Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, Maastricht University, PO Box 5800, 6202 AZ Maastricht, The Netherlands1
Author for correspondence: Cornelis Vink.Fax +31 43 3876643. e-mail kvi{at}lmib.azm.nl
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Abstract |
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Introduction |
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Of all HCMV virion proteins, pp150 is the strongest immunogen (Jahn et al., 1987b) . High antibody titres to this protein have been found, irrespective of the stage of infection, in nearly 100% of HCMV-seropositive individuals. Only during the early phase of naturally occurring primary HCMV infections was the antibody response to pp150 found to be either low or absent (Landini et al., 1988
). Consequently, (recombinant) pp150 is widely used in HCMV serological assays within diagnostic laboratories.
Currently, few tegument proteins of animal CMVs have been studied in detail. Among these are the products of murine CMV (MCMV) genes M82, M83 and M84, encoding homologues of the HCMV UL82 and UL83 tegument phosphoproteins (Cranmer et al., 1996) , and the product of the MCMV M99 gene, encoding the homologue of HCMV pp28 (Cranmer et al., 1994)
. However, putative homologues of the HCMV major tegument protein pp150 from animal CMVs have hitherto not been subjected to any detailed study. In this report, we describe the identification and characterization of the rat cytomegalovirus (RCMV) R32 gene, encoding the homologue of HCMV pp150.
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Methods |
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Cloning and sequencing of RCMV DNA.
RCMV genomic DNA was digested with SphI and separated on a 1% agarose gel. After transfer to a nylon membrane (Hybond-N+, Amersham), the DNA was hybridized to a probe that was generated from the RCMV EcoRI fragment A, which has a length of approximately 50 kb (Meijer et al., 1986) . Labelling, hybridization and detection experiments were performed with digoxigenin DNA-labelling and chemoluminescence detection kits (Boehringer Mannheim). Hybridizing SphI fragments were cloned as follows. RCMV DNA was digested with SphI and electrophoresed through agarose. RCMV EcoRI fragment A-specific SphI fragments were excised, purified and cloned into the SphI site of vector pUC119. The resulting constructs were sequenced and the presence of HCMV UL32-homologous regions was confirmed by alignment with sequences from the EMBL nucleic acid sequence database. Thus, a 3·6 kb RCMV SphI fragment was identified within plasmid pAS2B, which contains an ORF with significant identity to the HCMV UL32 gene. This novel RCMV ORF was termed R32. Both strands of the 3·6 kb SphI fragment were sequenced using the Thermosequenase cycle sequencing kit (Amersham) with Cy5-labelled primers and an ALFexpress automated DNA sequencer (Pharmacia Biotech). Sequence analysis was done using the program PC/Gene version 2.11 (Intelligenetics).
Poly(A)+ RNA isolation and Northern blot analysis.
Poly(A)+ RNA from RCMV- and mock-infected REF cells was isolated at immediate-early (in the presence of cycloheximide), early (in the presence of phosphonoacetic acid) and late times of infection and at 2, 8 and 48 h post-infection (p.i.) using a QuickPrep Micro mRNA purification kit (Pharmacia Biotech), as described previously (Beisser et al., 1998a) . Poly(A)+ RNA from mock-infected cells was isolated in a similar fashion as late mRNA, except that RCMV infection was omitted. Infections with RCMV were done with an m.o.i. of either 1 (for isolation of mRNA at immediate-early and early times and at 2 and 8 h p.i.) or 0·01 (for isolation of mRNA at late times and at 48 h p.i.). Electrophoresis of RNA and transfer to Hybond-N+ membranes has been described previously (Brown & Mackey, 1997)
. A 402 bp XbaIBamHI fragment, derived from plasmid p026 (Beisser et al., 1998a)
, was used as an R32-specific probe. Labelling, hybridization and detection experiments were performed using digoxigenin DNA-labelling and chemoluminescence detection kits (Boehringer Mannheim) and Dig Easy Hyb hybridization solution (Boehringer Mannheim).
Generation of pR32 bacterial expression constructs.
To generate a construct that expresses part of the R32-derived protein fused to glutathione S-transferase (GST), plasmid pAS2B was digested with SalI and the resulting 807 bp fragment was cloned into the SalI site of vector pRP269, generating plasmid p132. Vector pRP269 is derived from vector pGEX-1N (Pharmacia Biotech) and contains an extended polylinker-cloning site including more unique restriction endonuclease cleavage sites than pGEX- 1N. Plasmid p132 encodes a protein, termed GSTpR32, consisting of GST fused at its C terminus to amino acids 252522 of pR32.
To generate a construct that expresses a part of pR32 fused to thioredoxin and a tag consisting of six consecutive histidine residues (6xHis), an 842 bp BamHIHindIII fragment from p132 was cloned into the BamHI and Hin dIII sites of vector pET-32b (+) (Novagen), resulting in plasmid p135. This plasmid encodes a protein, termed 6HTrxpR32, containing amino acids 252522 of pR32 fused to the C terminus of the thioredoxin protein, which in turn is fused to the C terminus of a 6xHis tag.
Expression and purification of GSTpR32 and 6HTrxpR32.
Construct p132 was introduced into Escherichia coli strain AD202. The resulting strain was grown overnight at 37 °C in LB medium containing 50 µg/ml ampicillin. The culture was diluted 1:100 in 150 ml LB medium with ampicillin and grown at 37 °C to an OD600 of 0·8. Protein production was induced by addition of IPTG to a final concentration of 0·2 mM. After incubation for 3 h at 37 °C, the bacteria were harvested by centrifugation. The GSTpR32 protein was purified by single-step affinity chromatography using glutathioneSepharose 4b (Pharmacia Biotech), as recommended by the manufacturer.
Construct p135 was introduced into E. coli strain BL21(DE3) plysS. The resulting strain was grown overnight at 37 °C in LB medium containing 50 µg/ml ampicillin and 20 µg/ml chloramphenicol. The culture was diluted 1:100 in 150 ml LB medium containing ampicillin and chloramphenicol and grown at 37 °C to an OD600 of 0·8. Protein production was induced as described above. The 6HTrxpR32 protein was purified as follows. The bacterial pellet was resuspended in 10 ml buffer A (300 mM NaCl, 50 mM sodium phosphate buffer, pH 7·8). After sonication of the suspension on ice, Triton X-100 was added to a final concentration of 1 mM. The bacterial lysate was then centrifuged for 20 min at 12000 g (4 °C). Ten ml of a 50% slurry of Ni2+- nitroloacetic acid (NTA)agarose (Qiagen), equilibrated previously in buffer A containing 5 mM imidazole, was added to the supernatant. The suspension was stirred for 1 h at 4 °C and subsequently poured into a column. Non-specifically bound proteins were washed from the Ni-NTAagarose by three subsequent washes (of 4 ml each) with buffer A plus 5 mM imidazole, 10 mM imidazole and 20 mM imidazole, respectively. The 6HTrxpR32 protein was eluted from the column with 5 ml buffer A containing 300 mM imidazole. Fractions of 1 ml were collected, analysed by SDSPAGE, pooled and dialysed against PBS for 16 h at 4 °C.
Generation of a polyclonal antiserum directed against 6HTrxpR32.
A rabbit was immunized by intramuscular injection of 250 µg purified 6HTrxpR32 in Specol adjuvant (ID-DLO, Lelystad, The Netherlands). Two additional booster injections, each containing 250 µg purified 6HTrxpR32, were given at weeks 7 and 12. At week 16, blood was obtained and serum was prepared as described previously (Harlow & Lane, 1988) . This serum was found to be strongly reactive to both 6HTrxpR32 and GSTpR32 by Western blot analysis.
Generation of sera from RCMV- and mock- infected rats.
Male, specific-pathogen-free Lewis/N RT1 rats (Central Animal Facility, Maastricht University) were kept under standard conditions. The animals were immunocompromised by 5 Gy total-body Röntgen irradiation 1 day before infection, as described previously (Stals et al., 1990) . Rats (10 weeks old, 250300 g) were either mock-infected or infected with 5x106 p.f.u. RCMV. Serum was obtained from the rats at either day 28 or day 90 p.i.
SDSPAGE and Western blot analysis.
Proteins were separated on SDSpolyacrylamide gels, essentially as described by Laemmli (1970) . After electrophoresis, gels were either stained with Coomassie brilliant blue or transferred to nitrocellulose (PROTRAN, 0·2 µm; Schleicher & Schuell). Blots were developed by successive incubations with either rabbit anti-6HTrxpR32 antiserum (1:1000 diluted) followed by peroxidase-conjugated, goat anti-rabbit immunoglobulins (1:2000 diluted; P0448, Dako) or rat serum (1:50 diluted) followed by peroxidase-conjugated, rabbit anti-rat immunoglobulins (1:1000 diluted; P0450, Dako). The blots were stained by incubation in a solution containing diaminobenzidine.
Indirect immunofluorescence.
REF were grown in 96-well tissue culture plates (Costar). At several times after infection with RCMV, cells were fixed, permeabilized and washed. Rabbit anti-6HTrxpR32 antiserum, diluted 1:100 in PBS, was added and incubated for 30 min at 37 °C. After washing, the cells were incubated with FITC-conjugated swine anti- rabbit immunoglobulins (P0205, Dako), washed and examined with an Axiovert 100 fluorescence microscope (Zeiss). Staining of cells with monoclonal antibody RCMV8 was done as described previously (Bruning et al., 1987) .
Immunohistochemistry.
Serial tissue sections (4 µm) of the (submaxillary) salivary glands of RCMV- and mock-infected rats were mounted on glass slides and deparaffinized. Sections were incubated successively with the anti- 6HTrxpR32 antiserum, biotin-conjugated, swine anti-rabbit immunoglobulins (E0431, Dako) and streptavidinbiotinylated horseradish peroxidase complex (RPN 1051, Amersham). Staining was performed with diaminobenzidine. Staining of sections with monoclonal antibody RCMV8 was done as described previously (Bruning et al. , 1987) .
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Results |
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Generation of a polyclonal antiserum directed against recombinant pR32
In order to study the expression of the R32-encoded protein, we set out to generate rabbit polyclonal antibodies against recombinant pR32. For this purpose, we expressed a protein containing amino acids 252522 of pR32 fused to a combined 6xHisthioredoxin tag (6HTrxpR32). The region between amino acids 252 and 522 of pR32 was selected for expression because it largely overlaps the most hydrophilic part of the protein, located roughly between amino acid residues 280 and 600 (data not shown). Protein 6HTrxpR32 was expressed in E. coli and purified by affinity chromatography (Fig. 3a ). Purification resulted in a major, ~50 kDa protein that was approximately 80% pure, as judged by SDSPAGE and Coomassie blue staining (Fig. 3a
, lanes 912). The majority of the faster- migrating, minor species in lanes 912 was found to represent degradation products of the full-length ~50 kDa 6HTrxpR32 (see below). The fractions shown in lanes 912 were pooled, dialysed and used for immunization of a rabbit as described in Methods. The reactivity of the anti- 6HTrxpR32 antiserum generated against 6HTrxpR32 was tested by Western blot analysis. As shown in Fig. 3(b)
, the antiserum reacted with 6HTrxpR32 as well as with the GST fusion protein (GSTpR32), containing the same pR32 sequence as that in 6HTrxpR32 (Fig. 3b
, lanes 3 and 5). This shows that the anti-6HTrxpR32 antiserum contains antibodies that are directed specifically against the pR32 part of the fusion proteins. As might be expected, cross-reactivity of the antiserum was seen with another 6HTrx protein that contained part of the RCMV IE-1 protein (Beisser et al., 1998b
) (lane 2). Since the antiserum did not react with GST alone (lane 4), it is likely that the reactive, faster-migrating species from the GSTpR32 as well as the 6HTrx protein preparations are degradation products from the full-length fusion proteins. As shown in lanes 610 of Fig. 3(b)
, the rabbit preimmune serum did not react detectably with any of the recombinant proteins. These data indicate that the anti- 6HTrxpR32 antiserum contained antibodies directed against the pR32 part as well as the 6HTrx part of the 6HTrxpR32 protein.
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Discussion |
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Similar to its putative HCMV counterpart, the RCMV R32-derived protein (pR32) might be phosphorylated. This notion is based on two observations. First, the predicted amino acid sequence of the protein was found to contain 35 consensus phosphorylation sites. Second, the estimated molecular mass of pR32 (8085 kDa) was considerably larger than the theoretical molecular mass of the protein (73 kDa). It is, of course, possible that this difference in molecular mass is, at least in part, due to other post-translational modifications, like myristoylation or N- or O-linked glycosylation. Nevertheless, the pR32 virion protein was found to be resistant to digestion with N-glycosidase F (data not shown), which indicates that pR32 is probably not modified by N-linked glycosylation, similar to HCMV ppUL32 (Benko et al., 1988 ). It is also unlikely that the pR32 protein is modified by O-linked glycosylation. This was inferred from its insensitivity to alkaline ß-elimination (data not shown). In contrast to pR32, however, ppUL32 was reported to be modified by O -linked N-acetylglucosamine (Benko et al., 1988
).
In order to be able to study expression and localization of pR32 within RCMV-infected cells, a rabbit polyclonal antiserum was raised against an E. coli-produced part of pR32. In agreement with the transcription data, pR32 was found to be expressed at late times after infection of REF with RCMV in vitro, starting at 24 h p.i. The protein was localized predominantly within the cytoplasm of infected cells. In addition to diffuse cytoplasmic staining by the anti-pR32 antiserum, granular staining could also be observed, in particular at 24 h p.i. Although the granular staining was localized mainly in the cytoplasm, it was also apparent near nuclear membranes. A similar staining of `vesicle- like' structures has been reported previously for the HCMV counterpart of pR32, pp150 (Hensel et al., 1995 ). Hensel and co-workers reported pp150 to have both a cytoplasmic and nuclear distribution within HCMV-infected fibroblasts in vitro (Hensel et al., 1995)
. Other groups, however, reported pp150 to be localized predominantly to the cytoplasm (Sanchez et al. , 1998
; Scholl et al., 1988
), similar to our results for RCMV pR32 (Fig. 4
). Also, staining of pR32 was seen exclusively within the cytoplasm of salivary glands cells from RCMV-infected rats (Fig. 6a
). Since we only investigated the in vivo expression of pR32 at a single time after infection, it is possible that the protein is expressed within the nuclei of these cells at different times. The presence of two different consensus bipartite nuclear localization signals within pR32 indicates that the protein might indeed be transported to the nuclei of infected cells. It has been suggested previously that pp150 is acquired during nucleocapsid assembly in certain nuclear subcompartments within HCMV-infected cells (Hensel et al., 1995)
. This acquisition of pp150 may precede the packaging of DNA within the nucleocapsids. Since pp150 was found to be absent from nuclear dense body-like structures, the protein was hypothesized to play a role in maintaining a specific nucleocapsidnuclear membrane interaction (Hensel et al., 1995)
. Whether a similar function in virion assembly can be attributed to the RCMV pR32 protein will have to be investigated in future studies, which would include immunoelectron microscopy and biochemical experiments.
As has been reported for HCMV ppUL32, the RCMV pR32 protein is the target of a strong humoral immune response. Sera from seven infected rats were each found to contain antibodies that reacted with recombinant pR32. These antibodies could already be detected by Western blotting at day 28 p.i. Although we showed only that pR32 is a virion- associated protein, it is likely that it is located in the tegument of the virions. This notion is based on the similarity between pR32 and the HCMV pp150 tegument protein. Since antibodies against HCMV tegument proteins are generally non-neutralizing and non-protective, it is likely that the anti-pR32 antibodies from rat sera are similarly non- protective. However, as with the detection of anti-HCMV antibodies, the detection of anti-RCMV antibodies is extremely useful as a serological marker of the course of infection.
In conclusion, we have identified the RCMV R32 gene, which is likely to represent the homologue of the HCMV UL32 gene. The R32-encoded protein, pR32, is expressed predominantly within the cytoplasm of infected cells, both in vitro and in vivo, and is associated with RCMV virions. A strong humoral immune response is elicited against pR32 upon infection of rats with RCMV, similar to the response seen with pp150 in HCMV-infected individuals. Once again, these data demonstrate the close similarity between RCMV and HCMV.
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Acknowledgments |
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Footnotes |
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References |
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Received 29 March 1999;
accepted 1 July 1999.