1 Institute for Hygiene and Social Medicine, University of Innsbruck, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria
2 Institute of Molecular Biology, Austrian Academy of Sciences, Salzburg, Austria
3 Institute of Virology, University of Mainz, Germany
4 Department of Microbiology, CBD Porton Down, Salisbury, UK
Correspondence
Hartwig Huemer
hartwig.huemer{at}uibk.ac.at
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ABSTRACT |
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The DNA sequence of the gC gene of Cercopithecine herpesvirus type 1 reported in this paper has been deposited (30 October 1998) in EMBL under nucleotide accession no. SHE012474 and in GenBank under no. AJ012474.
Present address: Upper Austrian Research GmbH, Center for Biomedical Nanotechnology, Linz, Austria.
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INTRODUCTION |
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In contrast to the situation in its natural host, where disease symptoms resemble those of human herpes simplex virus (HSV), severe encephalitis associated with high mortality has been observed in humans (Davenport et al., 1994).
Interestingly, human HSV can also act as a killer virus' if transferred to certain new world monkey species (Huemer et al., 2002), indicating that the pathogenicity and virulence of herpesviruses are difficult to predict in different host environments.
Immunocompetent humans are not usually endangered by alphaherpesviruses from other species, a phenomenon that has been also observed in animals, where, in most cases, interspecies transmission seems to be rather restricted (Engels et al., 1992).
In contrast, a broad spectrum of cells from different species is susceptible to infection with alphaherpesviruses in vitro, which is most likely due to attachment to the heparan sulphate receptors' found on many cell types. Thus, it seems unlikely that a restriction at the cellular level is solely responsible for the observed host specificity of herpesviruses in vivo and a key topic is to investigate possible mechanisms for this widely observed natural immunity (reviewed by Skinner et al., 2001). Identification of virus and host factors that cause susceptibility or resistance to herpesviruses from other species may represent a possible way forward for future prophylactic or therapeutic measures.
In the early phase of virus infection, neither a specific humoral nor a cellular immune response is available. The fate of an infectious agent depends primarily on its ability to avoid the mechanisms of the so-called nonspecific immune response. One important antibody-independent system, acting without precedent priming with antigen, is the alternative complement pathway, a phylogenetically old system that represents a first barrier against invading microorganisms (Cooper, 1991). Its importance for complex viruses arises from the finding that several members of the Herpesviridae and also the Poxviridae express proteins on the virion and infected cell surface; these proteins are able to interact with proteins of the complement cascade at different levels (Ohmann et al., 1988
; Isaacs et al., 1992
; Huemer et al., 1992b
, 1993a
; Rother et al., 1994
).
Thus, HSV glycoprotein C (gCHSV-1/2) of both serotypes has been shown to bind to the central complement component C3 and thereby interfere with the antibody-independent alternative complement pathway (Fries et al., 1986; McNearney et al., 1987
). gCHSV-1 was found to be very similar to known human complement regulatory proteins, thus competing for binding with the alternative pathway activator properdin or regulator protein factor H, which was not observed using purified gCHSV-2 (Huemer et al., 1993b
). Absence of gCHSV-1 or pseudorabies virus (PRV) gIII (gCPRV) on virions has been shown to cause increased virus lysis via the alternative complement pathway and interestingly, species variations in the efficiency of lysis using different complement sources have been observed (Hidaka et al., 1991
; Ikeda et al., 2000
; Maeda et al., 2002
). This is in accordance with the finding of differences in the binding to complement C3 of herpesvirus gC proteins of different species (Huemer et al., 1993a
).
Expression of gC homologues is highly conserved and the gC proteins of different HSV serotypes are found in clinical isolates, with the exception of isolates originating from immunologically privileged locations (Hidaka et al., 1990). Moreover, the finding of an attenuated phenotype of other species isolates with deletions or diminished expression of the gC homologues (Mettenleiter et al., 1988
; Liang et al., 1992
; Moffat et al., 1998
) also suggests that these proteins perform vital functions in vivo.
Furthermore, the gC proteins have been shown to contribute to binding to cell surface heparan sulphate, which is also true for other herpesvirus glycoproteins such as gB (reviewed by Sawitzky et al., 1990; Liang et al., 1992
). However, it should be noted that binding to charged surface molecules, like heparan or chondroitin sulphate, appears to be a general mechanism of virus adsorption in nature, which is also found in many other viruses from different families.
gC binding to heparan sulphate is not essential for HSV-2 attachment (Gerber et al., 1995) and studies using gC-deficient HSV-1 mutants suggest that gC plays little role in the adsorption process (Griffiths et al., 1998
). Although this is still a controversial topic (Laquerre et al., 1998
), there is growing evidence that binding to heparan sulphate may not represent the predominant biological function of the gC homologues in vivo, at least in some of the strains tested. Additionally, the C3-binding sites on gCHSV-1 and the binding sites for heparin, dextran sulphate and other polysulphates seem to be related, as these substances block the C3 receptor function of gCHSV-1 (Huemer et al., 1992a
).
In this study, we have investigated the complement receptor of SHBV, and cloned and sequenced gCSHBV. Expression of the gene in eukaryotic expression systems facilitates characterization of the antigenic structure and furthers studies of possible immune regulatory properties. The constructs have also been used for diagnostic serology testing. Serodiagnosis of SHBV is difficult due to extensive cross-reactivity with HSV specimens (Falke, 1964; Hilliard et al., 1989
; Norcott & Brown, 1993
). In addition, vaccinia virus constructs using the established modified vaccine virus strain Ankara (MVA) have the potential for use in immunization studies.
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METHODS |
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DNA purification, identification of fragments and cloning procedures.
As isolation of the full-length gCSHBV gene by PCR amplification using HSV-specific primers was not successful, and because amplification of long SHBV fragments proved to be difficult due to the high G/C content of the SHBV genome, a conventional cloning strategy was chosen. Viral DNA was isolated from SHBV-infected roller bottles of Vero cells, which were lysed in 1 % lauryl sarcosine and digested with pronase E. Viral DNA was separated from genomic DNA by density centrifugation in CsCl gradients. Fractions containing viral DNA were precipitated with isopropanol and digested with rare-cutting restriction enzymes. Restriction digests were separated on 1 % agarose, blotted onto nylon membranes and hybridized with gCHSV-1- and gCEHV-1-specific probes under conditions of low stringency (50 °C, 0·5 % SSC wash). As probes, restriction fragments were excised from plasmids containing the coding sequences of HSV-1 strain KOS (Huemer et al., 1989) and EHV-1 strain Piber (Huemer et al., 1995
) and labelled by random priming using Klenow polymerase and digoxigenin-labelled UTP. Blots were developed using the anti-digoxigenin system from Boehringer Mannheim. Cross-hybridizing SHBV fragments were isolated, subcloned into cloning plasmids and subjected to sequence analysis. For comparison, the gC gene of HSV-1 strain Ang was isolated in a similar manner.
Sequence analysis.
Subcloning of several subfragments was necessary as the high G/C content of SHBV prevented long sequencing runs due to the formation of secondary structures. Constructed plasmids were analysed by automated sequencing in an ABI Prism DNA sequencer and a MWG LiCor using both dye labelling and dye primer sequencing techniques. Homology alignments with gC sequences obtained from EMBL were performed using the DNAStar program.
Construction of expression plasmids and expression of recombinant proteins.
After sequence analysis, a 1·4 kb BamHISacI fragment was excised and religated to a 570 bp NcoIBamHI fragment producing the complete gCSHBV-encoding sequence. The whole coding sequence following a NcoI site containing the start codon was inserted into the expression vector pCR3.1 (Invitrogen) under the control of the cytomegalovirus immediate-early promoter.
A construct lacking the putative transmembrane sequence was generated subsequently by PCR amplification of the 5'-terminal sequence, including the PstI site at position 1420 using kinase-treated forward (5'-GATGGAGTTCGGGAGCGGCGA-3') and reverse (5'-GACCCCGTGGGCCAGCAGGTGACCCACCATCACCATCACCATTGA-3') primers. The PCR fragment was cloned unidirectionally into pCR3.1 using a single-sided dephosphorylated vector (Invitrogen). The fragment was excised with PstI/EcoRI and inserted into the corresponding sites of pCR3.1SHBV, thus replacing the hydrophobic putative transmembrane sequences following aa 418 with six histidine residues and a stop codon.
For comparison, similar expression plasmids were constructed, inserting the full-length gC genes from EHV-1 (Huemer et al., 1995) and Marek's disease virus (MDV, kindly provided by K. Osterrrieder, IMB/BFAV, Insel Riems, Germany) into pCR3.1.
Plasmids were transfected into Vero and RK13 cells using Lipofection (Qiagen) and the expression of gCSHBV was monitored by immunofluorescence. Additionally, stable cell lines were generated by electroporation of RK13 cells using a Biorad GenePulser (0·4 cm cuvettes, 350 V, 500 µF). Recombinant cell lines were selected by outgrowth in medium containing 500 µg geneticin ml-1 (G418). These stable cell clones were finally moved to protein-free culture conditions, which enabled isolation of the secreted recombinant protein by filtration. Among the protein-free media tested, Cytoferr CHO medium (PAA Laboratories) supported growth of our transfected RK13 cells. Supernatants were concentrated to 100-fold by Amicon filtration (Millipore) using membranes with a 10 kDa cut-off pore. Filtrates were used as a source of recombinant protein.
Construction of vaccinia virus recombinants.
As insertion into the thymidine kinase gene leads to loss of replicative activity of MVA (Scheiflinger et al., 1996), insertion into the haemagglutinin (HA) gene of vaccinia virus was performed using recombination plasmid pHA11k-gpt, described recently by Huemer et al. (2000a)
. Full-length and truncated gCSHBV and the homologous sequences from HSV-1 and EHV-1 were ligated into the recombination vector pHA11k-gpt and transfected into CHF cells infected earlier with MVA. Recombinant viruses expressing gpt-resistance were selected in medium containing xanthine, hypoxanthine and mycophenolic acid and isolated by several rounds of plaque purification using semi-solid agar overlays.
Antisera and immunoassays.
A polyclonal anti-SHBV antiserum has been raised in rabbits (Falke, 1964). Sera against cynomolgus- and rhesus-derived isolates of SHBV have been kindly provided by M. Slomka (CPHL, Colindale, UK). Macaque sera were obtained from C. Coulibaly (Paul-Ehrlich-Institute, Germany). Production of antisera and monoclonal antibodies against gCHSV-1 has been described previously (Huemer et al., 1989
). Monoclonal and polyclonal antibodies against gC homologues from PRV, bovine herpesvirus type 1 (BHV-1) and EHV-1 have been used as described earlier (Huemer et al., 1992b
, 1993a
, 1995
). Antisera were tested by immunofluoresence using 12-well microscopic slides coated with acetone/methanol-fixed SHBV-infected Vero or RK13 cells stably transfected with gCSHBV or control constructs. Infected cell lysates, transfected cells and recombinant gCSHBV protein were reacted with polyclonal anti-SHBV rabbit sera for ELISA and for immunofluoresence assays, according to the methods described for gCHSV-1 (Huemer et al., 1989
, 1992a
).
Radioimmunoprecipitation of vaccinia virus lysates was performed by infecting 35S-labelled RK13 cells with recombinant gCSHBV-MVA constructs for 2 days. Cells were lysed with 0·5 % NP-40 and 0·5 % deoxycholate and supernatants were obtained by low-speed centrifugation. Lysates were then reacted with the indicated mono- or polyclonal antibodies and precipitated by formalin-fixed Staphylococcus aureus protein A (Sigma). Samples were washed in lysis buffer and analysed by SDS-PAGE. MVA constructs containing gCHSV-1, gCEHV-1 and the parental MVA were used for comparison.
Complement factors, C3-binding assays and polyanion binding.
The purification of complement component C3 from human serum and other species has been described elsewhere (Huemer et al., 1992b, 1993a
, b
). Macaque C3 was purified accordingly from cynomolgus monkey serum by ion exchange chromatography over Mono-Q and subsequent molecular size fractionation on CL4bSepharose (Pharmacia). Haemolytic activity of the purified C3 protein was tested by lysis of rabbit erythrocytes using C3-deficient human serum (Quidel).
Binding of C3 to surface-expressed gCSHBV was tested by FACS analysis of recombinant RK13 cells expressing full-length gCSHBV. Stable transformants producing the truncated gCSHBV lacking the transmembrane domain and the parental RK13 cell line were used as controls. Cells were incubated with complement component iC3 (100 µg ml-1 in PBS) on ice; iC3 represents haemolytically inactive C3 with the internal thioester hydrolysed, leading to C3b/iC3b-like properties of the molecule, including binding to different human complement receptors (Cole et al., 1985). This was confirmed by binding of the samples used to different complement receptor-carrying human cell lines, such as Raji and U937. Cells were washed with PBS and reacted with FITC-labelled antisera directed against C3d or C3c (Dako). After a final washing step, cells were fixed with formaldehyde and the intensity of fluorescence was determined using a FACScanIII (Beckton-Dickinson).
Binding of gCSHBV from virus-infected cell lysates was performed according to the immunoprecipitation described for gCPRV (Huemer et al., 1992b). In brief, purified C3 was coupled to CNBrSepharose and incubated with membrane extracts of SHBV- or HSV-1-infected Vero cells, which were labelled previously with [14C]glucosamine. Sepharose coupled with ovalbumin or purified mouse immunoglobulin served as negative controls. Washed precipitates were run on 10 % SDS-polyacrylamide gels and exposed to radiography.
Initial testing for binding of C3 to SHBV-infected Vero cells was performed by rosetting using sheep red blood cells (SRBC) coated with human complement component C3b. This has been found previously to be useful for detection of complement receptors expressed by HSV-1, EHV-1, EHV-4 and a guinea pig herpesvirus, but not for HSV-2, BHV-1 or PRV (Huemer et al., 1992a, 1993a
, 1995
). Inhibition of SRBC binding of SHBV-infected cells was performed using heparin sulphate, dextran sulphate and other polysulphates under conditions identical to those described for HSV-1 (Huemer et al., 1992a
).
Binding of recombinant gCSHBV from protein-free cell culture superatants to the polysulphates dextran sulphate and chondroitin sulphate was tested in ELISA. Thus, different concentrations of the substance were covalently coupled to CovaLink microtitre plates (Nunc). Plates were then blocked with 100 mM Tris/glycine followed by 1 % ovalbumin in PBS. Concentrated cell culture supernatants of stably transfected cells secreting gCSHBV were used as well as supernatant from control cells concentrated 100 times by membrane filtration. Bound gCSHBV was detected by polyclonal anti-SHBV serum and peroxidase-labelled secondary antibody. Plates were washed with PBS containing 0·1 % Tween-20, developed with ABTS [azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)] and read at a wavelength of 405 nm.
Properdin- and factor B-binding assays were performed as described previously for gCHSV-1/2 (Huemer et al., 1993b). C3b-coated plates were preincubated with 2-fold serial dilutions of gCSHBV, starting with 5 µg ml-1, and binding of purified properdin was tested using a specific monoclonal antibody (reagents from Quidel). Additionally, binding of serum-derived factor B to plate-bound C3b was tested using a polyclonal anti-factor B antiserum raised in goats (Atlantic Antibodies). Plates coated with BSA only and wells without viral glycoprotein served as background controls.
Complement lysis assay of transfected RK13 cells.
Human and porcine sera were obtained by clotting blood samples at 4 °C. Guinea pig serum, used for diagnostic complement fixation assays, was obtained from a commercial source. Haemolytic activity of sera was tested using antibody-coated SRBC, verifying high complement titres of the preparations.
RK13 cells were lysed by porcine serum but not by human and guinea pig sera, as could be detected by chromium-release assays. Proliferating RK13 cells were trypsinized, washed and 5x106 cells labelled at 37 °C for 1 h with 100 µCi (3·7 MBq) of sodium chromate containing 51Cr. Cells were washed to remove excess chromium, seeded into 96-well plates and after a further 1 h of incubation, serial dilutions of serum were added to the culture medium. The specific release of chromium was counted after 45 h by subtracting the background release of untreated cells. Percentage of cell lysis was compared to a maximum release value obtained by cell lysis using 2 % Triton X-100. Heat inactivation of sera or addition of EDTA inhibited complement-mediated lysis, completely reducing levels to background.
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RESULTS |
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Isolation and sequencing of the gCSHBV gene
SHBV DNA hybridized to gCHSV-1 but not to gCEHV-1-specific DNA probes under conditions of low stringency (data not shown).
The gCSHBV-encoding sequence was contained within two BamHI fragments of 960 bp and 3 kb. An open reading frame of 1401 bp encoding 467 aa was found and in contrast to the HSV proteins, two additional cysteine residues were identified in the leader sequence, in addition to the eight cysteines in the coding sequences at highly conserved positions (Fig. 2).
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Comparison with the homologous proteins of human varicella-zoster virus (VZV), PRV, BHV-1, EHV-1 and MDV revealed an overall amino acid identity of 21·2 % for PRV, 20·3 % for BHV-1, 20·1 % for EHV-1, 16·9 % for MDV and only 16·3 % for VZV.
Expression of gCSHBV in recombinant vaccinia virus
Cells infected with MVA constructs containing the gCSHBV-encoding sequence express the protein in the cell membrane, as detected by immunofluorescence with anti-SHBV-specific antiserum. No cross-reactivity was observed using anti-gCEHV-1 and anti-gCBHV-1 or gCPRV-specific polyclonal antisera or different anti-gCHSV-1 monoclonal antibodies (data not shown).
Expression of gCSHBV in recombinant MVA was compared with the homologous proteins from EHV-1 and HSV-1 in Fig. 3. Immunoprecipitation revealed a protein with a molecular mass of about 65 kDa, which indicates that the SHBV protein, similar to gCHSV-2, appears to be much less glycosylated than the HSV-1 homologue, although, underglycosylated gCHSV-1 precursor precipitated also. The stronger precipitations of gCHSV-1 and gCEHV-1 do not indicate a stronger rate of expression but reflect the high binding capacity of monoclonal antibodies as compared to polyclonal antisera directed against all SHBV proteins, with only a fraction of precipitated immunoglobulin representing anti-gCSHBV antibodies. No precipitations were observed with control MVA, thereby demonstrating the specificity of the antibodies used.
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Expression of recombinant proteins was detected on the surface and within transfected cells by immunofluorescence using a polyclonal anti-SHBV antiserum. Whereas the full-length protein is expressed homogeneously on the cell surface, constructs lacking the gC transmembrane domain show a protein staining mainly in the perinuclear (presumably Golgi) region (Fig. 4).
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Cells expressing gCSHBV on the cell surface were compared with gCSHBV constructs lacking the membrane anchor sequence in addition to the parental RK13 cell line for binding of complement C3 by FACS analysis. As depicted in Fig. 5, binding of human C3 was observed only to surface-bound gCSHBV but not to parental RK13 cells or gCSHBV constructs lacking the membrane anchor.
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As controls, the untransfected RK13 ancestor cell line and stable transformants expressing gCMDV on the cell membrane were used, with the latter also showing no protective effect against complement-mediated cell lysis. The percentage of lysis of the gCSHBV RK13 transfectants seemed to be largely dependent on the amount of gCSHBV protein expressed on the cell surface, as variations in lysis among different stable cell clones correlated with the rate of membrane expression of gCSHBV, as detected by immunofluorescence, and no inhibition was found with the truncated gCSHBV RK13 clone produced subsequently (data not shown).
Binding of recombinant gCSHBV to polysulphates and failure to block C3b binding of alternative pathway complement activator properdin
As depicted in Fig. 7, secreted gCSHBV bound to dextran sulphate coupled covalently to polystyrol surfaces in a dose-dependent manner. No binding was observed to microtitre plates coated with ovalbumin or BSA. Weak binding was also observed to sodiumhyaluronate, which is a charged substance found in connective tissue.
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Binding of factor B from human serum to our C3b-coated plates was rather weak compared to the strong binding of the purified properdin and also the use of a polyvalent antiserum led to considerable background. Furthermore, gCSHBV did not have any visible influence on this interaction (Fig. 7,
).
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DISCUSSION |
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The sequencing of the gCSHBV gene revealed a close relationship with the HSV homologues, which was expected from their comparable genome organization (Harrington et al., 1992). Regions with high amino acid identities and conserved cysteine residues indicate a similar structure, which is also reflected in the functional properties of the protein. Regarding the differences in the amino-terminal domain of gCSHBV, it should be noted that there are also major differences within the two human HSV subtypes in this region, with the deletion of 27 aa in the HSV-2 protein.
These differences in the amino terminus of the gC proteins possibly explain the different functional behaviour of gCHSV-1 and gCHSV-2 upon competition with complement regulatory proteins, including properdin, factor H or complement receptors CR1 and CR2, with the failure of gCHSV-2 to inhibit factor H or alternative pathway activator properdin (Huemer et al., 1993b). Therefore, it was not surprising that the recombinant gCSHBV was also unable to block binding of properdin but may even partially stabilize the properdinC3b interaction. Whether this observation has any significance for the function of gCSHBV and gCHSV-2 remains questionable, as these proteins might possibly exert their function only in their physiological (i.e. membrane-associated) conformation, which is also true for other membrane receptors.
Regarding the binding to surface-bound C3, SHBV, despite the HSV-2-like amino-terminal deletions, seems to be functionally similar to HSV-1, as gCHSV-2 does not mediate binding of C3-coated SRBC to HSV-2-infected cells. Whether this reflects influences such as differing densities or clustering of receptors, differences in lateral mobility and/or other membrane/receptor peculiarities of the behaviour of gCHSV-2 remain unclear. A suggested inhibitory influence of other HSV-2 glycoproteins in the cell membrane seems rather unlikely, as there were no detectable interferences of HSV-2 glycoproteins using intertypic recombinant strains expressing gCHSV-1 (Huemer et al., 1992a).
The sensitivity of the gCSHBVC3 interaction to heparin and dextran sulphate and the binding of gCSHBV to surface-bound polysulphates is also analogous to the situation found with gCHSV-1 (Huemer et al., 1992a), suggesting a comparable heparan sulphate receptor activity in SHBV. However, studies investigating adherence of gC-null HSV strains from both subtypes suggested that mediating adherence to heparan sulphate may not be the predominant function of gC (Gerber et al., 1995
; Griffiths et al., 1998
). Of interest, heparin also interacts with a variety of proteins in the clotting system, the immune system, growth factors, adherence molecules and receptors, including different endogenous complement regulatory proteins (Edens et al., 1993
).
From the sequence data, it appears that SHBV seems to be more closely related to HSV-2. Furthermore, PRV was found as the most closely related animal herpesvirus, which is in accordance with the neurotropic characteristics of both viruses. One of the characteristics of PRV is its broad host-range, which is unusual for the Alphaherpesvirinae, and it would be interesting to know more about the tropism of SHBV in this respect.
The interference of gCSHBV with cell lysis by porcine complement was somewhat surprising, as there was no binding to purified porcine C3, resisting the vigorous washing procedure in immunoprecipitation. Whether this indicates differences in affinity or the numbers of binding sites, etc., has to be clarified. Failure of direct binding on the surface does not necessarily correspond to a lack of function, like the situation with known human complement regulators such as decay-accelerating factor (DAF). DAF is a cell surface glycoprotein that regulates complement activity by accelerating the decay of C3/C5 convertases. Interacting directly with membrane-bound C3b/C4b, it prevents uptake of C2 and factor B but is not a C3b receptor detectable by the rosetting technique (Medof et al., 1984).
Furthermore, gCHSV-2 has been shown to interfere with the alternative complement pathway, although no direct binding of C3 was observed on the cell surface (McNearney et al., 1987). Similar observations have been made most recently with PRV, which has been shown to be protected against porcine serum by gCPRV (Maeda et al., 2002
), although binding to porcine C3 was only found with the purified protein and not by binding of complement-coated SRBC to PRV-infected cells (Huemer et al., 1992b
, 1993a
). This difficult scenario is complicated further by the fact that herpesviruses, as well as other enveloped viruses, are able to incorporate host cell-derived complement regulatory proteins into their membrane (Spear et al., 1995
). This has been shown to play an important role in variations of complement-mediated lysis of PRV grown in different host cells (Maeda et al., 2002
) and highlights further the complex differences among human and animal complement regulatory proteins, as well as possible influences of conformational requirements or varying affinities for different C3 species of the virally encoded complement receptor proteins.
Purified gCHSV-1 also has a reduced binding capacity for C3 derived from other species and binds very poorly to porcine C3 (Huemer et al., 1993a). This observation has been confirmed most recently by plasmon surface resonance analysis (Biacore), whereas human alternative complement pathway regulator factor H was able to bind to the porcine complement C3 in this set-up (M. Holmberg, Helsinki, personal communication).
Certain species-specific selectivity is observed among human complement regulators (Horstmann et al., 1985; Yu et al., 1997
) and marked differences in the efficiency of complement-mediated herpesvirus lysis using serum of different species as a complement source have been described (Hidaka et al., 1991
; Maeda et al., 2002
). The findings that HSV was lysed far more efficiently by serum of rodents as compared to human serum than earlier reports have suggested appears to play a crucial role in animal studies using herpesvirus vectors for anti-tumour therapy (Ikeda et al., 2000
). This suggests a close evolutionary relationship of microbial-encoded complement escape mechanisms and the unspecific host defence and highlights once more the importance of using appropriate animal hosts as models of herpesvirus infections.
Nevertheless, this class of molecules, acting as virus pathogenicity factors due to their conservation among herpesviruses, represents a good candidate for further vaccination studies, which are under way also with gC antigens from other species (Huemer et al., 2000b).
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ACKNOWLEDGEMENTS |
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Received 4 November 2002;
accepted 17 January 2003.
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