High-Level Expression of Marek's Disease Virus Gly
http://www.100md.com
病菌学杂志 2005年第10期
Department of Microbiology and Immunology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853
Laboratoire de Virologie Moléculaire et Immunologie, UR 086 BASE, Centre de Recherches INRA de Tours, 37380 Nouzilly, France
Lohmann Animal Health, 27472 Cuxhaven, Germany
ABSTRACT
Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in Escherichia coli. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the UL44 gene deleted was constructed. On the basis of this deletion mutant, the original or a modified UL44 gene with a mutated start codon (AUGACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC.
INTRODUCTION
Marek's disease virus (MDV), also referred to as gallid herpesvirus 2, is a member of the family Herpesviridae. Within this large virus family it is classified among the Alphaherpesvirinae and is the prototype member of the genus Mardivirus ("Marek's disease-like viruses") (6, 34, 68). MDV is the causative agent of Marek's disease (MD) in chickens (13), whereas its close relatives and other members of the genus, gallid herpesvirus 3 and herpesvirus of turkeys (HVT; meleagrid herpesvirus, MeHV-1), are completely apathogenic and have been used as MD vaccines since the early 1970s (7, 10, 52, 60). Following the first description of MD in 1907 by Jozef Marek (42), the clinical picture has changed from a polyneuritis associated with general wasting to one that is characterized by visceral lymphomas, transient paralysis, and early mortality within 2 weeks of infection. The virulence of MDV field isolates has increased over the years from mild (m), virulent (v), very virulent (vv), to very virulent plus (vv+) strains. While vv strains are able to break HVT vaccination, vv+ MDV are isolated from flocks vaccinated with a combination of SB-1 (a gallid herpesvirus 3 strain) and HVT (9, 74, 75).
In contrast to other alphaherpesviruses, MDV does not release detectable free enveloped, let alone infectious, virus into the supernatant of cultured cells (8). Similar to varicella-zoster virus (VZV), infectivity in vitro spreads directly only from an infected cell to a neighboring cell (29). In vivo, free infectious virus is released only from the feather follicle epithelium of infected chickens, while virus transfer from macrophages to epithelia, B cells, and finally activated T cells is thought to occur exclusively by direct cell-to-cell spread (8).
The roles that MDV tegument and envelope (glyco)proteins play in virus attachment and entry or in virus maturation, egress, and direct cell-to-cell spread have remained elusive to a large extent, but it is known that a number of MDV structural proteins are key players in these processes. With regard to the tegument proteins, it is known that the herpes simplex virus type 1 (HSV-1) tegument proteins named VP1/2, VP13/14, VP16, and VP22, which are encoded by UL36, UL47, UL48, and UL49, respectively, are major components of cell-free enveloped virus (25, 30, 65). While VP13/14 and VP22 are dispensable for HSV-1 growth, VP1/2 and VP16 were found to be essential for secondary envelopment and egress (21, 48, 71). Similarly, deletion of VP16 causes severe growth defects in relatives of HSV-1, such as equine herpesvirus type 1 (EHV-1) and pseudorabies virus (PRV) (27, 28, 70). In contrast, MDV VP11/12, VP13/14, and VP16 are dispensable for in vitro growth of MDV and VZV, whereas VP22 encoded by the UL49 homologue is essential for MDV replication in cultured cells (15, 24).
In addition to the differential requirement for certain tegument proteins, the role of membrane (glyco)proteins in the virus' life cycle also seems to be partially different in MDV compared to herpesviruses that undergo a complete secondary envelopment resulting in the release of free infectious virus. Sequence analyses have shown that, with the exception of glycoprotein G (gG) and gJ, all common alphaherpesviral glycoproteins, such as gB, gC, gD, gE, gH, gI, gK, gL, and gM as well as the UL49.5 product, the homologue of which is glycosylated and referred to as gN in PRV, are encoded in the MDV genome (34, 68). It could be shown that MDV mutants lacking gB were nonviable; however, unlike other related viruses, MDV is unable to replicate in the absence of two major membrane protein complexes, the gE-gI and the gM-pUL49.5 complex (61, 62, 67). While essentiality of gB has been shown for all herpesviruses analyzed thus far (53), most Alphaherpesvirinae are still able to grow in cultured cells in the absence of the other glycoproteins (2, 3, 22, 32, 37, 51, 56, 77, 78). Besides MDV, VZV is also an exception to the rule, because it requires expression of gE for growth in vitro and in vivo, while gI is required for VZV growth in Vero cells, but not in fibroblasts or melanoma cells (14, 39, 40, 62). Transcriptional analyses of the MDV unique short region and the gD locus, the expression of which is essential for HSV-1, PRV, EHV-1, and bovine herpesvirus 1 attachment and entry (33, 36, 43, 72), revealed that MDV gD is not expressed in cultured chicken embryo cells (66). This is consistent with the complete absence of a gD open reading frame (ORF) in VZV (20). These findings indicated different functions of tegument and glycoproteins in alphaherpesviruses that are able to produce cell-free infectious virions in vitro and those that are only capable of spreading directly from cell to cell.
Examinations of gC-like proteins of alphaherpesviruses, which are components of the viral envelope, suggest that they have multiple functions in vitro and in vivo. Generally speaking, gC orthologues have a central role in attachment of free virus to heparin- and chondroitin-like glycosaminoglycans on the surface of the plasma membrane, thereby conferring the first contact between the virion and cell (55). Independently from its initial binding function to the cell surface, a role of gC in penetration of PRV has been described (45, 57). Furthermore, gC is required for efficient egress of PRV and EHV-1, although both glycoproteins are dispensable for virus growth in vitro (45, 46). In the case of VZV, gC was shown to be a major determinant for virulence in skin, while gC-negative viruses exhibited accelerated and more efficient growth in cultured melanoma cells (16, 47). Another function of alphaherpesviral gC orthologues is binding to complement component C3b, thereby blocking the activation of the complement cascade and rendering the host unable to respond via this nonspecific defense mechanism (26).
MDV gC was previously referred to as the "A-antigen" and is encoded by the 1,503-bp UL44 orthologous gene. MDV gC is synthesized into a 47-kDa primary translation product. The primary product is cleaved at its signal peptide to a yield a 44-kDa precursor protein, which is co- and posttranslationally glycosylated to the mature glycoprotein of 57 to 63 kDa in size. In contrast to other herpesviruses, however, approximately 95% of all the gC produced by an infected cell is secreted. Besides secretion via the regular secretory pathway, a small portion of unglycosylated gC was also reported to reach the extracellular space by an alternative mechanism, although the mechanism of secretion was not described in further detail (31). Another striking feature of MDV gC is that its abundant expression in vitro and in vivo is dramatically reduced after serial passage in culture. Coinciding with reduced gC production, virulence of MDV decreases. Despite the greatly reduced gC expression levels after passage, no difference in the nucleotide sequence of UL44 or its promoter region between viruses with low and high gC expression levels were detectable, and the role of viral transcription factors has been discussed in the shutdown of gC expression (73). Although there is an apparent correlation between gC expression and virulence, there is no convincing evidence that gC is actually involved in virulence of MDV, because the glycoprotein has been found to be expressed at higher levels in some avirulent virus strains (4).
The aim of this study was to elucidate the growth properties of MDV in the absence or presence of gC in vitro. On the basis of a bacterial artificial chromosome (BAC) clone of the highly passaged MDV strain 584Ap80C, we generated a number of mutant viruses that either lacked the gC ORF, harbored point mutations in the gC start codon, or overexpressed the glycoprotein under the control of the human cytomegalovirus immediate-early promoter (PCMV). Our studies clearly demonstrated that there is a reciprocal relationship of MDV growth properties and the level of gC expression in vitro. In addition, the growth disadvantage was shown to be associated with gC present in the supernatant of infected cells. The results indicated that loss of gC function in cultured cells is a marked growth advantage and may explain its absence in highly passaged MDV strains and isolates.
MATERIALS AND METHODS
Viruses and cells. MDVs were propagated on primary or secondary chicken embryo cells (CEC) or on the permanent cell line SOgE, a QM7 (ATCC CRL-1962) derivative that constitutively expresses gE (63). All cells were maintained in Dulbecco's modified essential medium supplemented with 2 to 10% fetal bovine serum. MDV strain v20 was reconstituted from an infectious full-length BAC clone of 584Ap80C (BAC20) that lacks the US2 ORF (61). Both v20 and mutant viruses were recovered at days 5 to 7 after transfection of 1 to 5 μg of BAC DNA using the calcium phosphate precipitation method (61).
Plaque area determination and virus titration assays. For plaque size determinations, fresh CEC were cocultivated with 100 PFU of the various recombinant viruses or the parent virus and incubated for 72 h at 37°C. Cells were fixed with 90% acetone and analyzed by indirect immunofluorescence (IIF) using a convalescent-phase MDV-specific chicken serum (61). In some experiments, plaques were detected using a Sindbis virus-generated antiserum against MDV gC (64). Plaques were visualized with Alexa Fluor 488-conjugated goat anti-chicken immunoglobulin G (IgG) or Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes). Virus plaques were examined under a fluorescence microscope (Zeiss Axiovert 25). In two independent experiments 50 plaques each were photographed with a digital camera (Zeiss AxioCam HRc), and plaque sizes were determined using the ImageJ software that is freely available from the National Institutes of Health (http://rsb.info.nih.gov/ij/index.html). Statistical analyses of plaque sizes were performed using SAS software package, version 8.2 for Windows (SAS Institute) (59).
Virus titers were determined by infecting 1 x 106 CEC seeded in one well of six-well plates with 100 PFU per well of the various viruses, corresponding to a multiplicity of infection of 0.0001. At different times postinfection (p.i.), infected cells were trypsinized and coseeded with fresh CEC. Titers were determined 72 h later by counting plaques after IIF staining using the MDV-specific antiserum.
Construction of plasmids. To generate recombinant plasmid pTgC1-2, a PCR fragment, dubbed gC1-2, was amplified from BAC20 with primers 5'-TGAGCTCAATGTATCGTAGTAACCATG-3' and 5'-TGGATCCAGATATGTAGAGGGTTACGT-3', cleaved with SacI and BamHI (bold and italics), and cloned into vector pTZ18R (Pharmacia). Similarly, plasmid pTgC3-4 was constructed using primers 5'-GGATCCTGCAGTCTCATTGTTATGTAGTTGTG-3' and 5'-TTAGCATGCAATGGTGGGTAGTATACAG-3' that amplified a fragment that was cleaved with BamHI and SphI (bold and italics) before insertion into pTZ18R. The inserts in both plasmids were sequenced and combined after releasing the fragment gC3-4 from with BamHI and SphI and ligating it into pTgC1-2 digested with the same enzymes, finally resulting in plasmid pTgC1-4.
Recombinant plasmid pTUL44 was generated using primers 5'-TGGATCCCTCATGCTCACGCCGCG-3' and 5'-TTACTGCAGTTATAATCGAATATTTTTTCG-3' for amplification of the UL44 ORF. The PCR product was ligated into pTZ18R after digestion of the PCR fragment with BamHI and PstI (bold and italics) and sequenced. Similarly, a version of the ORF with a point mutation in the start codon (pTUL44ACG) was cloned using a different forward primer (5'TGGATCCCTCACGCTCACGCCGCG-3') that resulted in an ACG codon (underlined) instead of an ATG start codon. Both UL44 inserts were released from the recombinant plasmids with BamHI and PstI and introduced into pTRgC1-4, resulting in pTgC1-4_UL44 and pTgC1-4_UL44ACG. All three constructs (gC1-4, gC1-4_UL44, and gC1-4_UL44ACG) were released with SacI and SphI and cloned into plasmid pST76K_SR (1, 5) to create shuttle plasmids pST76K_SR-gC, pST76K_SR-gC-R, and pST76K_SR-MgC, respectively.
Finally, the human cytomegalovirus immediate-early promoter (PCMV) was released from pcDNA3 (Invitrogen) with BamHI and BglII and introduced into the BamHI site of pTgC1-4_UL44 and pTgC1-4_UL44ACG to create pTgC1-4_ExpUL44 and pTgC1-4_ExpUL44ACG, respectively. Both constructs were excised with SacI and SphI and transferred into pST76K_SR to generate pST76K_SR-ExpgC and pST76K_SR-ExpMgC. The shuttle plasmids were used for RecA-based mutagenesis of BAC20 (see below).
Mutagenesis of BAC20. To replace the UL44 sequence of BAC20 with an antibiotic resistance gene, a system employing homologous recombination at double-strand breaks of DNA in Escherichia coli was used. The products of phage genes exo, ?, and encoded by pKD46 under the control of an L-arabinose-inducible promoter allow recombination between 50 bp at both ends of a linear DNA fragment and their homologous sequences in BAC20 (17, 49, 50). E. coli strain DH10B harboring BAC20 and the temperature-sensitive plasmid pKD46 was grown at 30°C in Luria-Bertani (LB) medium with ampicillin (100 μg/ml), chloramphenicol (30 μg/ml), and 10 mM L-arabinose to an optical density at 600 nm of 0.6, washed three times with 10% glycerol, and concentrated 100-fold. Twenty-five microliters of competent cells were electroporated (1.25 kV/cm, 200 , 25 μF) with 100 ng of a PCR product of an aminoglycoside phosphotransferase gene (aphAI) derived from pACYC177 (12). The primers used for the PCR were 5'-GGAAAGTACCAATGAACATACCATAACAGAAACGACGGGCAAGACGCATCGATTTATTCAACAAAGCCACG-3' and 5'-CGTGTGGAGTTGTATAAACATAGGGCAGTCATGATTATCCCCATCCCTAAGCCAGTGTTACAACCAATTAACC-3'. After electroporation, E. coli was incubated for 1 h at 37°C in SOC (58) and plated onto LB agar containing kanamycin (30 μg/ml) and chloramphenicol (30 μg/ml). Colonies were grown in liquid LB with both antibiotics, and BAC DNA was isolated by a standard alkaline lysis protocol (58) or by column chromatography (QIAGEN plasmid MAXI kit) according to the manufacturer's protocol.
Shuttle mutagenesis was performed with pST76K_SR-derived plasmids. Electrocompetent E. coli DH10B bacteria harboring the appropriate BAC clone were transformed with 200 ng of the respective shuttle plasmid and shaken for 90 to 120 min at 30°C in SOC. Cells were plated onto LB agar with kanamycin (30 μg/ml) and chloramphenicol (30 μg/ml) and incubated overnight at 42°C to select for integration of the shuttle plasmid into BAC20 via recombination of one of the two homology flanks (Fig. 1). Colonies were grown overnight at 42°C in liquid LB containing both antibiotics. DNA from kanamycin- and chloramphenicol-resistant colonies was isolated, digested with appropriate restriction enzymes, and separated overnight by agarose gel electrophoresis (58). Positive clones were grown in LB containing only chloramphenicol for 4 h at 37°C, 10% (wt/vol) sucrose was added to the medium, and cultures were shaken for additional 4 to 8 h. Serial 10-fold dilutions were prepared and grown overnight at 30°C on LB plates containing chloramphenicol and 10% sucrose. The latter procedure selected for loss of sacB sequences of the shuttle plasmid via a second recombination in one of the flanks (Fig. 1). Larger individual colonies were replica plated onto LB agar plates containing either chloramphenicol or kanamycin. DNA was isolated from clones that were unable to grow in the presence of kanamycin but remained chloramphenicol resistant, and the restriction patterns of the clones were analyzed for the presence of predicted rearrangements (1, 5). Finally, the mutated regions of positive candidates were amplified by PCR, and nucleotide sequencing was performed to confirm that the desired mutations had been introduced.
Generation of anti-gC monoclonal antibodies (MAb) and Western blot analyses. For immunization of mice, recombinant gC was expressed in a commercially available baculovirus/Sf21 system exactly as described earlier (23). Briefly, UL44 was amplified by PCR from BAC20 with primers 5'-GGATCCATGCTCACGCCGCGTGTGTTACGAGC-3' and 5'-GAATTCTTATAATCGAATATTTTTTCGTGTGGAGTTG [BamHI and EcoRI sites are shown in bold and italic type] with thermostable rTth DNA polymerase XL (Applied Biosystems). The PCR product was cloned in pGEM (Promega), and the sequence of the insert was checked for integrity. The insert from pGEM-gC was cleaved with BamHI and EcoRI and cloned into pFastI plasmid (Bac-To-Bac baculovirus expression system; Invitrogen). The presence of UL44 in the recombinant baculovirus genome was ascertained by PCR. One BALB/c mouse was immunized with the equivalent of 106 infected Sf21which were collected 72 h p.i. The schedule of immunization and the fusion procedure were as described previously (23). Supernatants from growing hybridomas were tested on CEC infected with MDV and on Sf21 infected with the gC-expressing baculovirus recombinant. One MAb termed A6 was selected for its strong affinity to MDV-infected cells, and subcloning and amplification of the hybridoma was performed as described previously (69). Isotyping was performed with a mouse monoclonal antibody isotyping kit (Sigma), and MAb A6 was found to be of the IgG2b isotype.
Western blot analysis of MDV gC secreted into the supernatant of infected cells was done after concentrating supernatants using concanavalin A (ConA). Briefly, 3 ml of (infected) cell culture supernatant was mixed with an equal volume of ConA buffer (0.5% Zwittergent, 10 mM Tris HCl, pH 7.4, 0.5 mM MgCl2, 0.5 mM MnCl2, 500 mM NaCl), and 100 μl of ConA-Sepharose beads (Sigma) washed in ConA buffer was added. After gentle shaking at 4°C overnight, beads were centrifuged (12,000 x g, 1 min, 4°C), and the ConA slurry was washed three times. Finally, beads were resuspended in 100 μl of sample buffer and heated at 95°C for 5 min. Separation by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), transfer of proteins to nitrocellulose membranes, and Western blot detection using enhanced chemiluminescence (ECL kit; Amersham) was done exactly as previously described (56).
RESULTS
Absence of UL44 sequences results in larger virus plaques. In an attempt to generate a comprehensive library allowing a functional analysis of individual MDV proteins, several MDV ORFs were deleted from BAC20 by Red recombination (17, 61, 62, 67). The viral genes were deleted by replacing individual ORFs with the bacterial aphAI gene that encodes a protein conferring kanamycin resistance. In one of the resulting BAC20 clones, the majority of the UL44 gene encoding gC was deleted, resulting in the recombinant 20kangC BAC (Fig. 2A). After confirmation of the correct restriction enzyme pattern by agarose gel electrophoresis (Fig. 3B) and subsequent Southern blot analysis (data not shown), BAC DNA isolated from E. coli was used to transfect CEC to reconstitute mutant virus v20kangC or the parental v20. Infected cells were frozen as virus stocks in liquid nitrogen and titrated. At 72 h p.i., plaque areas of the gC-negative v20kangC were shown to be increased to more than 185% relative to those of the parental v20 (Fig. 2B). In addition, growth kinetics of both viruses on CEC were determined, and the approximately twofold difference in plaque areas was confirmed by increased growth kinetics of v20kangC on primary chicken cells compared to those of v20 (Fig. 2B).
Markerless manipulation of UL44 sequences using shuttle mutagenesis. The preliminary analysis of a deletion of MDV gC-encoding sequences in v20 had shown that absence of the ORF resulted in markedly increased plaque sizes and suggested a growth advantage of MDV in the absence of gC. These results prompted us to initiate a more comprehensive study that applied genetic modifications that would not result in the introduction and maintenance of a selectable marker, because MDV has a densely packed genome with regulative sequences often overlapping ORFs (34, 68).
First, and to corroborate the findings with the v20kangC mutant virus, plasmid pST76K_SR-gC was used to generate a markerless deletion mutant by shuttle mutagenesis, which led to removal of the complete UL44 ORF and resulted in recombinant BAC clone 20gC (Fig. 3A). Starting with the constructed 20gC deletion mutant, the same method of RecA-based shuttle mutagenesis was applied utilizing plasmid pST76K_SR-gC-R to reintroduce the UL44 sequence into the original locus. Virus reconstituted from this BAC clone (20gC-R) differed from BAC20 only in the introduced BamHI and PstI restriction sites that were used to clone the shuttle plasmids (Fig. 3A).
To create a gC-negative mutant that still contained UL44 sequences and to avoid interference with potential regulative elements, the gC start codon was replaced in the BAC20 infectious clone by shuttle mutagenesis using 20gC and pST76K_SR-MgC, which resulted in recombinant BAC clone 20MgC (Fig. 1 and 3A). Finally, two mutant BAC clones from 20gC were constructed, in which the PCMV promoter was inserted immediately upstream of UL44. Shuttle plasmids pST76K_SR-ExpgC and pST76K_SR-ExpMgC were used. While the former would result in expression of gC controlled by PCMV, the latter would be unable to express the viral protein, because the start codon was altered from AUG to ACG (Fig. 3A).
After generation and testing of all cointegrates by restriction enzyme analysis for correct insertion of the shuttle plasmid, the final mutant BAC genomes were analyzed after resolution of the cointegrates. Agarose gel electrophoresis of BamHI and PstI restriction enzyme digests of the individual BAC clones showed that the insertions and deletions were successful and that no spurious rearrangements had occurred (Fig. 3B). In the 20kangC mutant, replacement of UL44 with aphAI in 20kangC caused a shift of the BamHI-B fragment of approximately 250 bp towards the BamHI-A1 band that results from the introduction of the mini-F sequences into the BamHI-A fragment and the introduction of an additional BamHI site into BAC20 (61) (Fig. 3B, lane 2). In the case of 20gC (Fig. 3B, lane 3), due to the deletion of UL44 and the introduction of a BamHI site, the BamHI-B (18.0 kb) fragment present in BAC20 is divided into two bands of 7.5 kbp and 9.0 kbp in size. The latter band increased in size to 10.5 kbp in both the 20gC-R and 20MgC mutant (Fig. 3B, lanes 4 and 5) due to the reinsertion of the UL44 sequence. Finally, in the PCMV constructs (20ExpgC and 20ExpMgC) and due to the insertion of the promoter sequence, the 7.5-kb fragment increased to 8.4 kbp (Fig. 3B, lanes 6 and 7). These results were confirmed by nucleotide sequencing of the gC locus and adjoining sequences (data not shown), and both analyses clearly demonstrated the correct insertion or deletion of the desired sequences in the various clones.
Growth properties of UL44 mutant viruses. To analyze the generated UL44 virus mutants, primary CEC as well as SOgE cells were transfected with BAC DNA isolated from E. coli. Reconstituted viruses were propagated on the two cell types, and aliquots were frozen in liquid nitrogen. After determination of virus titers, fresh cells were coseeded with 100 PFU of the various viruses, fixed after 72 h, and analyzed by IIF. Whereas all the reconstituted viruses could clearly be detected using an MDV-specific chicken convalescent-phase serum, only cells infected with v20ExpgC exhibited robust IIF staining using a rabbit antiserum raised against MDV gC. A very faint reactivity of the gC-specific antibody with infected cells was also observed for parental v20 and the 20gC-R revertant virus (Fig. 4).
After determining the reactivity of the various virus plaques with different antibodies, plaque sizes on CEC were determined for all viruses as described above. The gC deletion mutant v20gC induced plaques that exhibited sizes that were more than twice the size of parental BAC20 virus plaques (203%) (Fig. 5A). Plaque sizes of the revertant v20gC-R virus were larger than parental plaques (114%), while the v20MgC that was unable to express gC exhibited plaque areas amounting to 148% of those of v20 (Fig. 5A). Areas measured for plaques of the gC-overexpressing mutant v20ExpgC were reduced to 44%, while virus plaques on CEC of the virus mutant that expressed the methionine mutant under the control of PCMV (v20ExpMgC) were as large (104%) as those induced by parental v20 (Fig. 5A). Data from the plaque area assays were analyzed with SAS software, and means of plaque areas induced by the generated recombinant viruses were compared with each other by analysis of variance to determine statistically significant differences (59). Since multiple comparisons were performed, only P values smaller than 0.0033 indicate significant differences using the Wilcoxon two-sample test or the Kruskal-Wallis test (59) (Table 1). Statistical analyses demonstrated that both gC-negative mutants, v20gC and v20MgC, induced significantly larger plaques than those induced by parental v20 as well as revertant v20gC-R virus, whereas the sizes of v20 and v20gC-R or v20gC and v20MgC were not significantly different from each other. With a P of <0.0033, plaque areas of the gC-overexpressing mutant v20ExpgC were statistically significantly smaller than those of parental virus; plaques induced by the virus overexpressing gC but harboring the mutated start codon (v20ExpMgC) were significantly larger than those induced by the true overexpression mutant (v20ExpgC) (Table 1). Plaque sizes of v20ExpMgC, however, were not significantly larger compared to parental v20 plaques, although this mutant was unable to express gC (Table 1).
To extend the findings of the plaque size determinations, multistep growth kinetics of the engineered mutant viruses were performed. The results of these experiments are summarized in Fig. 5B. Consistent with the findings of the plaque size determinations, the gC-negative v20gC virus grew slightly faster than parental v20, whereas v20ExpgC exhibited markedly reduced growth properties on CEC (Fig. 5B). All other mutant viruses exhibited comparable growth kinetics and were virtually indistinguishable from those of parental v20 (Fig. 5B). The results of MDV growth kinetics are largely a reflection of virus-induced plaque sizes, because infected cells are trypsinized and coseeded with fresh cells to determine virus titers, i.e., the larger the virus plaques, the higher the determined virus titers. Therefore, we concluded that the titration experiments corroborated the findings of the plaque size determinations (61), which provide a superior measure of the in vitro growth properties of MDV mutants.
Role of secreted MDV gC in cell culture supernatants. The variation in plaque sizes and growth kinetics of the different gC mutant viruses suggested that the differences in their ability to express gC accounted for their different abilities to spread from an infected cell to a neighboring uninfected cell. To analyze gC expression by v20 and its derivatives, supernatants of 5 x 106 cells infected with 1,000 PFU of the respective virus were harvested at day 3 p.i. gC was precipitated from supernatants (3 ml) using ConA-Sepharose beads, and precipitates were separated by SDS-10% PAGE before transfer to nitrocellulose membranes. Detection by Western blotting of secreted gC using MAb A6 demonstrated that expression of this form of the glycoprotein in cells infected by parental v20 or v20gC-R, unlike the cell-associated form (Fig. 4), was below the detection limit, even after 30-fold concentration by ConA precipitation (Fig. 6). As expected, no reactivity was observed in the supernatants of CEC infected with v20gC. In contrast, a strong gC-specific signal was obtained in supernatants infected with the overexpressing v20ExpgC virus (Fig. 6), indicating that a significant increase in gC production was achieved by the replacement of the original gC promoter with PCMV (Fig. 3). The v20ExpMgC mutant, in which the gC start codon was mutated but also contained PCMV upstream of the open reading frame, was unable to express the protein (Fig. 6). From these and the above results, we concluded that the amount of gC present in the supernatant of infected cells correlated well with the plaque sizes induced by different gC mutant viruses.
To further investigate whether the effect of MDV gC on virus growth was indeed caused by the secreted form of gC, supernatants were collected from CEC that were infected with wild-type v20 or the gC-negative mutant virus v20gC. Supernatants were used to overlay freshly infected cells, and plaque sizes were determined. In this experiment, 1 x 106 CEC were infected with 500 PFU of v20 or v20gC and grown for 48 h in medium containing 10% fetal calf serum. At that time point, infection had proceeded to a stage where approximately 20% of cells were infected. Cell culture supernatant was removed, and 2 ml of fresh medium was added. After a further 24-h incubation period, medium was collected and centrifuged for 10 min at 16,100 x g to remove cellular debris. Fresh CEC were then infected with 100 PFU of v20 or v20gC and incubated from 24 h p.i. with a 1:1 mixture of fresh medium and supernatants obtained from cells infected with either v20 or v20gC. After 48 h, cells were fixed with 90% acetone, virus plaques were visualized by IIF, and plaque sizes were determined. The area of v20gC plaques grown with conditioned medium that originated from the CEC infected with the gC-negative virus were significantly larger (144%) than those of v20 (100%). However, plaque sizes of viruses grown in the presence of culture medium originating from CEC infected with gC-positive v20 virus showed marked decreases (96% in the case of v20 and 82% in the case of v20gC) (Fig. 7). From the results, we concluded that a soluble factor present in the supernatant of MDV expressing UL44, probably the secreted form of gC, was responsible for the observed reduction in plaque sizes of both v20 and v20gC.
DISCUSSION
In the present study we conducted an in-depth analysis of the function of MDV gC for virus replication in vitro. Besides the observation that expression levels of the UL44 gene negatively correlate with attenuation of MDV and that gC expression is reduced by serial passage of MDV strains in cell culture, knowledge on the function of gC in highly cell-associated herpesviruses, such as MDV, has been sparse (73). Alignment of the gC amino acid sequence of MDV to that of its HSV-1 counterpart demonstrated that the carboxy-terminal domains of MDV and HSV-1 gC share high similarity, whereas the amino termini are completely different. However, all cysteine residues, which are important for maintaining the tertiary structure of gC (38, 41), are conserved in MDV. One important pair of cysteines at positions 127 and 144 flank a loop which forms a domain that contains a stretch of basic amino acids in HSV-1 gC that are thought to interact with heparan sulfate (41). Preliminary data suggest that MDV gC is unable to bind heparin (data not shown), which is supported by the fact that only two basic amino acids are present around the orthologous cysteines (Fig. 8). Furthermore, the whole protein harbors none of the described heparin-binding domains, XBBXBX or XBBBXXBX, where B is any basic amino acid and X is any hydrophobic amino acid (11).
In the case of VZV, another highly cell-associated alphaherpesvirus and close relative of MDV, deletion of orf14 encoding gC in the vaccine Oka strain did not interfere with heparin sensitivity (16), indicating that another envelope (glyco)protein is responsible for the first step of virus attachment that is mediated in all Alphaherpesvirinae analyzed to date by glycosaminoglycans on the plasma membrane bearing heparan or chondroitin sulfate moieties. The absence of the VZV orf14 gene product whose expression is—similar to the situation in MDV—reduced in attenuated strains had a minor effect on the growth of VZV in cultured cells. Deletion of gC, however, resulted in an increase of end-point viral titer of approximately twofold compared to the titer of the parental strain. Likewise, deletion of MDV UL44 led to this apparently marginal increase of virus growth, although mean plaque areas of v20gC or v20kangC were twice the size of that of the parental clone. As stated above, however, the twofold increase in end-point virus titers is an exact reflection of plaque sizes, because infected cells, whose numbers are dependent on the size of the plaques, are coseeded with fresh cells and the induced plaques are counted. The statistically significant difference in plaque size between gC-negative virus and parental virus showed that a clear phenotype is attributable to the presence or absence of UL44 sequences, although the 584Ap80C strain, from which BAC20 and consequently v20 are derived, were passaged 80 times in cultured cells and although expression of gC in v20-induced plaques was barely detectable and no significant amounts of gC seem to be secreted (Fig. 4 and 6). The deletion of only the gC start codon, which left the remainder of the coding sequences intact, also led to an increase of plaque sizes in cell culture. The difference in plaque size between the v20MgC mutant and the parental virus was also significant but was less than that after complete removal of UL44 sequences, which may suggest a second function of sequences harboring the gC ORF. Possible explanations for the observed differences are that regulation of the adjacent UL45 gene or that of a predicted 624-bp ORF, which is directed antisense to UL45 (LORF8) (34, 68), may be affected when the entire gC-encoding sequence is deleted. The functions of UL45 or its putative antisense ORF are completely unclear at present. Despite their positional conservation, they both show no homology to any other herpesvirus sequences outside of the Mardivirus genus (34, 68). A role of the long intergenic region between UL44 and UL45 is also possible, since a predicted promoter for UL45 overlaps with that of UL44 and as such would also be responsible for transcription of the entire 864-bp sequence that includes two smaller theoretical ORFs (34, 68) (Fig. 2).
In contrast to the deletion of gC, constitutive overexpression of gC in the v20ExpgC mutant, which was detected unequivocally by IIF and Western blot analyses (Fig. 4 and 6), resulted in significantly smaller virus plaques and reduced growth kinetics, clearly indicating that there is a negative effect of the UL44 gene product(s) on growth properties of MDV in cell culture. Consistent with theoretical predictions, the start codon deletion mutant driven by PCMV had significantly increased plaque sizes compared to the v20ExpgC mutant plaques, and gC—as expected—was not detected by IIF, convincingly showing that the point mutation of the AUG codon is sufficient to abrogate gC production even if transcription is controlled by a strong constitutive promoter. Surprisingly, however, the v20ExpMgC point mutant only grew to levels comparable to those of v20 and v20gC-R. Possible explanations for this observation are that either increased transcription of UL44 has a negative effect on virus growth by interference with regulatory sequences that overlap with the gC coding region, as discussed before, or that the strong PCMV affects not only expression of gC but also that of downstream genes (Fig. 2).
Most of the gC produced by MDV-infected cells is secreted into the supernatant, which has been attributed to unstable membrane anchoring that would be caused by the short predicted cytoplasmic domain (31, 73). Preliminary data obtained from reverse transcriptase PCR studies performed in our laboratory, however, indicate alternate splicing of gC that would result in the production of two gC species that lack a transmembrane domain. Moreover, the predicted molecular weights of proteins that would be encoded by the two identified mRNA species encoding truncated gC correspond nicely to the observed apparent sizes of gC moieties that are secreted into the supernatant of infected cells (B. K. Tischer and N. Osterrieder, preliminary data). Independent of the mode of gC secretion, our data using supernatants from CEC infected with v20 or the gC deletion mutant demonstrated that the secreted form of the UL44 gene product(s) indeed plays a role in plaque formation and virus growth. The large-plaque-size phenotype observed in the case of the gC-negative virus is present only in cells incubated with supernatants collected from cells that had been infected with the UL44 deletion mutant. In contrast, both parental and gC-negative virus grew at comparable rates when supernatants derived from v20-infected cells were added to CEC infected with either of the viruses, and even slightly smaller plaque areas were determined compared to those induced by v20 in the absence of the UL44 gene product in the culture medium. These results indicated that a component related to UL44 sequences in the supernatant of MDV-infected cells, probably secreted gC, prevents formation of larger plaque sizes usually induced by v20gC. One explanation of this effect is that soluble gC in cell culture can bind to an unknown receptor on the surfaces of neighboring cells, which may block interaction of membrane-bound gC that could play a role for cell-to-cell spread of MDV. This interpretation is supported by the fact that gC expression is reduced after only a few passages in vitro of highly virulent MDV strains (73). Irrespective of the mode of action of the secreted form of gC, our findings indicate a strong pressure for gC down regulation in cell culture, although some attenuated and passaged strains, such as CVI988-Rispens, still express significant amounts of gC and yet produce relatively large plaques in cultured cells (B. K. Tischer and N. Osterrieder, unpublished observations). It is also conceivable that secreted gC binds to membranous receptors and induces a subsequent signaling cascade, which may ultimately lead to inhibition or impairment of productive infection of those cells or that (soluble) gC is able to modify the expression or action of interferons that have been reported to suppress MDV plaque formation (35, 76). Since herpesviruses, especially chicken herpesviruses, and their hosts share a long history of coevolution (18, 19, 31), it does not seem impossible that the effect of small plaque sizes induced by the expression of soluble gC in cultured cells is caused by a cellular sensing for the presence of MDV and the induction of a subsequent antiviral reaction. Likewise, the in vitro effects of soluble gC may reflect a viral feedback mechanism orchestrated by gC in vivo to adapt its replication to different compartments or cell types in the chicken. Experiments are under way in the laboratory to elucidate these various possibilities by testing gC-negative MDV based on a virulent strain in vivo, and by an attempt to determine the cellular binding partner of MDV gC.
In summary, the integrity of the coding sequence even after extensive virus passage in culture on the one hand (73) and the significant larger plaques of a deletion mutant on the other hand indicate an intricately balanced function of MDV UL44 gene product(s) or associated sequences in the life cycle of MDV. We have demonstrated that gC expression is clearly detrimental to virus growth in vitro. Further experiments will focus on the function of gC in chickens to elucidate whether there is a correlation of the level of gC expression and MDV pathogenesis as discussed by other groups (31, 73) or whether the reduction of the UL44 expression in vitro is more a cell culture effect that is independent of virulence. In addition, future studies will aim at dissecting the effects of the soluble form versus the membrane-bound form of the glycoprotein by the introduction of targeted mutations in a predicted common splice acceptor site that would abrogate expression of two secreted gC products that result from two putative spliced mRNAs.
ACKNOWLEDGMENTS
We thank Jennifer L. Gardell for technical assistance, A. Karger for the ConA precipitation protocol, and Kerstin Osterrieder for performing statistical analyses.
This study was supported by USDA-NRI grant 2003-02234 to N.O.
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Laboratoire de Virologie Moléculaire et Immunologie, UR 086 BASE, Centre de Recherches INRA de Tours, 37380 Nouzilly, France
Lohmann Animal Health, 27472 Cuxhaven, Germany
ABSTRACT
Expression levels of Marek's disease virus (MDV) glycoprotein C (gC) are significantly reduced after serial virus passage in cell culture. Reduced gC expression coincides with enhanced MDV growth in vitro and attenuation. To analyze this phenomenon in detail, a full-length infectious MDV clone was modified by Red-based and shuttle mutagenesis in Escherichia coli. Besides a gC-negative deletion mutant harboring a kanamycin resistance gene, a markerless mutant with the UL44 gene deleted was constructed. On the basis of this deletion mutant, the original or a modified UL44 gene with a mutated start codon (AUGACG) was reinserted into the authentic locus. Similarly, mutants expressing authentic gC or the start codon mutation under the control of a strong constitutive promoter were generated. In vitro studies demonstrated that gC deletion mutants induced twofold-larger plaques than the parental virus did, whereas constitutive overexpression of the glycoprotein resulted in a more than twofold reduction in plaque size. In addition, plaque sizes of the gC deletion mutant were reduced when virus was grown using supernatants from cells infected with parental virus, but supernatants obtained from cells infected with the gC deletion mutant had no measurable effect on plaque size. The results indicated that (i) expression of MDV gC, albeit at low levels in a highly passaged virus, had a significant negative impact on the cell-to-cell spread capabilities of the virus, which was alleviated in its absence and exacerbated by its overexpression, and that (ii) this activity was mediated by the secreted form of MDV gC.
INTRODUCTION
Marek's disease virus (MDV), also referred to as gallid herpesvirus 2, is a member of the family Herpesviridae. Within this large virus family it is classified among the Alphaherpesvirinae and is the prototype member of the genus Mardivirus ("Marek's disease-like viruses") (6, 34, 68). MDV is the causative agent of Marek's disease (MD) in chickens (13), whereas its close relatives and other members of the genus, gallid herpesvirus 3 and herpesvirus of turkeys (HVT; meleagrid herpesvirus, MeHV-1), are completely apathogenic and have been used as MD vaccines since the early 1970s (7, 10, 52, 60). Following the first description of MD in 1907 by Jozef Marek (42), the clinical picture has changed from a polyneuritis associated with general wasting to one that is characterized by visceral lymphomas, transient paralysis, and early mortality within 2 weeks of infection. The virulence of MDV field isolates has increased over the years from mild (m), virulent (v), very virulent (vv), to very virulent plus (vv+) strains. While vv strains are able to break HVT vaccination, vv+ MDV are isolated from flocks vaccinated with a combination of SB-1 (a gallid herpesvirus 3 strain) and HVT (9, 74, 75).
In contrast to other alphaherpesviruses, MDV does not release detectable free enveloped, let alone infectious, virus into the supernatant of cultured cells (8). Similar to varicella-zoster virus (VZV), infectivity in vitro spreads directly only from an infected cell to a neighboring cell (29). In vivo, free infectious virus is released only from the feather follicle epithelium of infected chickens, while virus transfer from macrophages to epithelia, B cells, and finally activated T cells is thought to occur exclusively by direct cell-to-cell spread (8).
The roles that MDV tegument and envelope (glyco)proteins play in virus attachment and entry or in virus maturation, egress, and direct cell-to-cell spread have remained elusive to a large extent, but it is known that a number of MDV structural proteins are key players in these processes. With regard to the tegument proteins, it is known that the herpes simplex virus type 1 (HSV-1) tegument proteins named VP1/2, VP13/14, VP16, and VP22, which are encoded by UL36, UL47, UL48, and UL49, respectively, are major components of cell-free enveloped virus (25, 30, 65). While VP13/14 and VP22 are dispensable for HSV-1 growth, VP1/2 and VP16 were found to be essential for secondary envelopment and egress (21, 48, 71). Similarly, deletion of VP16 causes severe growth defects in relatives of HSV-1, such as equine herpesvirus type 1 (EHV-1) and pseudorabies virus (PRV) (27, 28, 70). In contrast, MDV VP11/12, VP13/14, and VP16 are dispensable for in vitro growth of MDV and VZV, whereas VP22 encoded by the UL49 homologue is essential for MDV replication in cultured cells (15, 24).
In addition to the differential requirement for certain tegument proteins, the role of membrane (glyco)proteins in the virus' life cycle also seems to be partially different in MDV compared to herpesviruses that undergo a complete secondary envelopment resulting in the release of free infectious virus. Sequence analyses have shown that, with the exception of glycoprotein G (gG) and gJ, all common alphaherpesviral glycoproteins, such as gB, gC, gD, gE, gH, gI, gK, gL, and gM as well as the UL49.5 product, the homologue of which is glycosylated and referred to as gN in PRV, are encoded in the MDV genome (34, 68). It could be shown that MDV mutants lacking gB were nonviable; however, unlike other related viruses, MDV is unable to replicate in the absence of two major membrane protein complexes, the gE-gI and the gM-pUL49.5 complex (61, 62, 67). While essentiality of gB has been shown for all herpesviruses analyzed thus far (53), most Alphaherpesvirinae are still able to grow in cultured cells in the absence of the other glycoproteins (2, 3, 22, 32, 37, 51, 56, 77, 78). Besides MDV, VZV is also an exception to the rule, because it requires expression of gE for growth in vitro and in vivo, while gI is required for VZV growth in Vero cells, but not in fibroblasts or melanoma cells (14, 39, 40, 62). Transcriptional analyses of the MDV unique short region and the gD locus, the expression of which is essential for HSV-1, PRV, EHV-1, and bovine herpesvirus 1 attachment and entry (33, 36, 43, 72), revealed that MDV gD is not expressed in cultured chicken embryo cells (66). This is consistent with the complete absence of a gD open reading frame (ORF) in VZV (20). These findings indicated different functions of tegument and glycoproteins in alphaherpesviruses that are able to produce cell-free infectious virions in vitro and those that are only capable of spreading directly from cell to cell.
Examinations of gC-like proteins of alphaherpesviruses, which are components of the viral envelope, suggest that they have multiple functions in vitro and in vivo. Generally speaking, gC orthologues have a central role in attachment of free virus to heparin- and chondroitin-like glycosaminoglycans on the surface of the plasma membrane, thereby conferring the first contact between the virion and cell (55). Independently from its initial binding function to the cell surface, a role of gC in penetration of PRV has been described (45, 57). Furthermore, gC is required for efficient egress of PRV and EHV-1, although both glycoproteins are dispensable for virus growth in vitro (45, 46). In the case of VZV, gC was shown to be a major determinant for virulence in skin, while gC-negative viruses exhibited accelerated and more efficient growth in cultured melanoma cells (16, 47). Another function of alphaherpesviral gC orthologues is binding to complement component C3b, thereby blocking the activation of the complement cascade and rendering the host unable to respond via this nonspecific defense mechanism (26).
MDV gC was previously referred to as the "A-antigen" and is encoded by the 1,503-bp UL44 orthologous gene. MDV gC is synthesized into a 47-kDa primary translation product. The primary product is cleaved at its signal peptide to a yield a 44-kDa precursor protein, which is co- and posttranslationally glycosylated to the mature glycoprotein of 57 to 63 kDa in size. In contrast to other herpesviruses, however, approximately 95% of all the gC produced by an infected cell is secreted. Besides secretion via the regular secretory pathway, a small portion of unglycosylated gC was also reported to reach the extracellular space by an alternative mechanism, although the mechanism of secretion was not described in further detail (31). Another striking feature of MDV gC is that its abundant expression in vitro and in vivo is dramatically reduced after serial passage in culture. Coinciding with reduced gC production, virulence of MDV decreases. Despite the greatly reduced gC expression levels after passage, no difference in the nucleotide sequence of UL44 or its promoter region between viruses with low and high gC expression levels were detectable, and the role of viral transcription factors has been discussed in the shutdown of gC expression (73). Although there is an apparent correlation between gC expression and virulence, there is no convincing evidence that gC is actually involved in virulence of MDV, because the glycoprotein has been found to be expressed at higher levels in some avirulent virus strains (4).
The aim of this study was to elucidate the growth properties of MDV in the absence or presence of gC in vitro. On the basis of a bacterial artificial chromosome (BAC) clone of the highly passaged MDV strain 584Ap80C, we generated a number of mutant viruses that either lacked the gC ORF, harbored point mutations in the gC start codon, or overexpressed the glycoprotein under the control of the human cytomegalovirus immediate-early promoter (PCMV). Our studies clearly demonstrated that there is a reciprocal relationship of MDV growth properties and the level of gC expression in vitro. In addition, the growth disadvantage was shown to be associated with gC present in the supernatant of infected cells. The results indicated that loss of gC function in cultured cells is a marked growth advantage and may explain its absence in highly passaged MDV strains and isolates.
MATERIALS AND METHODS
Viruses and cells. MDVs were propagated on primary or secondary chicken embryo cells (CEC) or on the permanent cell line SOgE, a QM7 (ATCC CRL-1962) derivative that constitutively expresses gE (63). All cells were maintained in Dulbecco's modified essential medium supplemented with 2 to 10% fetal bovine serum. MDV strain v20 was reconstituted from an infectious full-length BAC clone of 584Ap80C (BAC20) that lacks the US2 ORF (61). Both v20 and mutant viruses were recovered at days 5 to 7 after transfection of 1 to 5 μg of BAC DNA using the calcium phosphate precipitation method (61).
Plaque area determination and virus titration assays. For plaque size determinations, fresh CEC were cocultivated with 100 PFU of the various recombinant viruses or the parent virus and incubated for 72 h at 37°C. Cells were fixed with 90% acetone and analyzed by indirect immunofluorescence (IIF) using a convalescent-phase MDV-specific chicken serum (61). In some experiments, plaques were detected using a Sindbis virus-generated antiserum against MDV gC (64). Plaques were visualized with Alexa Fluor 488-conjugated goat anti-chicken immunoglobulin G (IgG) or Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probes). Virus plaques were examined under a fluorescence microscope (Zeiss Axiovert 25). In two independent experiments 50 plaques each were photographed with a digital camera (Zeiss AxioCam HRc), and plaque sizes were determined using the ImageJ software that is freely available from the National Institutes of Health (http://rsb.info.nih.gov/ij/index.html). Statistical analyses of plaque sizes were performed using SAS software package, version 8.2 for Windows (SAS Institute) (59).
Virus titers were determined by infecting 1 x 106 CEC seeded in one well of six-well plates with 100 PFU per well of the various viruses, corresponding to a multiplicity of infection of 0.0001. At different times postinfection (p.i.), infected cells were trypsinized and coseeded with fresh CEC. Titers were determined 72 h later by counting plaques after IIF staining using the MDV-specific antiserum.
Construction of plasmids. To generate recombinant plasmid pTgC1-2, a PCR fragment, dubbed gC1-2, was amplified from BAC20 with primers 5'-TGAGCTCAATGTATCGTAGTAACCATG-3' and 5'-TGGATCCAGATATGTAGAGGGTTACGT-3', cleaved with SacI and BamHI (bold and italics), and cloned into vector pTZ18R (Pharmacia). Similarly, plasmid pTgC3-4 was constructed using primers 5'-GGATCCTGCAGTCTCATTGTTATGTAGTTGTG-3' and 5'-TTAGCATGCAATGGTGGGTAGTATACAG-3' that amplified a fragment that was cleaved with BamHI and SphI (bold and italics) before insertion into pTZ18R. The inserts in both plasmids were sequenced and combined after releasing the fragment gC3-4 from with BamHI and SphI and ligating it into pTgC1-2 digested with the same enzymes, finally resulting in plasmid pTgC1-4.
Recombinant plasmid pTUL44 was generated using primers 5'-TGGATCCCTCATGCTCACGCCGCG-3' and 5'-TTACTGCAGTTATAATCGAATATTTTTTCG-3' for amplification of the UL44 ORF. The PCR product was ligated into pTZ18R after digestion of the PCR fragment with BamHI and PstI (bold and italics) and sequenced. Similarly, a version of the ORF with a point mutation in the start codon (pTUL44ACG) was cloned using a different forward primer (5'TGGATCCCTCACGCTCACGCCGCG-3') that resulted in an ACG codon (underlined) instead of an ATG start codon. Both UL44 inserts were released from the recombinant plasmids with BamHI and PstI and introduced into pTRgC1-4, resulting in pTgC1-4_UL44 and pTgC1-4_UL44ACG. All three constructs (gC1-4, gC1-4_UL44, and gC1-4_UL44ACG) were released with SacI and SphI and cloned into plasmid pST76K_SR (1, 5) to create shuttle plasmids pST76K_SR-gC, pST76K_SR-gC-R, and pST76K_SR-MgC, respectively.
Finally, the human cytomegalovirus immediate-early promoter (PCMV) was released from pcDNA3 (Invitrogen) with BamHI and BglII and introduced into the BamHI site of pTgC1-4_UL44 and pTgC1-4_UL44ACG to create pTgC1-4_ExpUL44 and pTgC1-4_ExpUL44ACG, respectively. Both constructs were excised with SacI and SphI and transferred into pST76K_SR to generate pST76K_SR-ExpgC and pST76K_SR-ExpMgC. The shuttle plasmids were used for RecA-based mutagenesis of BAC20 (see below).
Mutagenesis of BAC20. To replace the UL44 sequence of BAC20 with an antibiotic resistance gene, a system employing homologous recombination at double-strand breaks of DNA in Escherichia coli was used. The products of phage genes exo, ?, and encoded by pKD46 under the control of an L-arabinose-inducible promoter allow recombination between 50 bp at both ends of a linear DNA fragment and their homologous sequences in BAC20 (17, 49, 50). E. coli strain DH10B harboring BAC20 and the temperature-sensitive plasmid pKD46 was grown at 30°C in Luria-Bertani (LB) medium with ampicillin (100 μg/ml), chloramphenicol (30 μg/ml), and 10 mM L-arabinose to an optical density at 600 nm of 0.6, washed three times with 10% glycerol, and concentrated 100-fold. Twenty-five microliters of competent cells were electroporated (1.25 kV/cm, 200 , 25 μF) with 100 ng of a PCR product of an aminoglycoside phosphotransferase gene (aphAI) derived from pACYC177 (12). The primers used for the PCR were 5'-GGAAAGTACCAATGAACATACCATAACAGAAACGACGGGCAAGACGCATCGATTTATTCAACAAAGCCACG-3' and 5'-CGTGTGGAGTTGTATAAACATAGGGCAGTCATGATTATCCCCATCCCTAAGCCAGTGTTACAACCAATTAACC-3'. After electroporation, E. coli was incubated for 1 h at 37°C in SOC (58) and plated onto LB agar containing kanamycin (30 μg/ml) and chloramphenicol (30 μg/ml). Colonies were grown in liquid LB with both antibiotics, and BAC DNA was isolated by a standard alkaline lysis protocol (58) or by column chromatography (QIAGEN plasmid MAXI kit) according to the manufacturer's protocol.
Shuttle mutagenesis was performed with pST76K_SR-derived plasmids. Electrocompetent E. coli DH10B bacteria harboring the appropriate BAC clone were transformed with 200 ng of the respective shuttle plasmid and shaken for 90 to 120 min at 30°C in SOC. Cells were plated onto LB agar with kanamycin (30 μg/ml) and chloramphenicol (30 μg/ml) and incubated overnight at 42°C to select for integration of the shuttle plasmid into BAC20 via recombination of one of the two homology flanks (Fig. 1). Colonies were grown overnight at 42°C in liquid LB containing both antibiotics. DNA from kanamycin- and chloramphenicol-resistant colonies was isolated, digested with appropriate restriction enzymes, and separated overnight by agarose gel electrophoresis (58). Positive clones were grown in LB containing only chloramphenicol for 4 h at 37°C, 10% (wt/vol) sucrose was added to the medium, and cultures were shaken for additional 4 to 8 h. Serial 10-fold dilutions were prepared and grown overnight at 30°C on LB plates containing chloramphenicol and 10% sucrose. The latter procedure selected for loss of sacB sequences of the shuttle plasmid via a second recombination in one of the flanks (Fig. 1). Larger individual colonies were replica plated onto LB agar plates containing either chloramphenicol or kanamycin. DNA was isolated from clones that were unable to grow in the presence of kanamycin but remained chloramphenicol resistant, and the restriction patterns of the clones were analyzed for the presence of predicted rearrangements (1, 5). Finally, the mutated regions of positive candidates were amplified by PCR, and nucleotide sequencing was performed to confirm that the desired mutations had been introduced.
Generation of anti-gC monoclonal antibodies (MAb) and Western blot analyses. For immunization of mice, recombinant gC was expressed in a commercially available baculovirus/Sf21 system exactly as described earlier (23). Briefly, UL44 was amplified by PCR from BAC20 with primers 5'-GGATCCATGCTCACGCCGCGTGTGTTACGAGC-3' and 5'-GAATTCTTATAATCGAATATTTTTTCGTGTGGAGTTG [BamHI and EcoRI sites are shown in bold and italic type] with thermostable rTth DNA polymerase XL (Applied Biosystems). The PCR product was cloned in pGEM (Promega), and the sequence of the insert was checked for integrity. The insert from pGEM-gC was cleaved with BamHI and EcoRI and cloned into pFastI plasmid (Bac-To-Bac baculovirus expression system; Invitrogen). The presence of UL44 in the recombinant baculovirus genome was ascertained by PCR. One BALB/c mouse was immunized with the equivalent of 106 infected Sf21which were collected 72 h p.i. The schedule of immunization and the fusion procedure were as described previously (23). Supernatants from growing hybridomas were tested on CEC infected with MDV and on Sf21 infected with the gC-expressing baculovirus recombinant. One MAb termed A6 was selected for its strong affinity to MDV-infected cells, and subcloning and amplification of the hybridoma was performed as described previously (69). Isotyping was performed with a mouse monoclonal antibody isotyping kit (Sigma), and MAb A6 was found to be of the IgG2b isotype.
Western blot analysis of MDV gC secreted into the supernatant of infected cells was done after concentrating supernatants using concanavalin A (ConA). Briefly, 3 ml of (infected) cell culture supernatant was mixed with an equal volume of ConA buffer (0.5% Zwittergent, 10 mM Tris HCl, pH 7.4, 0.5 mM MgCl2, 0.5 mM MnCl2, 500 mM NaCl), and 100 μl of ConA-Sepharose beads (Sigma) washed in ConA buffer was added. After gentle shaking at 4°C overnight, beads were centrifuged (12,000 x g, 1 min, 4°C), and the ConA slurry was washed three times. Finally, beads were resuspended in 100 μl of sample buffer and heated at 95°C for 5 min. Separation by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), transfer of proteins to nitrocellulose membranes, and Western blot detection using enhanced chemiluminescence (ECL kit; Amersham) was done exactly as previously described (56).
RESULTS
Absence of UL44 sequences results in larger virus plaques. In an attempt to generate a comprehensive library allowing a functional analysis of individual MDV proteins, several MDV ORFs were deleted from BAC20 by Red recombination (17, 61, 62, 67). The viral genes were deleted by replacing individual ORFs with the bacterial aphAI gene that encodes a protein conferring kanamycin resistance. In one of the resulting BAC20 clones, the majority of the UL44 gene encoding gC was deleted, resulting in the recombinant 20kangC BAC (Fig. 2A). After confirmation of the correct restriction enzyme pattern by agarose gel electrophoresis (Fig. 3B) and subsequent Southern blot analysis (data not shown), BAC DNA isolated from E. coli was used to transfect CEC to reconstitute mutant virus v20kangC or the parental v20. Infected cells were frozen as virus stocks in liquid nitrogen and titrated. At 72 h p.i., plaque areas of the gC-negative v20kangC were shown to be increased to more than 185% relative to those of the parental v20 (Fig. 2B). In addition, growth kinetics of both viruses on CEC were determined, and the approximately twofold difference in plaque areas was confirmed by increased growth kinetics of v20kangC on primary chicken cells compared to those of v20 (Fig. 2B).
Markerless manipulation of UL44 sequences using shuttle mutagenesis. The preliminary analysis of a deletion of MDV gC-encoding sequences in v20 had shown that absence of the ORF resulted in markedly increased plaque sizes and suggested a growth advantage of MDV in the absence of gC. These results prompted us to initiate a more comprehensive study that applied genetic modifications that would not result in the introduction and maintenance of a selectable marker, because MDV has a densely packed genome with regulative sequences often overlapping ORFs (34, 68).
First, and to corroborate the findings with the v20kangC mutant virus, plasmid pST76K_SR-gC was used to generate a markerless deletion mutant by shuttle mutagenesis, which led to removal of the complete UL44 ORF and resulted in recombinant BAC clone 20gC (Fig. 3A). Starting with the constructed 20gC deletion mutant, the same method of RecA-based shuttle mutagenesis was applied utilizing plasmid pST76K_SR-gC-R to reintroduce the UL44 sequence into the original locus. Virus reconstituted from this BAC clone (20gC-R) differed from BAC20 only in the introduced BamHI and PstI restriction sites that were used to clone the shuttle plasmids (Fig. 3A).
To create a gC-negative mutant that still contained UL44 sequences and to avoid interference with potential regulative elements, the gC start codon was replaced in the BAC20 infectious clone by shuttle mutagenesis using 20gC and pST76K_SR-MgC, which resulted in recombinant BAC clone 20MgC (Fig. 1 and 3A). Finally, two mutant BAC clones from 20gC were constructed, in which the PCMV promoter was inserted immediately upstream of UL44. Shuttle plasmids pST76K_SR-ExpgC and pST76K_SR-ExpMgC were used. While the former would result in expression of gC controlled by PCMV, the latter would be unable to express the viral protein, because the start codon was altered from AUG to ACG (Fig. 3A).
After generation and testing of all cointegrates by restriction enzyme analysis for correct insertion of the shuttle plasmid, the final mutant BAC genomes were analyzed after resolution of the cointegrates. Agarose gel electrophoresis of BamHI and PstI restriction enzyme digests of the individual BAC clones showed that the insertions and deletions were successful and that no spurious rearrangements had occurred (Fig. 3B). In the 20kangC mutant, replacement of UL44 with aphAI in 20kangC caused a shift of the BamHI-B fragment of approximately 250 bp towards the BamHI-A1 band that results from the introduction of the mini-F sequences into the BamHI-A fragment and the introduction of an additional BamHI site into BAC20 (61) (Fig. 3B, lane 2). In the case of 20gC (Fig. 3B, lane 3), due to the deletion of UL44 and the introduction of a BamHI site, the BamHI-B (18.0 kb) fragment present in BAC20 is divided into two bands of 7.5 kbp and 9.0 kbp in size. The latter band increased in size to 10.5 kbp in both the 20gC-R and 20MgC mutant (Fig. 3B, lanes 4 and 5) due to the reinsertion of the UL44 sequence. Finally, in the PCMV constructs (20ExpgC and 20ExpMgC) and due to the insertion of the promoter sequence, the 7.5-kb fragment increased to 8.4 kbp (Fig. 3B, lanes 6 and 7). These results were confirmed by nucleotide sequencing of the gC locus and adjoining sequences (data not shown), and both analyses clearly demonstrated the correct insertion or deletion of the desired sequences in the various clones.
Growth properties of UL44 mutant viruses. To analyze the generated UL44 virus mutants, primary CEC as well as SOgE cells were transfected with BAC DNA isolated from E. coli. Reconstituted viruses were propagated on the two cell types, and aliquots were frozen in liquid nitrogen. After determination of virus titers, fresh cells were coseeded with 100 PFU of the various viruses, fixed after 72 h, and analyzed by IIF. Whereas all the reconstituted viruses could clearly be detected using an MDV-specific chicken convalescent-phase serum, only cells infected with v20ExpgC exhibited robust IIF staining using a rabbit antiserum raised against MDV gC. A very faint reactivity of the gC-specific antibody with infected cells was also observed for parental v20 and the 20gC-R revertant virus (Fig. 4).
After determining the reactivity of the various virus plaques with different antibodies, plaque sizes on CEC were determined for all viruses as described above. The gC deletion mutant v20gC induced plaques that exhibited sizes that were more than twice the size of parental BAC20 virus plaques (203%) (Fig. 5A). Plaque sizes of the revertant v20gC-R virus were larger than parental plaques (114%), while the v20MgC that was unable to express gC exhibited plaque areas amounting to 148% of those of v20 (Fig. 5A). Areas measured for plaques of the gC-overexpressing mutant v20ExpgC were reduced to 44%, while virus plaques on CEC of the virus mutant that expressed the methionine mutant under the control of PCMV (v20ExpMgC) were as large (104%) as those induced by parental v20 (Fig. 5A). Data from the plaque area assays were analyzed with SAS software, and means of plaque areas induced by the generated recombinant viruses were compared with each other by analysis of variance to determine statistically significant differences (59). Since multiple comparisons were performed, only P values smaller than 0.0033 indicate significant differences using the Wilcoxon two-sample test or the Kruskal-Wallis test (59) (Table 1). Statistical analyses demonstrated that both gC-negative mutants, v20gC and v20MgC, induced significantly larger plaques than those induced by parental v20 as well as revertant v20gC-R virus, whereas the sizes of v20 and v20gC-R or v20gC and v20MgC were not significantly different from each other. With a P of <0.0033, plaque areas of the gC-overexpressing mutant v20ExpgC were statistically significantly smaller than those of parental virus; plaques induced by the virus overexpressing gC but harboring the mutated start codon (v20ExpMgC) were significantly larger than those induced by the true overexpression mutant (v20ExpgC) (Table 1). Plaque sizes of v20ExpMgC, however, were not significantly larger compared to parental v20 plaques, although this mutant was unable to express gC (Table 1).
To extend the findings of the plaque size determinations, multistep growth kinetics of the engineered mutant viruses were performed. The results of these experiments are summarized in Fig. 5B. Consistent with the findings of the plaque size determinations, the gC-negative v20gC virus grew slightly faster than parental v20, whereas v20ExpgC exhibited markedly reduced growth properties on CEC (Fig. 5B). All other mutant viruses exhibited comparable growth kinetics and were virtually indistinguishable from those of parental v20 (Fig. 5B). The results of MDV growth kinetics are largely a reflection of virus-induced plaque sizes, because infected cells are trypsinized and coseeded with fresh cells to determine virus titers, i.e., the larger the virus plaques, the higher the determined virus titers. Therefore, we concluded that the titration experiments corroborated the findings of the plaque size determinations (61), which provide a superior measure of the in vitro growth properties of MDV mutants.
Role of secreted MDV gC in cell culture supernatants. The variation in plaque sizes and growth kinetics of the different gC mutant viruses suggested that the differences in their ability to express gC accounted for their different abilities to spread from an infected cell to a neighboring uninfected cell. To analyze gC expression by v20 and its derivatives, supernatants of 5 x 106 cells infected with 1,000 PFU of the respective virus were harvested at day 3 p.i. gC was precipitated from supernatants (3 ml) using ConA-Sepharose beads, and precipitates were separated by SDS-10% PAGE before transfer to nitrocellulose membranes. Detection by Western blotting of secreted gC using MAb A6 demonstrated that expression of this form of the glycoprotein in cells infected by parental v20 or v20gC-R, unlike the cell-associated form (Fig. 4), was below the detection limit, even after 30-fold concentration by ConA precipitation (Fig. 6). As expected, no reactivity was observed in the supernatants of CEC infected with v20gC. In contrast, a strong gC-specific signal was obtained in supernatants infected with the overexpressing v20ExpgC virus (Fig. 6), indicating that a significant increase in gC production was achieved by the replacement of the original gC promoter with PCMV (Fig. 3). The v20ExpMgC mutant, in which the gC start codon was mutated but also contained PCMV upstream of the open reading frame, was unable to express the protein (Fig. 6). From these and the above results, we concluded that the amount of gC present in the supernatant of infected cells correlated well with the plaque sizes induced by different gC mutant viruses.
To further investigate whether the effect of MDV gC on virus growth was indeed caused by the secreted form of gC, supernatants were collected from CEC that were infected with wild-type v20 or the gC-negative mutant virus v20gC. Supernatants were used to overlay freshly infected cells, and plaque sizes were determined. In this experiment, 1 x 106 CEC were infected with 500 PFU of v20 or v20gC and grown for 48 h in medium containing 10% fetal calf serum. At that time point, infection had proceeded to a stage where approximately 20% of cells were infected. Cell culture supernatant was removed, and 2 ml of fresh medium was added. After a further 24-h incubation period, medium was collected and centrifuged for 10 min at 16,100 x g to remove cellular debris. Fresh CEC were then infected with 100 PFU of v20 or v20gC and incubated from 24 h p.i. with a 1:1 mixture of fresh medium and supernatants obtained from cells infected with either v20 or v20gC. After 48 h, cells were fixed with 90% acetone, virus plaques were visualized by IIF, and plaque sizes were determined. The area of v20gC plaques grown with conditioned medium that originated from the CEC infected with the gC-negative virus were significantly larger (144%) than those of v20 (100%). However, plaque sizes of viruses grown in the presence of culture medium originating from CEC infected with gC-positive v20 virus showed marked decreases (96% in the case of v20 and 82% in the case of v20gC) (Fig. 7). From the results, we concluded that a soluble factor present in the supernatant of MDV expressing UL44, probably the secreted form of gC, was responsible for the observed reduction in plaque sizes of both v20 and v20gC.
DISCUSSION
In the present study we conducted an in-depth analysis of the function of MDV gC for virus replication in vitro. Besides the observation that expression levels of the UL44 gene negatively correlate with attenuation of MDV and that gC expression is reduced by serial passage of MDV strains in cell culture, knowledge on the function of gC in highly cell-associated herpesviruses, such as MDV, has been sparse (73). Alignment of the gC amino acid sequence of MDV to that of its HSV-1 counterpart demonstrated that the carboxy-terminal domains of MDV and HSV-1 gC share high similarity, whereas the amino termini are completely different. However, all cysteine residues, which are important for maintaining the tertiary structure of gC (38, 41), are conserved in MDV. One important pair of cysteines at positions 127 and 144 flank a loop which forms a domain that contains a stretch of basic amino acids in HSV-1 gC that are thought to interact with heparan sulfate (41). Preliminary data suggest that MDV gC is unable to bind heparin (data not shown), which is supported by the fact that only two basic amino acids are present around the orthologous cysteines (Fig. 8). Furthermore, the whole protein harbors none of the described heparin-binding domains, XBBXBX or XBBBXXBX, where B is any basic amino acid and X is any hydrophobic amino acid (11).
In the case of VZV, another highly cell-associated alphaherpesvirus and close relative of MDV, deletion of orf14 encoding gC in the vaccine Oka strain did not interfere with heparin sensitivity (16), indicating that another envelope (glyco)protein is responsible for the first step of virus attachment that is mediated in all Alphaherpesvirinae analyzed to date by glycosaminoglycans on the plasma membrane bearing heparan or chondroitin sulfate moieties. The absence of the VZV orf14 gene product whose expression is—similar to the situation in MDV—reduced in attenuated strains had a minor effect on the growth of VZV in cultured cells. Deletion of gC, however, resulted in an increase of end-point viral titer of approximately twofold compared to the titer of the parental strain. Likewise, deletion of MDV UL44 led to this apparently marginal increase of virus growth, although mean plaque areas of v20gC or v20kangC were twice the size of that of the parental clone. As stated above, however, the twofold increase in end-point virus titers is an exact reflection of plaque sizes, because infected cells, whose numbers are dependent on the size of the plaques, are coseeded with fresh cells and the induced plaques are counted. The statistically significant difference in plaque size between gC-negative virus and parental virus showed that a clear phenotype is attributable to the presence or absence of UL44 sequences, although the 584Ap80C strain, from which BAC20 and consequently v20 are derived, were passaged 80 times in cultured cells and although expression of gC in v20-induced plaques was barely detectable and no significant amounts of gC seem to be secreted (Fig. 4 and 6). The deletion of only the gC start codon, which left the remainder of the coding sequences intact, also led to an increase of plaque sizes in cell culture. The difference in plaque size between the v20MgC mutant and the parental virus was also significant but was less than that after complete removal of UL44 sequences, which may suggest a second function of sequences harboring the gC ORF. Possible explanations for the observed differences are that regulation of the adjacent UL45 gene or that of a predicted 624-bp ORF, which is directed antisense to UL45 (LORF8) (34, 68), may be affected when the entire gC-encoding sequence is deleted. The functions of UL45 or its putative antisense ORF are completely unclear at present. Despite their positional conservation, they both show no homology to any other herpesvirus sequences outside of the Mardivirus genus (34, 68). A role of the long intergenic region between UL44 and UL45 is also possible, since a predicted promoter for UL45 overlaps with that of UL44 and as such would also be responsible for transcription of the entire 864-bp sequence that includes two smaller theoretical ORFs (34, 68) (Fig. 2).
In contrast to the deletion of gC, constitutive overexpression of gC in the v20ExpgC mutant, which was detected unequivocally by IIF and Western blot analyses (Fig. 4 and 6), resulted in significantly smaller virus plaques and reduced growth kinetics, clearly indicating that there is a negative effect of the UL44 gene product(s) on growth properties of MDV in cell culture. Consistent with theoretical predictions, the start codon deletion mutant driven by PCMV had significantly increased plaque sizes compared to the v20ExpgC mutant plaques, and gC—as expected—was not detected by IIF, convincingly showing that the point mutation of the AUG codon is sufficient to abrogate gC production even if transcription is controlled by a strong constitutive promoter. Surprisingly, however, the v20ExpMgC point mutant only grew to levels comparable to those of v20 and v20gC-R. Possible explanations for this observation are that either increased transcription of UL44 has a negative effect on virus growth by interference with regulatory sequences that overlap with the gC coding region, as discussed before, or that the strong PCMV affects not only expression of gC but also that of downstream genes (Fig. 2).
Most of the gC produced by MDV-infected cells is secreted into the supernatant, which has been attributed to unstable membrane anchoring that would be caused by the short predicted cytoplasmic domain (31, 73). Preliminary data obtained from reverse transcriptase PCR studies performed in our laboratory, however, indicate alternate splicing of gC that would result in the production of two gC species that lack a transmembrane domain. Moreover, the predicted molecular weights of proteins that would be encoded by the two identified mRNA species encoding truncated gC correspond nicely to the observed apparent sizes of gC moieties that are secreted into the supernatant of infected cells (B. K. Tischer and N. Osterrieder, preliminary data). Independent of the mode of gC secretion, our data using supernatants from CEC infected with v20 or the gC deletion mutant demonstrated that the secreted form of the UL44 gene product(s) indeed plays a role in plaque formation and virus growth. The large-plaque-size phenotype observed in the case of the gC-negative virus is present only in cells incubated with supernatants collected from cells that had been infected with the UL44 deletion mutant. In contrast, both parental and gC-negative virus grew at comparable rates when supernatants derived from v20-infected cells were added to CEC infected with either of the viruses, and even slightly smaller plaque areas were determined compared to those induced by v20 in the absence of the UL44 gene product in the culture medium. These results indicated that a component related to UL44 sequences in the supernatant of MDV-infected cells, probably secreted gC, prevents formation of larger plaque sizes usually induced by v20gC. One explanation of this effect is that soluble gC in cell culture can bind to an unknown receptor on the surfaces of neighboring cells, which may block interaction of membrane-bound gC that could play a role for cell-to-cell spread of MDV. This interpretation is supported by the fact that gC expression is reduced after only a few passages in vitro of highly virulent MDV strains (73). Irrespective of the mode of action of the secreted form of gC, our findings indicate a strong pressure for gC down regulation in cell culture, although some attenuated and passaged strains, such as CVI988-Rispens, still express significant amounts of gC and yet produce relatively large plaques in cultured cells (B. K. Tischer and N. Osterrieder, unpublished observations). It is also conceivable that secreted gC binds to membranous receptors and induces a subsequent signaling cascade, which may ultimately lead to inhibition or impairment of productive infection of those cells or that (soluble) gC is able to modify the expression or action of interferons that have been reported to suppress MDV plaque formation (35, 76). Since herpesviruses, especially chicken herpesviruses, and their hosts share a long history of coevolution (18, 19, 31), it does not seem impossible that the effect of small plaque sizes induced by the expression of soluble gC in cultured cells is caused by a cellular sensing for the presence of MDV and the induction of a subsequent antiviral reaction. Likewise, the in vitro effects of soluble gC may reflect a viral feedback mechanism orchestrated by gC in vivo to adapt its replication to different compartments or cell types in the chicken. Experiments are under way in the laboratory to elucidate these various possibilities by testing gC-negative MDV based on a virulent strain in vivo, and by an attempt to determine the cellular binding partner of MDV gC.
In summary, the integrity of the coding sequence even after extensive virus passage in culture on the one hand (73) and the significant larger plaques of a deletion mutant on the other hand indicate an intricately balanced function of MDV UL44 gene product(s) or associated sequences in the life cycle of MDV. We have demonstrated that gC expression is clearly detrimental to virus growth in vitro. Further experiments will focus on the function of gC in chickens to elucidate whether there is a correlation of the level of gC expression and MDV pathogenesis as discussed by other groups (31, 73) or whether the reduction of the UL44 expression in vitro is more a cell culture effect that is independent of virulence. In addition, future studies will aim at dissecting the effects of the soluble form versus the membrane-bound form of the glycoprotein by the introduction of targeted mutations in a predicted common splice acceptor site that would abrogate expression of two secreted gC products that result from two putative spliced mRNAs.
ACKNOWLEDGMENTS
We thank Jennifer L. Gardell for technical assistance, A. Karger for the ConA precipitation protocol, and Kerstin Osterrieder for performing statistical analyses.
This study was supported by USDA-NRI grant 2003-02234 to N.O.
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