Institute of Molecular Biology, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany1
Author for correspondence: Walter Fuchs.Fax +49 38351 7151. e-mail Walter.Fuchs{at}rie.bfav.de
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Abstract |
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Introduction |
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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 US 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 UL 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 UL 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 UL 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 US 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 UL 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
).
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Methods |
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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
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 [
-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-
UL10G (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-
UL10G (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-
UL10G 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 [-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.
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Results and Discussion |
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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.
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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 TATAT/AAT/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).
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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. 4
a, 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).
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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-
UL10G; 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 UL10G 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
UL10G 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-
UL10G (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
UL10G 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
UL10G 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
UL10G (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.
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Acknowledgments |
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Footnotes |
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Received 27 January 1999;
accepted 28 April 1999.