DNA sequence of the UL6 to UL20 genes of infectious laryngotracheitis virus and characterization of the UL10 gene product as a nonglycosylated and nonessential virion protein

The molecular analysis of the genome of infectious laryngotracheitis virus (ILTV, gallid herpesvirus-1) is of particular interest for two reasons. First, infectious laryngotracheitis is an economically important respiratory disease of chickens, and prevention might be substantially improved by the development of genetically engineered vaccines (Bagust & Guy, 1997 ). Second, our knowledge of ILTV might contribute to a more profound understanding of herpesvirus evolution. Previous studies indicated that, although classified as a member of the Alphaherpesvirinae subfamily of the Herpesviridae (Roizman, 1996 ), ILTV exhibits a considerable phylogenetic distance to all other mammalian and avian herpesviruses investigated so far (Griffin & Boursnell, 1990 ; McGeoch & Cook; 1994 ; Johnson & Tyack, 1995 ). On the other hand, early DNA analyses demonstrated that ILTV possesses a typical alphaherpesvirus class D genome consisting of a long (U_L) and a short (U_S) unique region, which is flanked by inverted repeat (IR, TR) sequences (Leib et al., 1987 ; Johnson et al., 1991 ; Roizman, 1996 ).

Up to now, ca. 90 kbp of the 155 kbp ILTV genome have been analysed by DNA sequencing (Fig. 1a). The characterized genome parts include the entire U_S region, which encodes a cluster of conserved alphaherpesvirus glycoproteins (Wild et al., 1996 ), and the adjoining inverted repeat sequences encoding the ILTV homologue of the major immediate early protein ICP4 of herpes simplex viruses (Johnson et al., 1995 ). The DNA sequences of the left and right ends of the U_L genome region (Fuchs & Mettenleiter, 1996 ; Johnson et al., 1997 ) indicated a collinear arrangement of conserved alphaherpesvirus genes also within this part of the genome. As in the completely sequenced type D genomes of varicella-zoster virus (VZV; Davison & Scott, 1986 ) and equine herpesvirus-1 (EHV-1; Telford et al., 1992 ), the U_L region of ILTV is apparently fixed in the opposite orientation to the prototypic isomer of the herpes simplex virus type 1 (HSV-1) E genome (McGeoch et al., 1988 ). However, recent sequence analyses revealed several unique features of the ILTV genome. One of them is a large internal inversion within the U_L region, which includes the UL22 to UL44 gene homologues (Ziemann et al., 1998a ), and another is the translocation of a UL47 homologous gene to the U_S region (Wild et al., 1996 ). Even more salient was the identification of a considerable number of apparently expressed open reading frames (ORFs) which are presumably ILTV-specific, since they exhibit neither structural nor positional identity to any other known herpesvirus genes. A set of five unique ORFs was found to be clustered adjacent to a functional origin of replication between the conserved UL22 and UL45 genes (Ziemann et al., 1998a ). Two other ILTV-specific genes, which are located at the right end of the U_L region (Fuchs & Mettenleiter, 1996 ; Ziemann et al., 1998b ), are related to each other but not to the ICP0 genes found at a similar position within other alphaherpesvirus genomes (Everett et al., 1993 ).

(41K): Fig. 1. Genome structure of ILTV. (a) The herpesvirus type D genome of ILTV consists of a unique long (UL) and a unique short (US) region, which is flanked by inverted repeat (IR, TR) sequences. Previously sequenced segments are shaded, and locations of KpnI restriction fragments as well as selected genes are indicated. The depicted viral inserts of cosmids pCI-X12, -SB27,-E19 and -S28 were used for sequence analysis of the genomic DNA fragment KpnI A. (b) Enlarged map of the analysed genome part with KpnI and EcoRI (short vertical bars) restriction sites. Nucleotide numbers refer to GenBank entry AJ131832. The identified ORFs (pointed rectangles) are named according to their homologues in HSV-1. Identified viral mRNAs are indicated by dotted arrows. For mRNA detection (Fig. 2), the labelled cRNA probes AD were transcribed in vitro from plasmids pILT-E42 and -E38, respectively. The synthetic oligonucleotide primers U15-R (nt 72297247) and U15-F (reverse of nt 1018210164) were used for reverse transcription, amplification and sequencing of the spliced UL15 mRNA (Fig. 3). (c) Plasmid pBl-SX3.8 contains the authentic UL10 gene of ILTV. An EcoRVPvuII fragment comprising UL10 codons 164342 was replaced by a GFP expression cassette in pBl-ΔUL10G, which was used for generation of ILTV recombinants. For prokaryotic expression, an EcoRI fragment containing the 3'-terminal codons 320393 of the UL10 ORF was fused to the GST gene in pGEX-UL10.

To continue our investigations of the ILTV genome, we sequenced part of the U_L region located between the characterized UL5 (Fuchs & Mettenleiter, 1996 ) and UL21 (Ziemann et al., 1998a ) genes. This region includes the ILTV homologue of the UL10 gene of HSV-1, which was shown to encode a nonessential virion glycoprotein, gM (Baines & Roizman, 1993 ). The gM gene remained the last of the so far described alphaherpesvirus glycoprotein genes to be detected in the ILTV genome, since homologues of the genes encoding gB (Griffin, 1991 ), gC (Kingsley et al., 1994 ), gD, gE, gG, gI, gp60 which is a positional homologue of gJ (Wild et al., 1996 ), gK (Johnson et al., 1997 ), gL (Fuchs & Mettenleiter, 1996 ), gH and gN (Ziemann et al., 1998a ) were already described (Fig. 1a). However, only the gB, gC, gG and gp60 gene products of ILTV were identified and characterized up to now (Kongsuwan et al., 1993a ,b ; Kingsley et al., 1994 ; Poulsen & Keeler, 1997 ). To identify the predicted gM protein of ILTV, a part of the UL10 ORF was expressed in E. coli, and a rabbit antiserum was prepared against the fusion protein and tested in different immunochemical assays. Furthermore, a UL10-negative ILTV mutant was generated. Virus and cells.
A pathogenic ILTV strain (obtained from D. Lütticken, Boxmeer, NL) was propagated in primary chicken kidney cells as described earlier (Fuchs & Mettenleiter, 1996 ). Transfection experiments were performed with the chicken hepatoma cell line LMH (Kawaguchi et al., 1987 ), which was maintained in minimum essential medium supplemented with 10% foetal calf serum. For plaque assays, ILTV-infected LMH cells were overlaid with medium containing 0·8% methyl cellulose.

Cosmid and plasmid cloning of ILTV DNA.
Viral DNA was prepared from infected chicken kidney cells as described previously (Fuchs & Mettenleiter, 1996 ), partially digested with Sau3AI, and dephosphorylated at the 5' ends. The DNA was then ligated to BamHI-digested vector SuperCos I, packaged in vitro into λ phage particles (Gigapack III XL), and used for infection of E. coli strain XL-1 Blue MR (all purchased from Stratagene). In another approach, bacteria were transformed with EcoRI-digested ILTV DNA which had been ligated into plasmid pBS(-) (Stratagene). DNA was prepared from the obtained cosmid and plasmid clones (Qiagen plasmid kit, Qiagen) and characterized by restriction analyses, Southern blot hybridizations and sequencing of the insert termini with vector-specific T3 and T7 primers.

DNA sequencing.
The entire sequence of the 24 kbp KpnI fragment A of ILTV DNA was determined using cosmids pCI-E19, -S28, -SB27 and -X12 (Fig. 1a). Starting from the characterized flanking regions (Fuchs & Mettenleiter, 1996 ; Ziemann et al., 1998a ) and from the known internal sequences of plasmid-cloned EcoRI fragments of ILTV DNA (pILT-E38,-E42, -E46; Fig. 1b), custom-made primers (GibcoBRL) were derived. Sequencing reactions were perfomed with the Thermo Sequenase cycle sequencing kit (Amersham) and [α-³⁵S]dATP (ICN) for labelling. The products were separated on denaturing 5% polyacrylamide gels containing 7 M urea in Trisborate electrophoresis buffer, which were then dried and exposed to X-ray film (X-OMAT AR, Kodak). The obtained sequence data were assembled and analysed with the GCG software package in UNIX version 9.1 (Devereux et al., 1984 ).

Northern blot analyses.
Total RNA was prepared from noninfected chicken kidney cells and from cells harvested 16 h after ILTV infection at an m.o.i. of 5. The RNA was separated in agarose gels, transferred to nylon membranes and hybridized with ³²P-labelled RNA probes as described previously (Fuchs & Mettenleiter, 1996 ). Strand-specific probes were transcribed in vitro from plasmids pILT-E38 and -E42 (Fig. 1b) with T3 and T7 RNA polymerases, respectively.

Reverse transcription and PCR amplification of viral mRNA.
RNA of ILTV-infected and noninfected cells (5 µg each) was hybridized with 2·5 pmol of the primer U15-R (nt 72297247) and incubated for 1 h at 42 °C with 200 U reverse transcriptase (SuperScript II, GibcoBRL). After 15 min incubation at 70 °C template RNA was digested with RNases H and T1 for 30 min at 37 °C. Aliquots of the cDNAs, or 10 ng of genomic ILTV DNA as control, were amplified by PCR with 20 pmoles of primers U15-R and U15-F (reverse of nt 1016410182), 2·5 nmol of each dNTP and 1 U Deep Vent DNA polymerase (New England Biolabs). An initial denaturation step for 1 min at 97 °C was followed by 35 cycles of 95 °C and 55 °C for 30 s each, and 72 °C for 3 min (Primus 96 Thermocycler, MWG Biotech). After phenol extraction and ethanol precipitation, the PCR products were treated with Klenow polymerase and polynucleotide kinase, purified from agarose gels (Qiaquick gel extraction kit, Qiagen), cloned into SmaI-digested plasmid pBS(-), and analysed by DNA sequencing.

Generation of a UL10-negative ILTV mutant.
To obtain a transfer plasmid for deletion of the ILTV UL10 gene the green fluorescent protein (GFP) expression vector pEGFP-N1 (Clontech) was doubly digested with BglII and BamHI and religated to remove the multiple cloning site located between the GFP reading frame and the preceding human cytomegalovirus (HCMV) immediate early gene promoter. The modified expression cassette was excised as a 1581 bp AseIAflII fragment, blunt-ended with Klenow polymerase and inserted into the polylinker region of the SmaI-digested plasmid pBluescript SK(-) (Stratagene). In the same vector, a 3831 bp SpeIXhoI fragment of the ILTV genome was subcloned from cosmid pCI-E19. From the resulting plasmid pBl-SX3.8 (Fig. 1c), a 1973 bp SpeIEcoRV fragment and a 1324 bp PvuIIXhoI fragment were subsequently recloned in the novel GFP expression vector, which had been treated with BamHI, Klenow enzyme and SpeI, or EcoRV and XhoI, respectively. The final plasmid, pBl-ΔUL10G (Fig. 1c), carries a 534 bp deletion of ILTV DNA sequences (nt 1510315636) representing UL10 codons 164342, which are replaced by the GFP reporter gene. Subconfluent monolayers of LMH cells were transfected with pBl-ΔUL10G (ca. 10 µg per 10⁵ cells) by calcium phosphate coprecipitation (Mammalian transfection kit, Stratagene). After 24 h the inoculum was removed, the cells were infected with ILTV at an m.o.i. of 1, and further incubated at 37 °C until complete lysis occurred. Serial dilutions of the progeny virus and plaque assays were performed to determine the ratio between GFP-expressing recombinants and wild-type ILTV, which was monitored in a fluorescence microscope between 2 and 5 days after infection. The ILTV recombinants were then isolated by limiting dilutions of the transfection progeny on primary chicken kidney cells grown in microtitre plates. The procedure was repeated until the selected virus populations appeared homogeneous, and a single GFP-expressing isolate designated ILTV-ΔUL10G was further analysed.

Southern blot analyses.
Restriction fragments of ILTV DNA (1 µg per lane) were separated in 0·7% agarose gels and transferred to Nylon membranes (Hybond-N⁺, Amersham) by standard procedures. The blots were incubated overnight in 720 mM NaCl, 80 mM Na₂HPO₄, 4 mM EDTA, 1% SDS, 0·5% low-fat milk, 0·5 mg/ml denatured herring sperm DNA at 62 °C. After 6 h, the probes were added, which had been prepared by labelling (Rediprime system, Amersham) of plasmid DNA with [α-³²P]dCTP (ICN). Blots were washed at 62 °C twice with 2x SSC (300 mM NaCl, 30 mM sodium citrate), 0·5% SDS, and three times with 0·1x SSC, 0·5% SDS for 30 min each, and finally exposed to X-ray film.

Prokaryotic expression, preparation of antiserum and Western blot analysis.
A 414 bp EcoRI fragment encoding the C terminus (aa 320393) of the predicted UL10 protein was recloned from pBl-SX3.8 into expression vector pGEX-4T2 (Pharmacia). An expected 37 kDa fusion protein was expressed and purified as described (Fuchs et al., 1996 ). A rabbit was immunized four times at 2 week intervals by intramuscular injection of 100 µg of the fusion protein emulsified in mineral oil. Sera collected before and after immunization were analysed. For that purpose, lysates of ILTV-infected (m.o.i. of 5, 24 h after infection) and noninfected chicken kidney cells were separated on discontinuous SDSpolyacrylamide gels (Laemmli, 1970 ), and electrotransferred to nitrocellulose filters (TransBlot cell, Bio-Rad). The blots were subsequently incubated for 1 h each with 5% low-fat milk, rabbit antisera and peroxidase-conjugated secondary antibodies (Dianova), all diluted in TBS-T (10 mM TrisHCl pH 8·0, 150 mM NaCl, 0·25% Tween 20). After repeated washing, antibody binding was visualized by luminescence (ECL Western blot detection system, Amersham) and recorded on X-ray film.

Glycosidase treatment of virion proteins.
ILT and pseudorabies virions were sedimented from the supernatants of infected cells, and further purified by centrifugation through step-gradients of 30, 40 and 50% sucrose (Klupp et al., 1992a ). The particles collected from the interface between 40% and 50% sucrose were first treated with neuraminidase (1 mU/µg protein) in 50 mM sodium acetate (pH 5·2), 4 mM CaCl₂ for 2 h at 37 °C and then sedimented for 1 h at 45000 r.p.m. in a Beckman TLA45 rotor. For treatment with O-glycosidase (0·1 mU/µg) the pellet was resuspended in 20 mM Trisphosphate (pH 7·4), and incubation with N-glycosidase F (20 mU/µg) was performed in 50 mM potassium phosphate (pH 7·2), supplemented with 50 mM EDTA and 0·5% CHAPS (all enzymes were purchased from Boehringer Mannheim). After 20 h at 37 °C, proteins (ca. 5 µg per lane) were denatured for 5 min at 56 °C in sample buffer (Laemmli, 1970 ) containing 2% SDS and 5% ß-mercaptoethanol, and subjected to electrophoresis and Western blot analysis as described above.

Sequence of KpnI fragment A of ILTV DNA
Physical mapping of the ILTV genome (Johnson et al., 1991 ) revealed that the largest KpnI restriction fragment A is localized within the right part of the U_L region. We sequenced this fragment using cosmids which contain overlapping, randomly cloned ILTV DNA fragments (Fig. 1a). The sequence was verified by subsequent analysis of at least two independent clones for every part of the analysed genome region with ILTV-specific custom primers. To minimize band compressions and nonspecific termination, all sequence reactions were performed with a thermostable DNA polymerase at 72 °C in the presence of 7-deaza dGTP. Therefore, only very few sequence ambiguities had to be resolved by reverse-strand analysis. The total length of the assembled DNA sequence (GenBank accession no. AJ131832) is 24300 bp, and no differences were found between the duplicate clones used for characterization. The novel sequence overlaps with described parts of the ILTV genome at both ends. From position 1 to 22 the sequence corresponds to the 5' upstream region of UL21 (Ziemann et al., 1998a ; GenBank no. Y14301), and from position 24140 to 24300 the sequence overlaps with the UL5 gene (Fuchs & Mettenleiter, 1996 ; GenBank no. X97256). The KpnI fragment A itself is 24124 bp in size, and contains 44·9% G and C residues, which is in good agreement with the predicted overall G+C content of ILTV DNA of 45% (Plummer et al., 1969 ).

Localization and putative functions of conserved genes
The newly determined ILTV DNA sequence contains 14 ORFs sharing homologies with characterized alphaherpesvirus genes (Table 1) whose arrangement (Fig. 1b) is almost perfectly collinear to that found in HSV-1, VZV, EHV-1, bovine herpesvirus-1 (BHV-1) and pseudorabies virus (PrV) DNA (Davison & Scott, 1986 ; McGeoch et al., 1988 ; Telford et al., 1992 ; Vlcek et al., 1995 ; Klupp et al., 1992b ; Dijkstra et al., 1997a ). Within the analysed genome region no unique genes of ILTV could be detected between the conserved ORFs. However, one conserved ORF of the above-listed mammalian alphaherpesviruses, but also of beta- and gammaherpesviruses, like HCMV (Chee et al., 1990 ) and EpsteinBarr virus (EBV, Baer et al., 1984 ), is apparently absent from the corresponding position in the ILTV genome. In HSV-1, this gene was named UL16, and its product was identified as a capsid-associated virion protein which is dispensable for virus replication in vitro (Nalwanga et al., 1996 ). Therefore, absence of a corresponding ORF from the ILTV genome would be imaginable, but in analogy to UL47 (Wild et al., 1996 ), the UL16 gene of ILTV might also reside at an as yet unknown position.

Table 1. Homologous herpesvirus proteins

Using the GCG program `Gap', the predicted ILTV proteins were compared pairwise with their homologues to determine the percentage of positionally conserved, identical amino acid residues (Table 1). The results of these studies clearly confirmed the classification of ILTV as an alphaherpesvirus, since related proteins of beta- and gammaherpesviruses exhibited much lower degrees of identity, if detectable at all. However, comparison of conserved proteins of different mammalian alphaherpesviruses to each other, for example that of HSV-1 to that of VZV, in most cases revealed significantly greater homologies than that found with the ILTV gene products (Table 1). Only few sequence data are available from the corresponding genome regions of other avian alphaherpesviruses. The predicted products of UL19 of turkey herpesvirus (SWISS-PROT no. Q88528), and UL9 of Marek's disease virus (Wu et al., 1996 ) share only 37·3% and 40% of identical amino acids with the respective ILTV proteins, demonstrating that there is no close relationship between these viruses. Thus, in agreement with profound phylogenetic studies (McGeoch & Cook, 1994 ), our results confirm that ILTV should be considered as the only member of an alphaherpesvirus genus which evolved separately from the mammalian and other avian members of this herpesvirus subfamily.

Except for UL7 and UL14, gene functions were assigned to all HSV-1 counterparts of the newly identified ORFs of ILTV (Table 1; Roizman & Sears, 1996 ). In addition to their overall homologies, several of the predicted ILTV proteins contain distinct domains, indicating a similar virion localization or role during virus replication. For example, the deduced UL20 gene product of ILTV contains four extended hydrophobic regions which could represent transmembrane domains (not shown). The UL11 protein might be membrane-anchored by myristic acid, since an N-myristylation site was identified at amino acids 27 with the GCG program `Motifs'. Using the same program, a consensus sequence of serine/threonine protein kinases was found within the UL13 gene product of ILTV (aa 219231). Within the predicted origin binding protein (UL9) both an ATP/GTP binding site (aa 102109) and a leucine zipper (aa 171193) are conserved.

Transcriptional analysis
In Table 2 the precise locations and sizes of the identified ILTV ORFs are listed together with the positions of putative transcription signals. Assuming usage of the first in-frame ATG codons, several of the adjacent ORFs overlap by up to 224 nucleotides (Table 2). TATA box-like elements which fit the consensus sequence TATA^T/_AA^T/_A (Breathnach & Chambon, 1981 ) with maximally one mismatch could be identified upstream of all predicted genes, whereas putative polyadenylation signals (AATAAA; Wickens, 1990 ) were not found behind every ORF (Table 2). This finding indicates that UL6 and 7, UL8 and 9, UL11 and 12, UL13 and 14, as well as UL18, 19 and 20, are presumably expressed from 3'-coterminal sets of transcripts. The calculated minimum sizes of the expected viral mRNAs are listed in Table 2, but the addition of poly(A) tails has to be considered. Until now, the apparent transcript sizes were determined only for one segment of the analysed genome part by Northern blot analyses (Fig. 2). Total RNA harvested 16 h after ILTV infection of cultured chicken kidney cells was separated, blotted and hybridized with in vitro-transcribed RNA probes of plasmids pILT-E38 (Fig. 2c) or pILT-E42 (Fig. 2a,b; see also Fig. 1b). Probe A strongly reacted with presumably two slightly different RNAs of ca. 1·5 kb, which should both represent transcripts of the UL10 gene of ILTV. Probe B detected three much less abundant viral mRNAs, of which the 5·7 and 2·9 kb species fit the expected sizes of the coterminal UL9 and UL8 mRNAs, respectively (Table 2). An additional ILTV-specific RNA of 3·5 kb (UL8.5) might encode an amino-terminally truncated form of the UL9 protein, as was described for HSV-1 (Baradaran et al., 1994 ). The expression of coterminal transcripts of the UL12 and UL11 genes of ILTV could be verified by blot-hybridization with probe C, which detected viral RNAs of 1·9 and 0·5 kb (Fig. 2). The additional faint signal at 4 kb probably represents a read-through transcript of the upstream UL14 and UL13 genes. In accordance with the absence of complementary ORFs, with probe D (Fig. 1b) no viral RNAs could be identified (not shown).

Table 2. Properties of the identified ILTV genes

Table 2.

The highly conserved UL15 protein, which is required for cleavage and encapsidation of the viral genome, was shown to be expressed from spliced mRNAs in HSV-1 and other herpesviruses (Baines & Roizman, 1992 ). To verify that the homologous ILTV gene has a similar structure, and to determine the precise splice junction between exons UL15A and UL15B, a part of the UL15 mRNA from ILTV-infected cells was reverse-transcribed and amplified with synthetic primers U15-F and U15-R (Fig. 1b). Whereas the PCR product of genomic ILTV DNA is 2954 bp in size, a 587 bp fragment could be amplified from cDNA (Fig. 3a). The additional occurrence of the larger product is most likely caused by traces of viral DNA in the RNA preparation. Sequencing of the cloned cDNA fragment revealed that the UL15 mRNA is spliced between codons 369 and 370 (Fig. 3b), and that the entire ORF therefore consists of 764 codons (Table 2). As expected, both ends of the intron fit the consensus sequences for eukaryotic splice donor and acceptor sites (Breathnach & Chambon, 1981 ; Fig. 2b).

(20K): Fig. 3. Analysis of the ILTV UL15 mRNA. (a) Genomic ILTV DNA (lane 1), and reverse-transcribed RNAs of ILTV-infected (lane 2) and noninfected (lane 3) chicken kidney cells were amplified by PCR with primers U15-R and U15-F (Fig. 1b). DNA markers (M) and PCR products were separated in a 0·8% agarose gel. Sizes were determined by DNA sequencing. (b) Sequencing of the amplified UL15 cDNA revealed a large deletion between nt 10023 and 7655 of genomic ILTV DNA. Both intronexon boundaries match the consensus sequences of eukaryotic splice donor and acceptor sites, respectively (underlined and shaded nucleotides). Amino acid positions of the predicted UL15 protein are indicated.

Identification of the UL10 gene products of ILTV
The deduced UL10 gene product of ILTV consists of 393 amino acids (Table 2) with a predicted molecular mass of 43·1 kDa and an isoelectric point of 10·12, and it possesses homologues in all herpesvirus subfamilies (Table 1). All described UL10 homologues exhibit characteristics of multiply inserted membrane proteins containing six to eight hydrophobic domains and a more hydrophilic C-terminal region (Lehner et al., 1989 ). Hydrophilicity profiles of the predicted ILTV protein were determined with the GCG program `Peptidestructure' and revealed eight clusters of nonpolar amino acids, which may serve as transmembrane domains or signal sequences, respectively (data not shown). Furthermore, all previously characterized UL10 homologues contain at least one putative N-glycosylation site (N-X-T/S; Kornfeld & Kornfeld, 1985 ), which is located close to a conserved cysteine residue between the first and second hydrophobic region (Dijkstra et al., 1996 ). Within the respective ILTV gene product this cysteine residue is present at position 51, but no consensus motifs for the addition of N-linked glycans could be detected within the entire amino acid sequence. To verify whether this might be a particular feature of the investigated virus isolate, we cloned and sequenced the UL10 genes of six additional field and vaccine strains of ILTV after PCR amplification of viral DNA. Sequence comparison revealed few differences, but none of the deduced proteins specified an N-glycosylation site (data not shown).

The UL10 gene products of several herpesviruses, including HSV-1, PrV, EHV-1, BHV-1, HCMV and EBV were already characterized as glycosylated virion proteins, and were therefore designated as gM (Lehner et al., 1989 ; Baines & Roizman, 1993 ; Dijkstra et al., 1996 ; Pilling et al., 1994 ; Wu et al., 1998; Lake et al., 1998 ). To identify the homologous protein of ILTV, we expressed the hydrophilic C-terminal part of the UL10 gene (aa residues 320393) as a glutathione S-transferase (GST) fusion protein in E. coli (Fig. 1c). A rabbit antiserum raised against this fusion protein was tested in Western blot analyses and was shown to recognize two proteins with apparent molecular masses of 36 and 31 kDa in ILTV-infected cells (Fig. 4a, upper panel). These proteins were not detected by the respective preimmune serum, and the specific reactions of the anti-UL10 serum could be completely abolished by preincubation with the bacterial UL10 fusion protein, but not with other GST-fused expression constructs (data not shown). After metabolic labelling of ILTV-infected chicken cells with [³⁵S]methionine, both the 36 and 31 kDa UL10 gene products were also detectable by immunoprecipitation, and indirect immunofluorescence reactions of the anti-UL10 serum in infected cells, which had been fixed and permeabilized with a 1:1 mixture of methanol and acetone, revealed local accumulations of the detected proteins in the cytoplasm, which might represent virion-containing Golgi-derived vesicles (data not shown). Finally, the presence of the ILTV gM homologue in sucrose-gradient-purified virion preparations could be demonstrated (Fig. 4b, upper panel).

(52K): Fig. 4. Western blot analyses. Proteins were separated in SDS gels containing 12% (a; b, upper panel) or 10% (b, lower panel) polyacrylamide, and blotted to nitrocellulose membranes. Location of protein standards is indicated on the left. (a) Total proteins of noninfected chicken kidney cells (n.i.), and of cells infected with either wild-type (WT), or UL10-negative (ΔUL10G) ILTV at an m.o.i. of 5 for 24 h were incubated with monospecific rabbit antisera. The detected UL10 and UL10' (upper panel), or UL0 (lower panel) gene products of ILTV are indicated by arrowheads. (b) Purified ILT (upper panel) or pseudorabies (lower panel) virions were treated with either neuraminidase and O-glycosidase (O-Glyc), N-glycosidase F (N-Glyc) or no enzyme (undig). The separated ILTV proteins were incubated with a UL10-specific rabbit antiserum, and multiple forms of the PrV gB were detected by a monoclonal antibody (for details see text).

Two forms of the UL10 gene product of ILTV could be identified reproducibly. These might represent differentially processed proteins, or result from initiation of translation at different sites. The latter possibility is supported by the detection of presumably two distinct UL10 mRNAs (Fig. 2 a). Besides the predicted initiation codon, there are additional in-frame ATG codons at positions 20, 89 and 97 of the UL10 ORF. The calculated molecular masses of the deduced proteins are 43·1, 40·8, 33·6 or 32·6 kDa, respectively. Furthermore, there are putative signal peptidase cleavage sites (von Heijne, 1986 ) behind the first and second hydrophobic domains at positions 39 and 114 of the predicted protein. Cleavage would reduce the molecular masses to 38·6 or 30·7 kDa, which best correspond to the electrophoretic mobilities of the two detected proteins (Fig. 4). However, an aberrant electrophoretic behaviour of highly hydrophobic proteins, as implicated by in vitro translation studies of the UL10 homologue of PrV (Dijkstra et al., 1996 ), should also be considered.

To test for possible glycosylation of the UL10 proteins, gradient-purified ILT virions were treated with either N-glycosidase F or neuraminidase and O-glycosidase and subsequently analysed in Western blot with the anti-UL10 serum (Fig. 4b, upper panel). As expected from the known amino acid sequence, no N-linked sugars are present. Incubation with O-glycosidase does also not increase the electrophoretic mobility of any form of the UL10 protein. In contrast, analysis of the same samples with an ILTV-specific hyperimmune serum revealed alterations of several as yet uncharacterized proteins after treatment with either enzyme (data not shown). As an additional control, pseudorabies virions were prepared, treated similarly, and Western blots were incubated with a monoclonal antibody specific for gB. This protein is proteolytically cleaved during processing (Whealy et al., 1990 ), and besides traces of the unprocessed gBa (110 kDa), the chosen antibody detects the amino-terminal fragment gBb (68 kDa). Mobility shifts after glycosidase treatment revealed that PrV gB is N-, as well as O-glycosylated (Fig. 4b, lower panel).

Up to now, ILTV is the only herpesvirus species shown to generally express a nonglycosylated gM homologue. However, a similar phenomenon was observed in several PrV mutants, which exhibit spontaneous base changes within the conserved N-glycosylation consensus sequence and consequently express nonglycosylated UL10 proteins (Dijkstra et al., 1998 ). Another membrane protein, the UL49.5 gene product, which is glycosylated in several herpesviruses, was recently demonstrated to form a disulfide-linked complex with the gM protein in PrV, BHV-1 and EBV (Jöns et al., 1998 ; Lake et al., 1998 ; Wu et al., 1998 ). Within the ILTV genome a conserved UL49.5 gene was also identified (Ziemann et al., 1998a ), and characterization of its product is in progress.

Construction of a UL10-negative ILTV mutant
Although gM is structurally conserved throughout the herpesvirus family, little is known about its function. UL10 deletion mutants could be isolated from HSV-1, PrV and EHV-1 (Baines & Roizman, 1993 ; MacLean et al., 1993 ; Dijkstra et al., 1996 ; Osterrieder et al., 1996 ), demonstrating that gM is not required for replication of these viruses in cell culture. To investigate whether this is also the case for the homologous protein of ILTV we used a plasmid-cloned viral DNA fragment to substitute UL10 by a GFP expression cassette (pBl-ΔUL10G; Fig. 1c). Due to the overlapping UL9 gene (Table 2), only a part of the UL10 ORF comprising codons 164342 was deleted. As a consequence of this mutation the amino-terminal part including the first three hydrophobic domains of the UL10 protein might still be expressed, whereas the retained 3'-terminal gene fragment lacks an in-frame start codon. To generate an ILTV recombinant, chicken hepatoma cells (LMH) were transfected with the deletion plasmid, and infected with wild-type virus 24 h later. Virus progeny was screened for GFP-expressing recombinants and one of them was plaque-purified and further characterized.

Genomic DNA of the obtained ILTV mutant ΔUL10G and of wild-type virus was digested with EcoRI and analysed by Southern blot hybridization (Fig. 5). As expected, the radiolabelled plasmid pBl-SX3.8 (Fig. 1c) detected four 5·5, 1·1, 1·0 and 0·4 kbp fragments of wild-type ILTV DNA (Fig. 5b). In the genome of ILTV ΔUL10G the sizes of the 5·5 and 0·4 kbp fragments are altered to 5·1 and 2·0 kbp, respectively (Fig. 5b). This is the expected consequence of the deletion of one, and the introduction of another EcoRI site in the transfer plasmid pBl-ΔUL10G (Fig. 1c). Furthermore, a probe derived from the deleted UL10 gene reacted only with wild-type ILTV DNA (Fig. 5c), and a GFP gene probe hybridized only with the 2·0 kbp EcoRI fragment of the ILTV ΔUL10G genome (Fig. 5d). Analysis of the ethidium-bromide-stained gels (Fig. 5a) revealed no other than the described alterations of restriction patterns, and even by over-exposure of the blots no traces of the wild-type genome could be detected in ILTV ΔUL10G DNA. The successful UL10 gene deletion was further confirmed by Western blot analyses. Equal amounts of the ILTV-specific UL0 protein (Ziemann et al., 1998b ) were detectable 24 h after infection of chicken kidney cells with either wild-type virus, or ILTV ΔUL10G (Fig. 4a, lower panel). In contrast, no UL10 protein was detectable in cells infected with the virus mutant (Fig. 4a, upper panel). Strictly speaking, this result only demonstrates the absence of the C-terminal part of the ILTV UL10 protein, which was used for prokaryotic expression and antiserum preparation (Fig. 1c). Although the 5'-terminal fragment of UL10 is maintained in our virus mutant, it appears unlikely that this fragment, if stably expressed at all, still encodes a functional protein. We therefore conclude that, like its homologues in other alphaherpesviruses, the UL10 gene of ILTV is not required for replication in cell culture.

(59K): Fig. 5. DNA analysis of the ILTV UL10 deletion mutant. EcoRI-digested DNA of recombinant (ΔUL10G) and wild-type (WT) ILTV was separated on 0·7% agarose gels, stained with ethidium bromide (a) or transferred to nylon membranes. Blots were hybidized with the 32P-labelled plasmid pBl-SX3.8 (b, see Fig. 1c), and with probes derived from the ILTV UL10 (c) or GFP (d) genes, respectively. When compared to the DNA markers indicated on the left, the electrophoretic mobilities of the detected DNA fragments are in good agreement with their expected sizes denoted on the right.

Preliminary investigations of the in vitro growth properties of ILTV ΔUL10G indicated deficiencies with respect to both plaque sizes and virus titres (data not shown). However, trans complementing cell lines and virus revertants have to be generated to determine the phenotypic effects of the UL10 gene deletion precisely. Since recent studies demonstrated that gM-negative PrV is attenuated in its natural host, the pig (Dijkstra et al., 1997b ), in vivo experiments with ILTV ΔUL10G might also contribute to the development of improved ILTV vaccines for chickens. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, FU 395/1-1) and a grant from Intervet Intl BV. We thank D. Lütticken and O. Werner for providing ILTV strains, R. Riebe for preparation of primary chicken cell cultures, E. Mundt for help with the rabbit immunization, and B. G. Klupp and E. Weiland for providing the PrV-specific antibody. The technical assistance of Ch. Ehrlich, J. Jürgensen, H. Ludwig and D. Zühlke is greatly appreciated.

Footnotes

The nucleotide sequence reported here has been deposited in GenBank and assigned the accession no. AJ131832.