Cloning and expression of the complement receptor glycoprotein C from Herpesvirus simiae (herpes B virus): protection from complement-mediated cell lysis

Hartwig P. Huemer1,2, Christian Wechselberger2,{dagger}, Alice M. Bennett4, Dietrich Falke3 and Lesley Harrington4

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Simian herpes B virus (SHBV) is the herpes simplex virus (HSV) homologue for the species Macaca. Unlike in its natural host, and unlike other animal herpesviruses, SHBV causes high mortality in accidentally infected humans. SHBV-infected cells, like those infected with HSV-1 and equine herpesvirus types 1 and 4, express complement C3 receptor activity. To study immunoregulatory functions involved in susceptibility/resistance against interspecies transmission, the SHBV glycoprotein C (gCSHBV) gene (encoding 467 aa) was isolated. Sequence analysis revealed amino acid identity with gC proteins from HSV-2 (46·9 %), HSV-1 (44·5 %) and pseudorabies virus (21·2 %). Highly conserved cysteine residues were also noted. Similar to gCHSV-2, gCSHBV is less glycosylated than gCHSV-1, resulting in a molecular mass of 65 kDa if expressed in replication-deficient vaccinia virus Ankara. Stable transfectants expressing full-length gCSHBV on the cell surface induced C3 receptor activity and were substantially protected from complement-mediated lysis; no protection was observed with control constructs. This suggests that expression of the gC homologues on infected cell surfaces might also contribute to the survival of infected cells in addition to decreased virion inactivation. Interestingly, soluble gCSHBV isolated from protein-free culture supernatants did not interfere with the binding of the alternative complement pathway activator properdin to C3b, which is similar to our findings with gCHSV-2 and could be attributed to major differences in the amino-terminal portion of the protein with extended deletions in both gCSHBV and gCHSV-2. Binding of recombinant gCSHBV to polysulphates was observed. This, together with the heparin-sensitivity of the gCSHBV–C3 interaction on the infected cell surface, suggests a role in adherence to heparan sulphate, similar to the gC proteins of other herpesviruses.

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.

{dagger}Present address: Upper Austrian Research GmbH, Center for Biomedical Nanotechnology, Linz, Austria.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
As a rare exception among animal members of the family Herpesviridae, which do not usually infect humans, severe disease leading to high mortality in humans is caused by the primate simian herpes B virus (SHBV, Cercopithecine herpesvirus type 1). Accidental infection of humans has been described on several occasions with SHBV, originating from old world monkeys of the genus Macaca.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cells and viruses.
The macaque isolate from SHBV used in this study has been described (Falke, 1961). HSV-1 strain Ang and equine herpesvirus type 1 (EHV-1) strain Piber were used for comparison. Virus stocks of SHBV were produced in Vero cells (ATCC, #CCL-81). For expression experiments, the rabbit kidney RK13 cell line (ATCC, #CCL-37) was used. Chicken-adapted MVA was kindly provided by A. Mayr, LMU-Munich, Germany. Wild-type virus and recombinants were raised in primary chicken fibroblasts (CHF).

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 BamHI–SacI fragment was excised and religated to a 570 bp NcoI–BamHI 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 CL4b–Sepharose (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 CNBr–Sepharose 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 4–5 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.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression of complement receptor activity on SHBV-infected cells sensitive to polyanions
SHBV-infected Vero cells showed binding of complement component C3 coupled to SRBC and CNBr–Sepharose. Comparable to gCHSV-1 (Huemer et al., 1992a), this binding was inhibited by heparin or dextran sulphate (Fig. 1). SRBC, lacking C3 only, showed no binding to SHBV-infected cells; binding was also inhibited by anti-C3 antibodies (data not shown).



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Fig. 1. C3 receptor function in SHBV-infected Vero cells and inhibition by heparin and related polyanions. (a) Binding of [14C]glucosamine-labelled SHBV and HSV-1 lysates to C3–Sepharose. The binding of SHBV- and HSV-1-infected cell lysates labelled with [14C]glucosamine to Sepharose-coupled, purified C3 from the indicated species. Lanes: 1, human C3; 2, porcine C3; 3, cynomolgus C3; 4, IgG–Sepharose. (b) Binding of SHBV-infected cells to complement C3 on SRBC (upper panel); binding can be inhibited by the addition of heparin (10 µg ml-1) or related polyanions such as dextran sulphate (lower panel).

 
Precipitations of SHBV-infected Vero cells labelled with [14C]glucosamine revealed a C3-binding protein of about 65 kDa precipitated by human and macaque C3–Sepharose, whereas the fully glycosylated form of gCHSV-1 appeared at 120 kDa. Furthermore, no binding was observed of gCHSV-1 and gCSHBV to the porcine complement component as well as to the control–Sepharose (Fig. 1).

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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Fig. 2. Comparison of the gCSHBV protein sequence with gCHSV-1 and gCHSV-2. Conserved amino acids are boxed and cysteine residues are highlighted. Gaps are included to maximize alignment.

 
There are six potential N-glycosylation sites in gCSHBV and comparison of the protein sequence with the HSV homologues revealed major structural differences in the amino-terminal domain. Both gCHSV-2 and gCSHBV show deletions in the amino-terminal domain when compared with gCHSV-1, with the extended SHBV deletions preceding the deletion of the HSV-2 protein. Despite these differences, the overall amino acid identity was about 44·5 % for gCHSV-1 and 46·9 % for gCHSV-2. The highly conserved central region of the molecule suggests a comparable structure, which is also reflected by C3 binding.

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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Fig. 3. Production of gCSHBV in MVA and RK13 transfectants. (a) Expression of gCSHBV in recombinant vaccinia virus. Immunoprecipitations of 35S-labelled supernatants of RK13 cells infected with MVA constructs and the parent MVA virus are shown. Lysates were precipitated with anti-HSV-1 gC and anti-EHV-1 gC monoclonal antibodies and anti-SHBV polyclonal rabbit serum. Relative molecular masses in kDa are shown on the right. (b) Secretion of recombinant gCSHBV in supernatants of transfected cells. gCSHBV produced by constructs lacking the transmembrane region was detected by Western blotting (lane 1), whereas only trace amounts could be detected in the supernatant of stable RK13 transfectants expressing the membrane-associated protein (lane 2). In lane 3, gCEHV-1 is used as a control, which, due to glycosylation, shows up as a diffuse band. The amido black-stained blot of the protein marker is depicted on the right with bars indicating the relative molecular mass in kDa.

 
Detection of C3 receptor activity on transfected cells expressing surface-bound gCSHBV
Construction of stable cell lines expressing full-length and truncated gCSHBV.
Soluble gCSHBV was detected by Western blotting in the supernatant of cells lacking the transmembrane anchor sequence as a protein of approximately 65 kDa, whereas no secretion was detected in cells expressing the full-length protein (Fig. 3).

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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Fig. 4. gCSHBV expression on the cell surface and secretion by transfected RK13 cells. RK13 cells transfected with full-length gCSHBV (left panels) and a truncated version lacking the transmembrane (TM) region of the gC gene (right panels) were tested for recombinant protein expression by immunofluorescence using a SHBV-specific antiserum (a). Untransfected RK13 cells served as the negative control (b). Note the perinuclear Golgi-type staining of secreted gCSHBV in comparison with the surface staining of the membrane-bound protein.

 
Stable RK13 cell lines were obtained expressing full-length and truncated gCSHBV as well as clones expressing gCMDV. Interestingly, no clones showing sufficient expression of gCHSV-1 were obtained, presumably due to toxicity of the HSV-1 protein in eukaryotic cells; this presents a further difference between the simian and the human herpesvirus gC protein.

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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Fig. 5. Binding of C3 to cell lines expressing surface-bound gCSHBV. Binding of complement factor C3 was determined by FACS analysis. Filled curves represent cells incubated with complement component C3. Open curves indicate antibody controls without addition of C3. Stable transfectants and controls are shown as follows: S+, RK13 cells expressing gCSHBV, including the transmembrane region; S-, RK13 cells expressing truncated gCSHBV without the transmembrane region; RK13, negative control (untransfected ancestor RK13 cell line); Raji, positive control (complement receptor-positive Burkitt's lymphoma tumour B-cell line).

 
Inhibition of complement-mediated cell lysis by surface-expressed gCSHBV
Protection from alternative complement pathway lysis of the stable RK13 cell lines expressing surface-bound gC constructs was studied in chromium-release assays. As shown in Fig. 6, complement lysis of untransfected RK13 cells occurred in a concentration-dependent manner up to serum dilutions of 1 : 80.



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Fig. 6. Expression of gCSHBV on the cell surface protects from complement-mediated cell lysis. Stably transfected RK13 cells showing high expression of gCSHBV on the cell membrane ({bullet}) were compared with untransfected RK13 cells ({circ}) and cells expressing gCMDV on the cell surface ({blacktriangleup}). Means ±SE of triplicate experiments are shown. Lysis of cells by the indicated serum dilutions was measured by release of chromium and expressed as the percentage of total cell lysis achieved with detergent. Heat inactivation of serum completely inhibited complement-mediated lysis of RK13 cells ({lozenge}).

 
Of interest in RK13 cells expressing gCSHBV on the cell membrane was that complement lysis was diminished substantially at different serum concentrations, with something similar to a plateau or saturation point being reached around dilutions of 1 : 20–1 : 40. Higher serum concentrations exceeded the complement regulatory capacity of the membrane protein, although a significant reduction was also observed at lower dilutions.

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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Fig. 7. Secreted gCSHBV binds to polysulphates but does not inhibit properdin binding to C3b. (a) Binding of soluble gCSHBV to polysulphates. Microtitre plates were coated with the indicated substances by covalent binding. Serial 2-fold dilutions of recombinant gCSHBV were added in lanes 1–3 and binding detected in ELISA. Lane 4 indicates the background control with no gC added. (b) No inhibition of alternative complement pathway activator properdin by gCSHBV. Serial 2-fold dilutions of soluble gCSHBV were added to C3b-coated plates in lanes 1–3. Lane 4 served as positive reference with no gC added. Binding of purified properdin to C3b- ({bullet}) and BSA-coated plates ({circ}) was monitored by monoclonal antibody. Binding of complement factor B from human serum to plate-bound C3b ({blacktriangleup}) and BSA ({triangleup}) was examined for comparison.

 
Soluble gCSHBV did not inhibit the binding of purified alternative complement pathway activator properdin to surface-bound C3b, which is similar to gCHSV-2. The different concentrations of the viral protein represented by the data-points (Fig. 7, lanes 1–3) did not lead to a reduction in properdin binding to C3b (Fig. 7,{bullet}), but, interestingly, even led to slightly enhanced levels of properdin binding as compared to the data-point without viral protein added (Fig. 7, lane 4). BSA-coated wells were used as a comparison; no binding of properdin was observed, as detected by monoclonal anti-properdin antibody (Fig. 7, {circ}).

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, {blacktriangledown}).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Infection of humans with SHBV occurs after animal bites among animal care workers but infected organ or cell culture material is also hazardous for laboratory staff and the disease can be transferred to close contacts (Davenport et al., 1994). Serological testing for SHBV is rather difficult due to extensive cross reactivity with human HSV (Falke, 1964; Hilliard et al., 1989; Norcott & Brown, 1993). The recombinant constructs and proteins produced in this study may consequently provide a useful tool for serological testing of infected animals and human populations that may be at risk.

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 properdin–C3b 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 gCSHBV–C3 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).


   ACKNOWLEDGEMENTS
 
We thank Dr Y. Li (Oswell Laboratory, University of Birmingham, UK) for help with automated sequencing and Drs M. Slomka (CPHL, Collindale, UK) and C. Coulibaly (Paul Ehrlich Institute, Langen, Germany) for donation of antisera. The authors also appreciate the technical help provided by Bridget Rickard, Janet Drake and Steven Hibbs (all from the Chemical and Biological Defence Establishment, Porton Down, UK). The work was supported by the Austrian Academy of Sciences programme for the advancement of research and technology (APART), the Jubiläumsfonds of the Austrian National Bank and the Biotechnology Programme of the European Commission.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cole, J. L., Housley, G. A., Jr, Dykman, T. R., MacDermott, R. P. & Atkinson, J. P. (1985). Identification of an additional class of C3-binding membrane proteins of human peripheral blood leukocytes and cell lines. Proc Natl Acad Sci U S A 82, 859–863.[Abstract]

Cooper, N. R. (1991). Complement evasion strategies of microorganisms. Immunol Today 12, 327–331.[Medline]

Davenport, D. S., Johnson, D. R., Holmes, G. P., Jewett, D. A., Ross, S. C. & Hilliard, J. K. (1994). Diagnosis and management of human B virus (Herpesvirus simiae) infections in Michigan. Clin Infect Dis 19, 33–41.[Medline]

Edens, R. E., Linhardt, R. J. & Weiler, J. M. (1993). Heparin is not just an anticoagulant anymore: six and one-half decades of studies on the ability of heparin to regulate complement activity. In Complement Today (Complement Profiles), pp. 96–120. Edited by J. M. Cruse & R. E. Lewis, Jr. Basel: Karger.

Engels, M., Palatini, M., Metzler, A. E., Probst, U., Kihm, U. & Ackermann, M. (1992). Interactions of bovine and caprine herpesviruses with the natural and the foreign hosts. Vet Microbiol 33, 69–78.[Medline]

Falke, D. (1961). Isolation of two variants with different cytopathic properties from a strain of herpes B virus. Virology 14, 492–495.[Medline]

Falke, D. (1964). Serologic relationship between B virus and herpes simplex virus using complement fixation assay. Z Hyg 150, 185–193 (in German).

Fries, L. F., Friedman, H. M., Cohen, G. H., Eisenberg, R. J., Hammer, C. H. & Frank, M. M. (1986). Glycoprotein C of herpes simplex virus 1 is an inhibitor of the complement cascade. J Immunol 137, 1636–1641.[Abstract/Free Full Text]

Gerber, S. I., Belval, B. J. & Herold, B. C. (1995). Differences in the role of glycoprotein C of HSV-1 and HSV-2 in viral binding may contribute to serotype differences in cell tropism. Virology 214, 29–39.[CrossRef][Medline]

Griffiths, A., Renfrey, S. & Minson, T. (1998). Glycoprotein C-deficient mutants of two strains of herpes simplex virus type 1 exhibit unaltered adsorption characteristics on polarized or non-polarized cells. J Gen Virol 79, 807–812.[Abstract]

Harrington, L., Wall, L. V. & Kelly, D. C. (1992). Molecular cloning and physical mapping of the genome of simian herpes B virus and comparison of genome organization with that of herpes simplex virus. J Gen Virol 73, 1217–1226.[Abstract]

Hidaka, Y., Sakuma, S., Kumano, Y., Minagawa, H. & Mori, R. (1990). Characterization of glycoprotein C-negative mutants of herpes simplex virus type 1 isolated from a patient with keratitis. Arch Virol 113, 195–207.[Medline]

Hidaka, Y., Sakai, Y., Toh, Y. & Mori, R. (1991). Glycoprotein C of herpes simplex virus type 1 is essential for the virus to evade antibody-independent complement-mediated virus inactivation and lysis of virus-infected cells. J Gen Virol 72, 915–921.[Abstract]

Hilliard, J. K., Black, D. & Eberle, R. (1989). Simian alphaherpesviruses and their relation to the human herpes simplex virus. Arch Virol 109, 83–102.[Medline]

Horstmann, R. D., Pangburn, M. K. & Müller-Eberhard, H. J. (1985). Species specificity of recognition by the alternative pathway of complement. J Immunol 134, 1101–1104.[Abstract/Free Full Text]

Huemer, H. P., Bröker, M., Larcher, C., Lambris, J. D. & Dierich, M. P. (1989). The central segment of herpes simplex virus type 1 glycoprotein C (gC) is not involved in C3b binding: demonstration by using monoclonal antibodies and recombinant gC expressed in Escherichia coli. J Gen Virol 70, 1571–1578.[Abstract]

Huemer, H. P., Larcher, C., Dierich, M. P. & Falke, D. (1992a). Factors influencing the interaction of herpes simplex virus glycoprotein C with the third component of complement. Arch Virol 127, 291–303.[Medline]

Huemer, H. P., Larcher, C. & Coe, N. E. (1992b). Pseudorabies glycoprotein III derived from virions and infected cells binds to the third component of complement. Virus Res 23, 271–280.[CrossRef][Medline]

Huemer, H. P., Larcher, C., Van Drunen Littel-van den Hurk, S. & Babiuk, L. A. (1993a). Species selective interaction of Alphaherpesvirinae with the ‘unspecific’ immune system of the host. Arch Virol 130, 353–364.[Medline]

Huemer, H. P., Wang, Y., Garred, P., Koistinen, V. & Oppermann, S. (1993b). Herpes simplex virus glycoprotein C: molecular mimicry of complement regulatory proteins by a viral protein. Immunology 79, 639–647.[Medline]

Huemer, H. P., Nowotny, N., Crabb, B. S., Meyer, H. & Hübert, P. H. (1995). gp13 (EHV-gC): a complement receptor induced by equine herpesviruses. Virus Res 37, 113–126.[CrossRef][Medline]

Huemer, H. P., Strobl, B., Shida, H. & Czerny, C. P. (2000a). Induction of recombinant gene expression in stably transfected cell lines using attenuated vaccinia virus MVA expressing T7 RNA polymerase with a nuclear localisation signal. J Virol Methods 85, 1–10.[CrossRef][Medline]

Huemer, H. P., Strobl, B. & Nowotny, N. (2000b). Use of apathogenic vaccinia virus MVA expressing EHV-1 gC as basis of a combined recombinant MVA/DNA vaccination scheme. Vaccine 18, 1320–1326.[CrossRef][Medline]

Huemer, H. P., Larcher, C., Czedik-Eysenberg, T., Nowotny, N. & Reifinger, M. (2002). Fatal infection of a pet monkey with human herpesvirus. Emerg Infect Dis 8, 639–642.[Medline]

Ikeda, K., Wakimoto, H., Ichikawa, T., Jhung, S., Hochberg, F. H., Louis, D. N. & Chiocca, E. A. (2000). Complement depletion facilitates the infection of multiple brain tumors by an intravascular, replication-conditional herpes simplex virus mutant. J Virol 74, 4765–4775.[Abstract/Free Full Text]

Isaacs, S. N., Kotwal, J. & Moss, B. (1992). Vaccinia virus complement-control protein prevents antibody-dependent complement-enhanced neutralization of infectivity and contributes to virulence. Proc Natl Acad Sci U S A 89, 628–632.[Abstract/Free Full Text]

Laquerre, S., Argnani, R., Anderson, D. B., Zucchini, S., Manservigi, R. & Glorioso, J. C. (1998). Heparan sulfate proteoglycan binding by herpes simplex virus type 1 glycoproteins B and C, which differ in their contributions to virus attachment, penetration, and cell-to-cell spread. J Virol 72, 6119–6130.[Abstract/Free Full Text]

Liang, X., Babiuk, L. A. & Zamb, T. J. (1992). An in vivo study of a glycoprotein gIII-negative bovine herpesvirus 1 (BHV-1) mutant expressing {beta}-galactosidase: evaluation of the role of gIII in virus infectivity and its use as a vector for mucosal immunization. Virology 189, 629–639.[Medline]

Maeda, K., Hayashi, S., Tanioka, Y., Matsumoto, Y. & Otsuka, H. (2002). Pseudorabies virus (PRV) is protected from complement attack by cellular factors and glycoprotein C (gC). Virus Res 84, 79–87.[CrossRef][Medline]

McNearney, T. A., Odell, C., Holers, V. M., Spear, P. G. & Atkinson, J. P. (1987). Herpes simplex virus glycoproteins gC-1 and gC-2 bind to the third component of complement and provide protection against complement-mediated neutralization of viral infectivity. J Exp Med 166, 1525–1535.[Abstract]

Medof, M. E., Kinoshita, T. & Nussenzweig, V. (1984). Inhibition of complement activation on the surface of cells after incorporation of decay-accelerating factor (DAF) into their membranes. J Exp Med 160, 1558–1578.[Abstract]

Mettenleiter, T. C., Schreurs, C., Zuckermann, F., Ben-Porat, T. & Kaplan, A. S. (1988). Role of glycoprotein gIII of pseudorabies virus in virulence. J Virol 62, 2712–2717.[Medline]

Moffat, J. F., Zerboni, L., Kinchington, P. R., Grose, C., Kaneshima, H. & Arvin, A. M. (1998). Attenuation of the vaccine Oka strain of varicella-zoster virus and role of glycoprotein C in alphaherpesvirus virulence demonstrated in the SCID-hu mouse. J Virol 72, 965–974.[Abstract/Free Full Text]

Norcott, J. P. & Brown, D. W. G. (1993). Competitive radioimmunoassay to detect antibodies to herpes B virus and SA8 virus. J Clin Microbiol 31, 931–935.[Abstract]

Ohmann, H. B. & Babiuk, L. A. (1988). Induction of receptors for complement and immunoglobulins by herpesviruses of various species. Virus Res 9, 335–342.[CrossRef][Medline]

Rother, R. P., Rollins, S. A., Fodor, W. L., Albrecht, J. C., Setter, E., Fleckenstein, B. & Squinto, S. P. (1994). Inhibition of complement-mediated cytolysis by the terminal complement inhibitor of herpesvirus saimiri. J Virol 68, 730–737.[Abstract]

Sawitzky, D., Hampl, H. & Habermehl, K. O. (1990). Comparison of heparin-sensitive attachment of pseudorabies virus (PRV) and herpes simplex virus type 1 and identification of heparin-binding PRV glycoproteins. J Gen Virol 71, 1221–1225.[Abstract]

Scheiflinger, F., Falkner, F. G. & Dorner, F. (1996). Evaluation of the thymidine kinase (tk) locus as an insertion site in the highly attenuated vaccinia MVA strain. Arch Virol 141, 663–669.[Medline]

Skinner, G. R. B., Ahmad, A. & Davies, J. A. (2001). The infrequency of transmission of herpesviruses between humans and animals: postulation of an unrecognised protective host mechanism. Comp Immunol Microbiol Infect Dis 24, 255–269.[CrossRef][Medline]

Spear, G. T., Lurain, N. S., Parker, C. J., Ghassemi, M., Payne, G. H. & Saifuddin, M. (1995). Host cell-derived complement control proteins CD55 and CD59 are incorporated into the virions of two unrelated enveloped viruses. Human T cell leukemia/lymphoma virus type I (HTLV-I) and human cytomegalovirus (HCMV). J Immunol 155, 4376–4381.[Abstract]

Yu, J., Dong, S., Rushmere, N. K., Morgan, B. P., Abagyan, R. & Tomlinson, S. (1997). Mapping the regions of the complement inhibitor CD59 responsible for its species selective activity. Biochemistry 36, 9423–9428.[CrossRef][Medline]

Received 4 November 2002; accepted 17 January 2003.



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