-TGTSVNCIVEEMDARSVFPYSYFAMANGDIANI~FYGLSPPEA
UEI-HHINKFAPCYSSYSRVIGGTVFVAYHRDSrEYWTMP9UHSRGSTUL---- YR-ETCNLNCMLTITTARSKYPYHFFATSTGDWYISPFY--NGTNR
YET-DPMDTIYPCYNAVKMTKDGLTRVYVDRDG-VNITVNLKPTGGLANGVRRYAS~TELYDAPG--ULIU-TYR-TRTTVNCLITDnnAKSYSPFDFFVTTTG~TVEMSPFY--DGKNK
l . . . * . . * .*. * . . *** l . * . l . *** . . . . .
361 . . 480
PRRHSVYRDYRFLEIANY-PVRDLETG-~IRPPKKR-NFLTDEPFTIGUDAMEEKESVCTLSKUIEVPEAVRVSYKNS-YHFSLKDnTnTFSSGKPPFYlSRLHLAECVPTIASEAIDGI
HTEHTSYMDRFK9VDGF-YARDLTTKARATAPTTR-NLLTTPKFTVA~~PKRPSVCTMT~E~EMLRSEY-GGSFRFSSDAISTTFTTNLTEYPLSR~L~CIGKDARDAMDRI HSEHNAYMORFKQVDGF-FPRDFGTGRRHGSPVTY-NLLTTPMFTVGUNUAPKRPSVCTMT~REVPEMLRAEY-GSSFRFTSNALSATFTTNLT~YSLSR~LGDCVGKEAREAIDRl
HGEHIGYAPGRFPPVEHY-YPIOLOSRLRASESVTR-NFLRTPHFTVAWDVAPKTRRVCSLAKUREAEEMTRDETROGSFRFTSRAL~SFVSDVTQLDL~RVHL~CVLREASEAIDAI
YREHSNYAMDRFHPFEGY-RPRDLDTR-ALLEPMR-NFLVTPHLTVGUNWKPKRTEVCSLVKUREVEDWRDEYAH-NFRFTMKTLSTTFISETNEFNLNQIHLS~CVKEEARAIINRI
RIEHNSYAPERFRQVEGY-RPRDLDSKLQAEEPVTK-NFITTPHVTVSU~EKKVEACTLTKUKEVDELVRDEFR-GSYRFTIRSISSYFISNTT~FKLESAPLTECVSKEAKEAIDSI
CP--WATDVLSVPIDLNHTW-DYGNRATSPPHKKRI-FAHTLDYSVSUEAVNKSASVCSMVFUKSFPRAIQTE-HDLTYHFIANEITAGFSTVKEPLANFTSDY-NCLMTHINTTLEOK
AAEPMGYPPDNFKPLDSY-FSMDLDKRRKASLPVKR-NFLITSHFTVGUDWAPKTTRVCSMTKUKEVTEMLRATV-NGRYRFNARELSATFISNTTEFDPNRIILGPCIKREAEMIEPI
llASYFGENADKFFIFPNYTIVSDFGRPNMPETHRLVAFLERADSVIS~I~DEK~CQLTFUEASERTIRSE-AEDSYHFSSAKMTATFLSKK~EV~~SAL-DCVROEAINKL~~I
ET--FHERADSFHVRTNYKIV-DYDNRGTNPPCERRA-FLDKGTYTLSUK-LENRTAYCPL9HU~TFOSTIATE-TGKSIHFVTOEGTSSFVTNT~GIELPDAF-KCIEE~VN~MHEK
l . * . . . . *. *. .* .*. .*. . .
481 600
FARKYSSTHVRSGD-IEYYLGSGGFLIAFPKLMSHGLAEMYLEEAQRP)(HL---------------PRGRERRQMG---------------------RRTASLQSGP~GDRIT------ FARRYIUTHIKVGP-PQYYLANGGFLIAYaPLLSNTLAELYVREHLREQS---------------------RKPPNPTPPPPGASA~~ERI---------------------------
YLEKYNNTHLRVGS-VPYYLATGGFLIAYPPLLSNNLADLYVKELNREaA------- --------------LKPEERK----- LIIATTDGKV,---------------------------
YRRRYNSTHVLAGDRPEVYLARGGFVVAFRPLISNELAPRRARRSPGPAGTPEPPAVNGTGH------------------------------L
YTTRYNSSHVRTGD-IPTYLARGGFVVVFPPLLSNSLARLYLQELVRENTN-------HSPaKHPTRNTRSRRSV-------PVELRANRT----------------------------- YKKPYESTHVFSCD-VEYYLARGGFLIAFRPMLSNELARLYLNELVRSNRTYDLKNLLNPNANNNNNTTRRRRSLLSVPEP~PT~DGVHRE~ILHRLHKRAVEATAGTDSSYYTAKPLEL
IARVNNT-HTPNGTA-EYYGTEGGHILVUQPLIAI---------- -----------------ELEEA~LEATTSpVTpSApTSSS---------- RSKR-----AIRSIRDVSAGSENNV
FRTKYNDSHVKVG-HVPYFLALGGFIVAYQPVLSKSLAHHAP---NRKITLDDTTA
FNTSYNP-TYEKYGNVSVFETSGGLWF~GIKPKSLVEL---------------------ERLANRSSLNITH---------------------RTRRST~NNTTHLSSMESVHNLVY YEAVPDR-YTKGPEAITYFITSGGLLLAULPLTPRSLATV---------------------KNLTELTTPTSSPPSSPSPPAPSMRGSTPATPVPPTAPGKSLGT
..** . . . . .
[LTV
HSV
BHV-2
PRV vzv
EHV-1
HVS
MDV
HCNV
EBV
ILTV
HSV BHV-2
PRV
vzv
EHV- 1
HVS MDV
HCMV
EBV
I LTV
HSV
BHV-2
PRV
vzv EHV-1
HVS
MDV
HCMV
EBV
I LTV
HSV BHV-2
PRV
vzv
EHV- 1 HVS
MDV
HCMV
EBV
SHORT COMMUNICATIONS 409
601 . . . . . 720
--lHSSAlFAIILOFAYDKlPAHVYELlGNLLEAVCELONROLl~HENKKLNPNSLMlSLFGOPVSARLL~~VAVSKClE~p~EN-IRCW)~RVP~plNCYlRPVLlFRVSSSPESP
-KlTSSIEFARLOFTYNHIORHVWDnLCRVAIAVCELONHELlLUNEARKLNPNA~ASAlVGRRVSARML~~VSlC~v~N-V~vGN~ISsRP~CYSRPLVSF~--RYEDOC -TTlSSVEFARLOFTYNHlGKHVNENFGR~VSUCELONOELlL~EAKKl~~ASVlLHRRVSACMLWVLA~SlCVAVPAEN-VI~NSNRIPSKPGlCYSRPLLSF---KH~GE
RllTGSAEFARLOFTYDHlPH~DNLGRl~UCELONKDRlLUSENSRL~VAlMLGORVSARML~~lSRCVEVRGG--WVONSnRVPGERGTCYSRPLVTF----EHNGT lTTTSSVEFAnLOFTYDHlOEHVNENLARlSSSVCOLONRERALUSGLFP~~~STILDORV~RlL~V~SVSNCPELG~TRllLQNSllRVSGSllRCYSRPLlS~V---SLNGS
lKTTSSIEFAllLOFAYDHIOSH~ENLSRlAlA~PLONKERPLUNEMVK~TPSAlVSATLDERVMRVL~VfA~THCAKIEGN--VYLONUIR-SEY)SNlCYSRPP~FTTlKNANNR
FLS----O---lOYAYDKLRPSlNNVLEELAIT~REOVROTMWElAKl~S~TAIYGKPVSR~L~VISVTEC~N~-OSSVSIHKSLKlENND-ICYSRPPVTFKFV------
lKSlSSVOFAnLOFLYDHIOlHlNDMFSRIATAYCELONRELVLUHEG~KI~TASATLGRRVMKML~VMVSSCTAIDAES-VlLON~RV~lSlNTCYSRPLVLFS---YGENO
------AO---LOFTYDTLRCYINRALAOlAEAUC~ORRTLEVFKELSKIN~lLSAIYNKPlMRFM~VLGLASCVllN-~lS~LRDMNVKESPGRCYSRPWlFNFA------ - - LNNPAlVO---IOFAYDSLRROINRHL~LARAUCLEPKRONMVLRELTKI~T~SSIYG~VMKRL~VISVSOCVPVN-OATVlLRKSNRVPGSETNCYSRPLVSFSFl------
.*. . . . c .* . . . .** *. . . ..*.. . . . l +. ..: . . . . . . **.- . .
721 . 640 FSA~ENHNLGILGOLGEHNEILO~NLlEPCM~NHRRYFLLGENYLLYEDYTF~OV~IEEVSTFINL~ILEDLDFVPVEVIlREELRDTGTLNYDDWRYONIYNKRFRDID
----------PLVEGOLGENNELRLlRDAIEPClVGHRRYFTFGGGY~FEEYAYSHOLSR~llTVSTF~DL~LEDHEF~LEWTRHEl~SGLLDYlEVORRNOLHDLRF~~D
----------ELNEGOLCENNEIRLDRDAVEPCSVGHKRYFLF~GY~FEEYTYSHOLSR~lTAVSTF~DL~LEDHEFVPLEWlROE~~SGLLDYAEVORRNOL~LRF~~D
----------GVIEGOLU)DNELLISRDLIEPCTGNHRRYFKLGSGYVYYEDYNY~H~VPET--ISTRVlL~LLEDREFLPLEVTTREELADTCLLDYSElORRNOLHALKFYDfD
----------GlVEGPLGlDNELINSRDLLEPCVANHKRYFLFGHHY~YEDYRY~EIAVHDV~lSTY~L~LL~REFNPLO~TRDELRDTGLLDYSEIORRN~HSLRFYDID
----------GSIEGOLCEENEIFTERKLlEPUILlPKRYFKFGKEY~YENYTF~~PPTE~EV~STY~L~LLEDREFLPLEWlRAELEDTGLLDYSElORRNOLHALRFYDID
-------~LFKWLGARNEILLSESLVENCHONAETFFTAKNElYHFKNYVH~TLPVN~lLDTFLAL~FIEN~DF~VELYSSGERKLANVFDLETNFREYNYYAOS~SGLR ----------CNIOGOLGENNELLPTLEAVEPCSANHRRYFLFGSGYALFENYNF~~~~OIASlFVEL~LLEDRElLPLSWlKEELRDVGVLDYAEVARRNOLHELKFYD~N
-------~VOYCOLGEDNEILLGNHRlEECOLPSLKIFlAGNSAYEYVDYLFKRMIDLSSISTVDSWlALDlDPLENTDFRVLELYSOKELRSSNVFDLEEIMREFNSYKORV----
-------Y)TKTYEGOLGTDNEIFLTKKnlEVCOATSOYYFOSGNE~HWNDYHHFKTIELDGIATLOTFlSL~lENIDFASLELYSRDEORASNVFDLEGIFREYNF~ONIAGLR l **** * t . . l l l . . l * . . . . . * . l l . . . . . . . . . . . . . . I ‘. .
841 I 1 - *I I s s 960 lVIR---GDRGDAIFRAIADFFGNTLGEVGI(ALCTVVIITAAMSIILCLLAFKYVIINLKSNPVOVLFP---------GAVPPACTPPRPSRRY
TVIH---ADANMI(FACLGAFF-EWIGDLGRAVGKWWClVGGWSAVSGVSSFNSNPF~LAVGLLVLAGL~FFAFRY~RLOSNPM~LYP---------LlTKELK~NPDAS
TVIK---ADPNMIFAGLHCFF-EGLCDVtRAVGRWLGWGGWATVSGVSSFLSNPFGALA~GLLVLGGLVMFFAFRY~RLORNPN~LYP---------LlTKDLKH-P--SECG
RWK---VDHNWLLRGIANFF-OGLCDVWUVCKWLCATCAVISAVG~VSFLSNPFGALA~GLLVLAGLVMFLAYRHlSRLRRNPN~LYP---------VTTKTLKE--------
L(WO---YDSGTAII(PWVIOFF-OGLGTAGOAVGHWLGAT~LLSTVHGFlTFLSNPF~LAVGLLVLAGLVMFFAYRYVLKLKlS~~LYP---------LTTKGLKOLPE~PF SWN---VDNTAVIIRGSPAFS-RAVVKVGRPVERS-FSARGAWSTVSGIACFLNNPFGGLA~GLLVIAGLVMFFAYRY~lRSNPM~LYP---------IlTYALKN--I(AKTSY
KDFDNSORNNRDRlIWFSEILA-DLGSICKVIVRVASCAFSLFGGlVTGILNF~KNPLG~FTFLLlGAVIlLVlLLVRRlN~APlRMIYP-------------------------
KVIE---VDTNYAFHNGLAELF-NOlCPVCQAlGKVWGAACAlVPFGALA~GLIIIAGLVMFLAYRYVNKLKSNPM~LYP---------NTTEVLYAMTRELHG
KYVEDKWDPLPPYLKGLDDLNS-GLCMGKAVGVAItAVGGAVASWEGVAlFLKNPFCAFTIILVAIAWIITYLIYlRORRLClOPLONLFPYLVSADGTTVTSGST~TSLMPPS
WDLDNAVSNCRNOFVDCLGEL~-SLGSVGOSIlNLVSlVGGLFSSLVSGFISFF~PFGGNLlLVLVAGWlLVISLTRRTRONSOOPV~LYP------------------------- L I I I I I
. l l . . . . . . . l ***et . . . . . . . . . . . . .
. . . .
FIG. 4. Homology of ILTV “gB” with those of other herpesviruses. Multiple alignment of the amino acid sequences predrcted for the “gB”-like proteins of 10 different herpesviruses. Sequences were aligned using the CLUSTAL program (39). Asterisks indicate identical amino acids and dots represent conserved amino acid substitutions. Putative N-linked glycosylation sites are shown in bold and underlined. The signal se- quences are double underlined and the triple transmembrane domains are boxed. Conserved cysteine and proline residues are shown bym and =, respectively.
functions, namely viral entry and cell fusion (31). lines constitutively expressing the gB homologue (gl) of Whether the product of ILTV gB homologue shares BHV-1 (32). these functions remains to be seen. There is evidence Finally, of special interest is the importance of ILlV that some of the proteins are functionally equivalent. gB in eliciting protective immunity in infected chickens. The gll-negative PRV mutant was able to grow on cell There is good evidence that gB, as well as other viral
410 SHORT COMMUNICATIONS
glycoproteins, plays an important role in the host im- mune response. HSV gB has been demonstrated, ei- ther by temperature-sensitive mutant studies (33) or as an immunopurified protein (34, to invoke circulating antibody and cell-mediated responses which pro- tected mice against lethal challenge with the virus. HSV gB expressed by the recombinant vaccinia vector (35) or by the recombinant adenovirus vector (36) pro- tected mice against lethal challenge. Vaccination of rabbits with a recombinant vaccinia virus expressing the HCMV gB homologue produced antiserum which could neutralize HCMV infectivity in vitro (28). Injection of the purified gNgB glycoprotein complex from HCMV induced both humoral and cellular immune responses in humans (37). A subunit vaccine containing essen- tially only glycoproteins of the 205K complex protected 100% of chickens against clinical disease and also against viral replication (J. J. York and K. J. Fahey, per- sonal communication). This suggests that gB could prove to be a major protective immunogen of ILlV and therefore is a prime candidate for inclusion in a subunit or recombinant vaccine.
12. 13.
14.
15.
16. 17.
18.
19.
20.
ACKNOWLEDGMENTS
21.
22.
23.
24.
25.
The authors thank Steven Rhodes for his technical assistance, Julie Harris for preparing CK cells, Peter McWaters for providing the monoclonal antibody supernates, and Sandra McAuliffe for prepara- tion of the manuscript. This work was supported by Arthur Webster Pty Ltd, Sydney, Australia.
26.
27.
28.
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