Institutes of Molecular Biology1, of Infectology2 and of Diagnostic Virology3, Friedrich-Loeffler-Institutes, Federal Research Centre for Virus Diseases of Animals, D-17498 Insel Riems, Germany
Author for correspondence: Walter Fuchs. Fax +49 38351 7219. e-mail walter.fuchs{at}rie.bfav.de
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Abstract |
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Introduction |
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As a prerequisite for the development of genetically engineered vaccines, most of the ILTV genome has been characterized by DNA sequencing over the last few years. These studies confirmed that ILTV possesses a herpesvirus type D genome (Johnson et al., 1991 ; Leib et al., 1987
; Roizman, 1996
) consisting of a long (UL) and a short (US) unique region; the latter is flanked by inverted repeat sequences (IR, TR). In ILTV, as in other alphaherpesvirus genomes, the US region contains a conserved cluster of viral glycoprotein genes (Wild et al., 1996
), and the major immediate early (IE) protein ICP4 is encoded within the adjoining IR and TR sequences (Johnson et al., 1995a
). Gene content and arrangement of the not yet completely analysed UL region of ILTV DNA (Fuchs & Mettenleiter, 1996
, 1999
; Griffin & Boursnell, 1990
; Griffin, 1991
; Johnson et al., 1995b
, 1997
; Kingsley et al., 1994
; Ziemann et al., 1998a
, b
) also exhibits great similarities to the respective parts of other alphaherpesvirus genomes, and open reading frames (ORFs) were designated according to their herpes simplex virus 1 (HSV-1) homologues (McGeoch et al., 1988
). However, recent sequence analyses revealed several unique features of the ILTV genome, including an internal inversion within the UL region, the translocation of UL47 to the US region, the absence of a UL16 homologue, and the presence of several presumably ILTV-specific genes (Fuchs & Mettenleiter, 1999
; Wild et al., 1996
; Ziemann et al., 1998a
, b
).
Continuing the sequence analysis of ILTV DNA we have now closed a small gap located between the characterized UL53 (Johnson et al., 1995b ) and UL49.5 (Ziemann et al., 1998a
) genes. Besides UL52 and UL51, the novel DNA sequence contains the entire UL50 ORF of ILTV. The corresponding genes of HSV-1 and -2, varicella-zoster virus (VZV), bovine herpesvirus-1, pseudorabies virus (PrV), EpsteinBarr virus (EBV) and Kaposis sarcoma-associated herpesvirus were shown to encode functional viral homologues of the ubiquitous enzyme deoxyuridine triphosphatase (dUTPase), which is required during dTTP synthesis and for prevention of uracil-incorporation into DNA (Jöns & Mettenleiter, 1996
; Kremmer et al., 1999
; Liang et al., 1993
; Ross et al., 1997
; Williams et al., 1985
; Wohlrab et al., 1982
). In all herpesviruses investigated in this aspect, the UL50 gene is non-essential for virus replication in cell culture. However, UL50-deletion mutants of both HSV-1 and PrV were found to be significantly attenuated in animals: HSV-1 in mice and PrV in pigs (Jöns et al., 1997
; Pyles et al., 1992
). Based on these results dUTPase-negative ILTV might be a suitable live vaccine for chickens.
Only very few genetically engineered ILTV mutants carrying deletions in the thymidine kinase or UL10 genes have been described up to now (Fuchs & Mettenleiter, 1999 ; Okamura et al., 1994
; Schnitzlein et al., 1995
), probably because cotransfection of permissive cells with ILTV DNA and shuttle plasmids is rather inefficient. Since it was shown in other alphaherpesvirus systems that infectivity of naked virus DNA is significantly increased by viral transactivator proteins (Dargan & Subak-Sharpe, 1997
; Moriuchi et al., 1993
, 1994
), expression constructs of the ILTV UL48 and ICP4 genes were generated and used to improve DNA transfection of a chicken hepatoma cell line which permits ILTV replication (Kawaguchi et al., 1987
; Schnitzlein et al., 1994
). Thus, we were able to generate UL50-deletion mutants of ILTV.
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Methods |
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DNA sequencing.
From plasmids pILT-ED1 and -ED2 (Fig. 1B), which are independently obtained clones of the genomic 9·8 kbp EcoRI-fragment D in pBS- (Stratagene), the 4471 bp EcoRISalI fragments were first subcloned and digested with EcoRI and NheI or SalI and BstEII, and then unidirectionally shortened by nested deletion mutagenesis (nested deletion kit, Pharmacia). Sequencing with vector-specific primers was performed as described (Fuchs & Mettenleiter, 1996
), and remaining gaps were closed using custom-made ILTV-specific primers (GibcoBRL). DNA sequences were assembled and analysed with the GCG software package in UNIX version 9.1 (Devereux et al., 1984
).
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Transactivator protein expression plasmids.
The UL48 and ICP4 genes of ILTV (Fig. 1 A, B
) were isolated from cosmid-cloned virus DNA (Fuchs & Mettenleiter, 1999
) as 2259 bp NcoISpeI, or 6608 SnaBISpeI fragments, respectively. The fragments were inserted into HindIIIXbaI doubly digested pRc-CMV (Invitrogen) after Klenow fill-in of non-compatible 5'-overhangs. In the obtained plasmids pRc-ICP4 and pRc-UL48 the predicted initiation codons of the ILTV proteins are the first ATG motifs downstream from the human cytomegalovirus IE (PHCMV-IE) and T7 promoters, which permit constitutive expression in eukaryotic cells, as well as in vitro transcription and translation (Fig. 1D
).
Cotransfection experiments.
Virus DNA was prepared from ILTV-infected CEK cells as described (Fuchs & Mettenleiter, 1996 ), and plasmids were purified with the Qiagen Maxi kit (Qiagen). For calcium phosphate cotransfection (Graham & van der Eb, 1973
) 1 µg ILTV DNA and 10 µg of the desired plasmids were dissolved in 438 µl of 10 mM TrisHCl (pH 7·4). Subsequently, 62 µl of 2 M CaCl2, 500 µl of 50 mM HEPES, 1·5 mM Na2HPO4 and 280 mM NaCl (pH 7·13) were added slowly, and after gentle mixing the suspension was incubated at room temperature for 1 h. Then the DNA was added to subconfluent LMH cell monolayers in 5 cm Petri dishes containing 2 ml minimum essential medium (MEM) supplemented with 5% foetal calf serum. After 24 h at 37 °C the transfection solution was replaced by 5 ml of fresh medium and incubation was continued for 46 days until virus plaques became visible.
Isolation and characterization of UL50 mutants.
For generation of ILTV UL50G, LMH cells were cotransfected with ILTV WT DNA, transactivator plasmids pRc-UL48 and -ICP4, and transfer plasmid pILT-CSG (Fig. 1 C
). GFP-expressing virus recombinants were isolated by limiting dilutions of the transfection progeny on CEK cells grown in microtitre plates. The rescue mutant ILTV UL50R and the deletion mutant ILTV
UL50 were obtained after transfections with ILTV
UL50G DNA, transactivator plasmids and pILT-CS or -CSD, respectively (Fig. 1 C
). In these cases, virus progeny was screened for non-fluorescent plaques. ILTV recombinants were further analysed by Southern blot hybridizations of enzyme-digested virus DNA (Fuchs & Mettenleiter, 1999
). Furthermore, the UL50 gene regions of all recombinant ILTV genomes were PCR-amplified with primers UL50-F (reverse of nt 56895707) and UL50-R (nt 41694188), plasmid-cloned and sequenced as described (Fuchs & Mettenleiter, 1999
).
dUTPase assays.
Activity of the viral dUTPase was determined essentially as described (Jöns & Mettenleiter, 1996 ; Wohlrab et al., 1982
). Confluent monolayers of ca. 106 LMH cells were infected with either ILTV WT, or recombinants ILTV
UL50G, ILTV
UL50 and ILTV UL50R at an m.o.i. of 1. After 16 h at 37 °C infected and non-infected cells were washed once with PBS, scraped into 500 µl hypotonic solution (20 mM HEPES, 1 mM dithiothreitol, 1 mM MgCl2, pH 7·8) and incubated on ice for 30 min after addition of 0·2% IGEPAL CA-630 (ICN). The nuclei were sedimented by centrifugation (500 g for 10 min), washed once with hypotonic solution and finally resuspended in 100 µl of the same buffer supplemented with 80 mM potassium acetate. Five µl of a reaction mixture containing 100 mM MgCl2, 10 mM dithiothreitol, 10 mM EGTA, 20 mM ATP and 30 nM [5-3H]dUTP (16 Ci/mmol; Amersham) were added to 45 µl of the extracts and incubated for 1 h at 4 °C. The reaction was terminated by subsequent addition of 5 µl of 0·5 M EDTA and 100 µl methanol. For thin-layer chromatography, 5 µl of the probes was mixed with 2·5 µl each of 100 mM solutions of unlabelled dUTP and dUMP, and applied dropwise to polyethyleneimine cellulose sheets containing a fluorescence indicator (Merck). After development in 1 M formic acid, 0·5 M LiCl, the separated spots of dUTP and dUMP were excised under UV light and measured by liquid scintillation counting. Finally, for each sample the ratio between [3H]dUTP and [3H]dUMP was calculated.
Plaque assays and one-step growth kinetics.
Two hours after infection of LMH cells with serial dilutions of ILTV in MEM, or 24 h after transfection with virus DNA the inoculum was removed and replaced by semi-solid MEM containing 5% foetal calf serum and 6 g/l methyl cellulose. After 46 days at 37 °C, the cells were fixed for 1 h with 5% formalin, washed and stained for 15 min with 1% crystal violet in 50% ethanol. For quantification of cell-to-cell spread, the average diameters of 30 microscopically measured plaques each of ILTV WT and the different UL50 mutants were calculated. One-step growth of WT and recombinant ILTV was monitored on CEK cells, which were infected at an m.o.i. of 5. After 1 h at 4 °C followed by 1 h at 37 °C, the inoculum was removed and non-penetrated virus was inactivated by a 2 min incubation with 40 mM citric acid, 10 mM KCl, 135 mM NaCl (pH 3). After repeated washing with PBS, fresh medium was added and the cells were further incubated at 37 °C. At indicated time-points the cells were scraped into the medium, lysed by freezethawing and progeny virus was titrated on LMH cells.
Animal experiments.
In vivo studies were performed with 20-weeks-old, pathogen-free White Leghorn chickens (fertilized eggs purchased from Lohmann, Cuxhaven, Germany). Five animals per group were intratracheally infected with 5x105 p.f.u. of either ILTV WT, ILTV UL50G, ILTV
UL50 or ILTV UL50R. During the following week, the chickens were examined daily for clinical symptoms (gasping, coughing), and tracheal swabs were taken to titrate shed virus by plaque-assays. After 3 and 4 days, one animal of each group was necropsied and tissues were sampled for histopathological investigations. Before and 14 days after infection, sera were collected and tested for ILTV-specific antibodies by indirect immunofluorescence analyses of infected CEK cells (Ziemann et al., 1998b
). Four weeks after primary infection, the remaining animals, as well as four non-infected chickens, were challenged by intratracheal application of 5x105 p.f.u. of virulent ILTV WT. Again, clinical symptoms and virus shedding were monitored, and 4 days after challenge infection all surviving animals were killed and necropsied.
Histopathology and in situ hybridization.
Immediately after necropsy, tissue samples of larynx, trachea and lung were fixed for 24 h in 4% neutral-buffered formalin and paraffin-embedded. Serial sections (3 µm) were dewaxed, mounted on organosilane-coated coverslips and stained with haematoxylin and eosin (HE) for light microscopy. Adjacent sections were investigated by non-radioactive in situ hybridization. As a probe, a 512 bp fragment representing the UL1 gene of ILTV was PCR-amplified from virus DNA in the presence of digoxigenin-labelled dUTP (Boehringer Mannheim) instead of dTTP (Fuchs & Mettenleiter, 1996 , 1999
). Hybridization and detection reactions were performed as described (Teifke et al., 1998
).
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Results |
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Generation and genetic characterization of UL50 mutants
To facilitate the isolation of UL50-negative ILTV, recombination plasmid pILT-CSG was constructed in which nearly the entire UL50 ORF (codons 10366) was replaced by a GFP expression cassette in reverse orientation (Fig. 1C). After cotransfection of LMH cells with ILTV WT DNA, pILT-CSG and transactivator plasmids, plaques of recombinant progeny virus could be easily identified in a fluorescence microscope, and purified to homogeneity by repeated limiting dilution in CEK cells. In a similar manner, the resulting recombinant ILTV
UL50G was used as parental virus of a UL50 rescue mutant (ILTV UL50R), and of a second deletion mutant (ILTV
UL50) which contains no foreign DNA sequences. To this end, non-fluorescent virus plaques were selected from the progenies of cotransfection experiments performed with ILTV
UL50G DNA and plasmids pILT-CS or -CSD, respectively (Fig. 1C
).
To verify whether all obtained ILTV recombinants contain the expected mutations, Southern blot analyses of EcoRI-digested virus DNA were performed (Fig. 3). Hybridization with the labelled plasmid pILT-ED (Fig. 3B
) revealed that, instead of the 9·8 kbp EcoRI-fragment D of ILTV WT DNA, two novel DNA fragments of ca. 5 kbp each were detected in ILTV
UL50G DNA as a consequence of an additional EcoRI site within the GFP expression cassette (Fig. 1C
). As expected, the authentic EcoRI-fragment D is restored in the genome of ILTV UL50R, whereas ILTV
UL50 possesses a ca. 1 kbp shorter fragment (Fig. 3B
). Further hybridization experiments confirmed the absence of the deleted UL50 gene fragment from the ILTV
UL50G and
UL50 genomes (Fig. 3C
), as well as the presence of the GFP expression cassette in ILTV
UL50G DNA (Fig. 3D
). The observed alterations of DNA restriction patterns are in good agreement with the calculated fragment sizes (indicated to the right of Fig. 3
). Accuracy of the mutations and purity of the virus stocks were further confirmed by PCR amplification of the UL50 gene region of all ILTV recombinants with primers UL50-F and UL50-R (Fig. 1C
), followed by cloning and sequencing of the products (data not shown).
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The UL50 gene product of ILTV exhibits dUTPase activity
Nuclear extracts of LMH cells, which had been infected at an m.o.i. of 1 for 16 h with either ILTV WT, the UL50-negative virus recombinants ILTV UL50G and ILTV
UL50, or the rescue mutant ILTV UL50R, were tested by dUTPase assays (Fig. 4
). After incubation of extracts of ILTV WT- or ILTV UL50R-infected cells with [3H]dUTP, ca. 45% of the provided radionucleoside-triphosphate was transformed into [3H]dUMP. In contrast, in similarly prepared lysates of cells infected with ILTV
UL50G or
UL50, only 1017% dUMP was detectable (Fig. 4
). These amounts are apparently produced by cellular dUTPase, since nearly 20% dUMP was also found after incubation of [3H]dUTP with the nuclei of non-infected LMH cells (Fig. 4
), whereas no conversion was observed after incubation without any extract. Very similar results were obtained from two independent experiments (Fig. 4
).
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In summary, under our experimental conditions both dUTPase-negative ILTV recombinants, as well as WT ILTV, were able to protect chickens from the fatal consequences of a subsequent infection. However, the deletion mutant ILTV UL50 was still pathogenic, and the avirulent substitution mutant ILTV
UL50G was genetically unstable in vitro and in vivo. Besides the GFP-expressing input virus, increasing amounts of non-fluorescent ILTV could be reisolated from all chickens infected with ILTV
UL50G after 4 and 5 days. Since the genomes of these viruses exhibit different deletions within the inserted foreign sequence (see above), they probably arose independently in each animal.
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Discussion |
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Both UL51 and UL50 were shown to be non-essential for replication of HSV-1 in cell culture (Barker & Roizman, 1990 ). The UL50 homologue of ILTV is apparently also not required for virus growth in vitro, since we isolated viable virus recombinants carrying deletions of nearly the entire gene. Probably because of the almost ubiquitous presence of cellular dUTPases, deletion of the UL50 gene of ILTV has no detectable effects on virus replication in cell culture, as quantified by one-step growth kinetics and plaque sizes. Similarly, UL50-negative HSV-1, PrV and VZV mutants were also phenotypically inconspicuous in vitro (Barker & Roizman, 1990
; Jöns & Mettenleiter, 1996
; Ross et al., 1997
). However, the same recombinants of HSV-1 and PrV were shown to be attenuated in mice or pigs, respectively (Jöns et al., 1997
; Pyles et al., 1992
). In contrast, virulence of ILTV
UL50 in chickens was not significantly reduced. An obvious explanation for this difference might be the different target organs of these viruses. Whereas clinical symptoms and mortality caused by HSV-1 in mice, and by PrV in its natural host are predominantly a consequence of neuroinvasion, lytic ILTV infection is restricted predominantly to the respiratory tract of chickens (Bagust & Guy, 1997
). This was confirmed by our studies, since none of the animals infected with either
UL50 or WT ILTV exhibited any histopathological lesions in the brain (data not shown). Because expression of cellular dUTPase is regulated in a cell-cycle-dependent manner (Duker & Grant, 1980
), it was suggested that the redundant herpesvirus enzyme is required for efficient lytic replication in non-dividing cells like neurons (Jöns & Mettenleiter, 1996
; Pyles et al., 1992
). Although ILTV is not neurovirulent, it is able to establish latent infections in the trigeminal ganglia of chickens (Williams et al., 1992
). Therefore, it will be interesting to investigate whether the deletion of the ILTV UL50 gene has any effects on establishment of, or reactivation from, latency.
Although the UL50-deleted ILTV recombinant UL50 is virulent, a similar recombinant, ILTV
UL50G, which differs from the other mutant only in the insertion of a GFP expression cassette, is not. Moreover, ILTV
UL50G exhibits a small-plaque phenotype in cultured cells. Probably due to this negative effect, expression of GFP from the UL50 locus of ILTV was unstable, and various deletion mutants spontaneously arose in vitro and in vivo, which all result in abolishment of GFP expression. Obviously, there is a strong selective pressure against GFP expression in these viruses. Similar phenomena were also observed with other virus recombinants expressing GFP from the thymidine kinase (UL23) or the unique UL0 gene locus of ILTV (unpublished results). Since one-step growth kinetics of any of these virus recombinants are not affected, we speculate that only after infection at low multiplicity does the overexpressed GFP accumulate to concentrations that impair productive virus replication, or virus spread. Thus, the value of GFP under control of the strong and constitutively active HCMV IE gene promotor as an insertional marker for ILTV is questionable.
Up to now, construction of ILTV recombinants suffered from the low infectivity of viral DNA, which is also difficult to purify in large enough quantities. Since the UL48 gene product of HSV-1, TIF, and the IE ICP4 protein function in subsequent transactivation of viral genes and, thus, enhance infectivity of viral DNA (Dargan & Subak-Sharpe, 1997
), we tested whether plasmids from which expression of ILTV UL48 and ICP4 is driven by the HCMV IE promotor have any effect on virus yield in DNA cotransfection assays. Our results show that both ILTV proteins increased the infectivity of ILTV DNA ca. fivefold. Interestingly, their effects are not additive and inclusion of either plasmid gave similar results. This is in contrast to results with the homologous transactivators of VZV, where the ICP4 homologue showed a much more pronounced effect on infectivity of virion DNA than
TIF (Moriuchi et al., 1994
). We therefore speculate that in our assays infectivity might still be limited by the low input amount of replication-competent virus DNA. Nevertheless, inclusion of the UL48 and ICP4 expression plasmids significantly facilitated the generation of ILTV recombinants. Thus, this technique will be used to generate further candidates for an improved ILTV vaccine.
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Acknowledgments |
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Footnotes |
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References |
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Received 24 September 1999;
accepted 12 November 1999.