Department of Medical Microbiology, Cardiovascular Research Institute Maastricht, University of Maastricht, 6202 AZ Maastricht, The Netherlands
Correspondence
Cornelis Vink
kvi{at}lmib.azm.nl
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ABSTRACT |
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INTRODUCTION |
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The biological significance of the UL33 family members has been demonstrated previously in studies using recombinant CMVs that carry either a disrupted UL33 (Margulies et al., 1996), M33 (Davis-Poynter et al., 1997
) or R33 gene (Beisser et al., 1998
) in their genomes. In cell culture, each of these mutant viruses replicated with similar efficiency as the corresponding wt viruses (Beisser et al., 1998
; Davis-Poynter et al., 1997
; Margulies et al., 1996
). However, during in vivo infection, significant differences were observed between animals infected with the recombinants and those infected with the wt viruses. In contrast to their wt counterparts, M33- and R33-deleted viruses could not be detected within the salivary glands of infected mice and rats, respectively (Beisser et al., 1998
; Davis-Poynter et al., 1997
). This indicated that M33 and R33 play a role in virus dissemination to or replication in the salivary glands (Beisser et al., 1998
; Davis-Poynter et al., 1997
). Furthermore, it was shown in the RCMV/rat model that R33 plays an important role in the pathogenesis of RCMV disease, since a significantly lower mortality was seen among rats infected with R33-deleted RCMV (RCMV
R33) than among those infected with wt RCMV (Beisser et al., 1998
).
It has been established firmly that the members of the UL33 gene family encode GPCRs. The human herpesvirus type 6B (HHV-6B) member of the UL33 family, pU12, was reported to be a calcium-mobilizing receptor for several CC chemokines (Isegawa et al., 1998). In addition, we found the RCMV R33-encoded protein to signal in a ligand-independent, constitutive fashion (Gruijthuijsen et al., 2002
). Recently, similar activities have also been attributed to the murine CMV (MCMV) and HCMV counterparts of these proteins (Waldhoer et al., 2002
). Like the UL33 family members, the UL78 gene family members were found to have important roles in the pathogenesis of infection. A significantly lower mortality was observed among rats infected with R78-deleted RCMV strains (RCMV
R78a and RCMV
R78c) than among animals infected with wt RCMV (Beisser et al., 1999
). Additionally, cells infected with these recombinant viruses produced virus titres that were 10- to 100-fold lower than their wt counterpart. Similar observations have been made in the MCMV/murine model (Oliveira & Shenk, 2001
). Despite the relatively low sequence similarity with known chemokine receptors, the HHV-6A pUL78 homologue (pU51) was reported to bind several CC chemokines, such as CCL2, CCL5, CCL7, CCL11 and CCL13, as well as an HHV-8-encoded chemokine, vMIP-II (Milne et al., 2000
). These binding characteristics strongly resemble those of the HHV-6B homologue of pUL33, pU12. Nevertheless, signalling activities have hitherto not been identified for any other member of the UL78 family.
To monitor the expression of both pR33 and pR78 in vitro and in vivo, we set out to generate recombinant RCMV strains expressing either pR33-enhanced green fluorescent protein (EGFP) or pR78-EGFP instead of native pR33 and pR78, respectively. Here, we show that these recombinant viruses (RCMV33G and RCMV78G, respectively) differ from wt RCMV in various aspects of replication in vitro and in vivo. Most notably, while strain RCMV33G is defective in producing infectious virus in the salivary glands of infected rats, strain RCMV78G is incapable of producing virus progeny in the spleen. In all other organs and tissues tested, these strains replicate in a fashion indistinguishable from that of wt virus.
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METHODS |
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RCMV33G recombinant plasmid construction.
To generate an RCMV strain in which the R33 ORF is fused in-frame to the 5' end of the EGFP ORF, a recombinant plasmid was generated. This plasmid, designated p388 (Fig. 1A), was constructed by cloning a 2·4 kb NheIXbaI fragment, containing both the EGFP ORF and a neomycin resistance gene (neo), into the XbaI site of plasmid p384. This fragment was designated the EGFP-neo cassette. A detailed description of the construction of p384 has been described previously (Gruijthuijsen et al., 2002
). Plasmid p384 contains the complete R33 gene with its translation termination codon changed into a leucine codon and an XbaI site. The 2·4 kb NheIXbaI insert of p388 was derived from plasmid p374, which was generated as follows. First, a 1·4 kb fragment containing the simian virus 40 (SV40) early promoter and neo was amplified by PCR, using primers NEO.C-F and NEO.C-R (Table 1
) and, as template, plasmid Rc/CMV (Invitrogen). The amplified fragment was digested with EcoRI/XbaI and cloned into EcoRI- and XbaI-digested pUC119, generating plasmid p370. Subsequently, the EGFP ORF was cloned upstream of the neo ORF. To this purpose, the EGFP ORF was amplified by PCR using primers GFP.CN-F and GFP.C-R (Table 1
) and, as template, plasmid p368. Plasmid p368 was derived from vector pEGFP-N1 (Clontech) by deletion of the 51 bp BamHIBglII fragment. The 1·1 kb PCR fragment containing the EGFP ORF was digested with EcoRI and cloned into EcoRI-digested p370, resulting in plasmid p374. The integrity of all DNA constructs was verified by sequence analysis.
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Generation of strains RCMV33G and RCMV78G.
Approximately 1x107 Rat2 cells were trypsinized and centrifuged for 5 min at 500 g. The cells were resuspended in 0·5 ml culture medium, after which 10 µg of either plasmid p388 or p390 was added. The suspension was transferred to a 0·4 cm electroporation cuvette (Bio-Rad) and pulsed at 0·25 kV and 500 µF in a Bio-Rad Gene Pulser electroporator. Cells were then seeded in T75 culture flasks. At 14 h after transfection, the cells were infected with low-passage RCMV at an m.o.i. of 1. The culture medium was supplemented with 50 µg G418 ml-1 at 16 h post-infection (p.i.). Recombinant viruses were cultured on REF monolayers and plaque-purified as described earlier (Beisser et al., 1998, 1999
, 2000
).
Southern blot hybridization.
DNA isolated from wt RCMV, RCMV33G and RCMV78G was digested with NcoI, electrophoresed through a 1 % agarose gel and blotted onto a Hybond-N+ nylon membrane (Amersham Pharmacia Biotech), as described previously (Brown, 1993). The integrity of genomic DNA from strain RCMV33G was checked using plasmids p375 (containing R32, R33 and R34 sequences) and p374 (containing EGFP and neo sequences) as probes. The integrity of the RCMV78G DNA was verified using p377 (which contains R77, R78 and R79 sequences) and p374 as probes. Hybridization and detection experiments were performed with digoxigenin DNA-labelling and chemiluminescence detection kits (Roche).
Isolation of poly(A)+ RNA and Northern blot hybridization.
Poly(A)+ RNA was isolated from wt RCMV-, RCMV33G- or RCMV78G-infected REFs (m.o.i. of 0·01) at day 6 p.i. using a QuickPrep Micro mRNA Purification kit (Amersham Pharmacia Biotech), according to the manufacturer's protocol. Electrophoresis of RNA under denaturing conditions and transfer to Hybond-N membranes have been described previously (Brown & Mackey, 1997). The blots were hybridized with probes specific for R32, R33, R34, R77, R78, R7980, EGFP, neo or the SV40 early antigen promoter. The 426 bp BamHI, 976 bp SacI, 501 bp Asp718HindIII, 1432 bp BamHIBglII, 734 bp NcoIEcoRI and 1380 bp EcoRIXbaI fragments from plasmid p388 were used as R32-, R33-, R34-, R7980-, EGFP- and neo-specific probes, respectively. R77- and R78-specific probes were generated as described previously by Beisser et al. (1999)
. Hybridization and detection experiments were performed with digoxigenin DNA labelling and chemiluminescence detection kits (Roche).
Western blot analysis.
REF cells were infected with wt RCMV, RCMV33G or RCMV78G at an m.o.i. of 0·01. At day 6 p.i., cells were harvested and resuspended in lysis buffer [150 mM NaCl, 50 mM NaF, 25 mM Tris/HCl pH 7·5, 2 mM EDTA, 1 % (w/v) NP-40]. Subsequently, lysates from 3x104 infected cells were separated by 12 % SDS-PAGE, essentially according to the Laemmli method. The gel was transferred to a nylon filter (NYTRAN NY 12 N; Schleicher & Schuell) and incubated successively with rabbit anti-EGFP polyclonal antiserum (Living Colours A.v. Peptide Antibody; Clontech) and peroxidase-conjugated, goat anti-rabbit immunoglobulins (Dako). The blot was developed using a luminescent detection system (ECL; Amersham Pharmacia Biotech).
Confocal laserscan microscopy.
Rat2 cells were grown on glass coverslips and infected with wt RCMV, RCMV33G or RCMV78G. At several time-points p.i., the cells were fixed for 20 min with 3·7 % paraformaldehyde in PBS. Confocal laserscan microscopy images were collected at wavelengths of 488 nm using an MRC 600 confocal microscope equipped with an oil immersion objective (40x magnification, numerical aperture=1·3; Bio-Rad), as described previously (Broers et al., 1999). Digital images were processed using Confocal Assistance software from Bio-Rad.
Dissemination of wt RCMV, RCMV33G and RCMV78G in vivo.
Male specific-pathogen-free Lewis/M rats (Central Animal Facility, Maastricht University, Maastricht, The Netherlands) were kept under standard conditions (Stals et al., 1990). All experimental protocols mentioned in this paper were approved by the Maastricht University Animal Experiments Committee and were consistent with the Dutch Laboratory Animal Care Act. Five groups of 12 male specific-pathogen-free Lewis/M rats (7 weeks old, 250300 g body weight, immunosuppressed 1 day before infection) were infected with 9x105 p.f.u. wt RCMV, RCMV33G, RCMV
R33, RCMV78G or RCMV
R78. On days 5 and 28 p.i., six rats from each group were sacrificed and their internal organs were collected. These organs were subjected to immunohistochemical analysis, plaque assay (as described previously by Bruggeman et al., 1982
) and quantitative PCR as described below. The plaque test and PCR data were analysed statistically by applying the MannWhitney U-test using SPSS (SPSS International).
Real-time quantitative PCR.
Total cellular DNA was extracted from the salivary glands, spleen, kidneys, liver, lungs, heart, pancreas and thymus as follows. Frozen tissue (approximately 4 mm3 in size) was lysed in lysis buffer (100 mM NaCl, 10 mM Tris/HCl pH 8·0, 25 mM EDTA, 0·5 % SDS) supplemented with 50 ng proteinase K ml-1 (Roche) and 5 µg RNase A ml-1 (Amersham Pharmacia Biotech), followed by homogenization and incubation for 30 min at 56 °C. Next, DNA was extracted with phenol/chloroform (1 : 1) and ethanol-precipitated. Before the samples were subjected to real-time PCR, they were analysed by both agarose gel electrophoresis, to establish their integrity, and spectrophotometry, to determine their DNA concentrations. The sequences of the TaqMan primers (RCMVfor and RCMVrev; Table 1) and that of the TaqMan probe (RCMVpro; Table 1
) used to quantify CMV were selected from the immediate-early 1 (IE1) gene with Primer Express software, version 2·0 (Perkin Elmer). The TaqMan probe selected between the primers was fluorescently labelled at the 5' end with FAM, as the reporter dye, and at the 3' end with TAMRA, as the quencher (Table 1
). PCR was performed with 12·5 µl TaqMan Universal PCR Master mix (Perkin Elmer), 900 nM forward primer, 300 nM reverse primer, 125 nM TaqMan probe and 100 ng sample DNA in a total volume of 25 µl. PCR was performed in 96-well microtitre plates under the following conditions: 2 min at 50 °C and 10 min at 95 °C, followed by 42 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were analysed using the ABI PRISM 7000 Sequence Detection System software (Perkin Elmer). For quantification, standard curves were generated using dilutions of RCMV DNA preparations of known concentration.
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RESULTS |
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Transcription of R33-EGFP, R78-EGFP and neighbouring genes
To evaluate transcription of R33-EGFP, R78-EGFP and their neighbouring genes, Northern blot analysis was performed with poly(A)+ RNA extracted from RCMV-, RCMV33G- and RCMV78G-infected cells. Two different sets of Northern blots were prepared. The first set contained RNA from wt RCMV- and RCMV33G-infected cells. These blots were treated with probes specific for R32, R33, R34, EGFP or neo (Fig. 2A, B). We found previously the RCMV genomic region containing R33 to be transcribed in a highly complex fashion (Beisser et al., 1998
). Whereas R32 was reported to be transcribed as a single, major 2·5 kb mRNA (Beuken et al., 1999
), numerous cotranscripts were identified that contained both R33 and R34 sequences (Beisser et al., 1998
). In light of the expression of these large, 46 kb cotranscripts, it was to be expected that insertion of both the EGFP ORF and the neo expression cassette into the RCMV genome would have a significant impact on the transcription patterns of the R33 and R34 genes. Indeed, Fig. 2(C)
shows clear differences between strain RCMV33G and wt RCMV in the expression of these genes. Most notably, strain RCMV33G expresses two unique transcripts, with lengths of 3·1 and 3·5 kb, respectively, which hybridize with both the R33- and EGFP-specific probe (Fig. 2C
, lanes 4 and 8). These mRNAs are likely to represent transcripts for the pR33-EGFP fusion protein. In contrast to the R33 and R34 genes, the R32 gene was transcribed by both RCMV33G and wt virus in a similar fashion (Fig. 2C
, lanes 1 and 2). Given the complexity of transcription of the RCMV R33R34 genomic region, and in the absence of antibodies directed against either pR33 or pR34, it is difficult to predict the physiological consequences of the transcriptional differences between RCMV33G and wt RCMV. Therefore, we cannot exclude the possibility that potential differences in replicative potential between RCMV33G and wt virus, either in vitro or in vivo, can be attributed to differences in transcription of genes other than, and downstream of, the R33 gene.
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Strain RCMV78G replicates less efficiently than wt RCMV in vitro
In previous experiments, it was shown that deletion of the R33 gene from the RCMV genome does not affect virus replication in vitro (Beisser et al., 1998). In contrast, the R78 gene was found to play a crucial role during in vitro replication, as 10- to 100-fold lower virus titres were recovered from cultures of cells infected with R78 null mutant RCMV strains (RCMV
R78a and RCMV
R78c) (Beisser et al., 1999
). To determine the impact of replacing the R33 and R78 genes in the RCMV genome with R33-EGFP and R78-EGFP, respectively, multi-step growth curves were generated for wt RCMV, RCMV33G and RCMV78G. As a control for attenuation due to R78 dysfunction, RCMV
R78a was also included in these experiments. REFs were infected with either of these virus strains at an m.o.i. of 0·01. Subsequently, culture medium samples were taken at days 1, 3, 5 and 7 p.i. and subjected to plaque titration assays. Since R33-deleted virus was shown previously to replicate with a similar efficiency as wt RCMV in vitro, it was not surprising to find that strain RCMV33G also replicates in a similar fashion as wt virus (Fig. 3
A). In contrast, strain RCMV78G was found to produce virus titres 3- to 4-fold lower than wt virus titres at days 5 and 7 p.i., respectively (P<0·05; Fig. 3B
). Although the level of virus production of strain RCMV78G is significantly higher than that of RCMV
R78a, which produces virus titres approximately 50-fold lower than wt virus at days 5 and 7 p.i. (P<0·05; Fig. 3B
), these data indicate that strain RCMV78G may be attenuated, albeit moderately, in comparison with wt RCMV. Therefore, it is likely that the R78-EGFP gene can only partly functionally replace the R78 gene.
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To characterize further the expression of pR33-EGFP and pR78-EGFP by the recombinant viruses, Western blot analysis was performed. First, protein extracts were made from REFs infected with either RCMV33G or RCMV78G. Subsequently, extracts were electrophoresed and blotted onto nitrocellulose filters, which were developed using commercial anti-EGFP antibodies. Contrary to the green fluorescent signal evident in RCMV33G-infected cells, as seen by confocal microscopy (Fig. 4), we observed a weak EGFP-specific signal in extracts of these cells using Western blot analysis (Fig. 5
, lane 2). Accordingly, we were previously unable to detect any EGFP signal in extracts of transfected cells expressing pR33-EGFP by Western blot analysis, whereas the expression of this protein could be monitored easily under the microscope (Gruijthuijsen et al., 2002
). We hypothesize that pR33-EGFP forms large, insoluble aggregates which do not enter polyacrylamide gels efficiently. Nevertheless, the weak EGFP band in Fig. 5
corresponds to a protein with an estimated molecular mass of approximately 73 kDa, which is similar to the predicted molecular mass of pR33-EGFP (71 kDa). This suggests that the R33-EGFP fusion gene within the RCMV33G genome expresses a full-length pR33-EGFP fusion protein.
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Dissemination and replication of RCMV33G and RCMV78G in immunocompromised rats at 5 days after infection
To monitor the dissemination and replication of strains RCMV33G and RCMV78G, the following animal experiment was performed. Five groups of 7-week-old immunocompromised rats were infected with wt RCMV, RCMV33G, RCMVR33, RCMV78G or RCMV
R78a. At days 5 and 28 p.i., six rats from each group were sacrificed and dissected. The salivary glands, spleen, kidneys, liver, lungs, heart, pancreas and thymus were extracted and analysed by immunohistochemistry, plaque assay and quantitative PCR.
First, we set out to examine expression of either pR33-EGFP or pR78-EGFP in RCMV33G- and RCMV78G-infected rats, respectively. This was done by direct fluorescence microscopy and immunohistochemical analysis using both anti-pR44 antibodies (directed against an RCMV early phase protein; Kaptein et al., 2001) and anti-EGFP antibodies. We were unable to detect pR33-EGFP expression in solid organs, such as spleen and liver, at day 5 p.i. However, pR33-EGFP fluorescence was evident in cells from femoral bone marrow suspensions (Fig. 6
A). These suspensions yielded 69±50 (mean±SD) pR33-EGFP-positive cells per 106 bone marrow cells, similar to bone marrow suspensions from RCMV78G-infected rats, which yielded 121±72 pR78-EGFP-positive cells per 106 bone marrow cells. In contrast to the absence of pR33-EGFP-specific signals in spleen and liver tissue, overt expression of pR78-EGFP could be observed in these tissues in RCMV78G-infected rats (Fig. 6C, D
). In additional, in double-stained sections of the spleen, pR78-EGFP could be detected not only in each pR44-positive cell (Fig. 6E
) but also in pR44-negative cells in both spleen (Fig. 6F
) and liver (Fig. 6G
). Similar to what was observed in RCMV78G-infected REFs in vitro, the subcellular distribution of pR78-EGFP in either bone marrow cells or spleen or liver sections appeared to be granular and predominantly perinuclear.
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At day 28 p.i., the salivary glands were the only sites to harbour infectious virus (Table 2). High virus titres were found in the salivary glands of wt RCMV-, RCMV78G- and RCMV
R78a-infected rats (Fig. 7C
). These titres did not differ significantly between the three experimental groups. In accordance with previous findings (Beisser et al., 1998
), infectious virus could not be detected in salivary gland samples from RCMV
R33-infected rats (Table 2
and Fig. 7C
). Surprisingly, we were also unable to detect infectious virus in salivary gland samples of RCMV33G-infected animals (Table 2
and Fig. 7C
). Thus, like RCMV
R33, RCMV33G is unable to either enter or replicate efficiently in the salivary glands. To study further the presence of recombinant viruses in the salivary glands, viral genomic DNA levels were assessed by quantitative PCR (Table 2
and Fig. 7D
). Fig. 7(D)
shows that approximately 4x105 copies of viral DNA µg-1 tissue were found in the salivary glands from wt RCMV-, RCMV78G- and RCMV
R78a-infected rats. Viral DNA loads between the three experimental groups did not differ significantly. Interestingly, viral DNA could only be detected in one of the six RCMV33G-infected rats and in none of the RCMV
R33-infected animals (Fig. 7D
). Moreover, the virus titre within the single, RCMV33G-positive salivary gland sample was very low, i.e. 27 copies of viral DNA µg-1 tissue (Fig. 7D
). Taken together, these data show that strain RCMV33G is defective in replication in the salivary glands.
We conclude that both strain RCMV33G and strain RCMV78G have similar in vivo replication characteristics as their corresponding null mutant viruses (RCMVR33 and RCMVR78, respectively). Although this conclusion largely disqualifies RCMV33G and RCMV78G as useful tools for studying pR33 and pR78 expression in vivo, the characterization of these recombinant strains has revealed the crucial role of the R78 gene, which is conserved among all betaherpesviruses, in the production of infectious RCMV in the spleen. In addition, these studies have underlined the essential function of the second conserved GPCR gene of RCMV, R33, in salivary gland tropism.
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DISCUSSION |
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Previously, we reported that pR33 and pR33-EGFP possess similar constitutive signalling properties in vitro (Gruijthuijsen et al., 2002). Based on these results, we anticipated that the fusion of both pR33 and pR78 to EGFP would not affect the function of these proteins. However, we found that strain RCMV33G differed from wt virus in one major, but significant, characteristic: this recombinant strain is unable to either enter or replicate in the salivary glands of infected rats. A similar phenotype has been attributed previously to strain RCMV
R33, which lacks a functional R33 gene (Beisser et al., 1998
). Our current data, therefore, corroborate the notion that RCMV R33, like its homologue from MCMV (Davis-Poynter et al., 1997
), is essential for salivary gland tropism of the virus. Additionally, we conclude that the R33-EGFP gene is not able to functionally replace its native counterpart in vivo. Putative causes for the deficit of strain RCMV33G are the following: (i) the transcription of R33-EGFP and surrounding genes may be different from that of their native counterparts. Indeed, transcriptional analysis of the genomic region of R33-EGFP and R33 indicated that there are clear differences between RCMV33G and wt virus, not only in the expression of R33(-EGFP) but also in the transcription of the R34 gene, which is located downstream of R33. However, given the similarity between strains RCMV33G and RCMV
R33 in their in vivo phenotypes, it is unlikely that these phenotypes are caused by aberrant expression of R34; (ii) another difference between wt RCMV and RCMV33G in salivary gland tropism may be an altered subcellular distribution of pR33-EGFP in comparison with pR33. Due to the unavailability of anti-pR33 antibodies, this hypothesis cannot be tested yet; (iii) in comparison with pR33, pR33-EGFP may be hindered in its interaction with cellular proteins due to the presence of the EGFP moiety at its C terminus. As noted previously, we did not observe any differences in the signalling properties of pR33 and pR33-EGFP in vitro (Gruijthuijsen et al., 2002
). However, it is possible that these proteins carry out other, or additional, signalling activities and bind to different protein partners in vivo. An example of a process in which pR33 and pR33-EGFP may differ potentially is desensitization, which serves to modulate GPCR activity by modification of amino acid residues at the (intracellular) C terminus (Sibley & Lefkowitz, 1985
). The C-terminal EGFP tag of pR33-EGFP could mask critical residues within the pR33 sequence from regulatory proteins responsible for desensitization of the receptor; (iv) finally, the expression of the pR33-EGFP fusion proteins may induce immune mechanisms that do not occur during wt RCMV infection. Similar mechanisms may also be induced by the expression of the neo gene by RCMV33G. Nevertheless, while each of the recombinant RCMV strains in our laboratory has been generated using the same neo expression cassette, each of these strains exhibits clear phenotypic differences. In addition, one of these mutants, RCMV
r144 (Beisser et al., 2000
), could not be distinguished phenotypically from wt RCMV, both in rat survival and virus dissemination experiments. Therefore, we deem it unlikely that the expression of the neo gene would induce additional immune mechanisms that are not seen during wt RCMV infection.
Although RCMV33G replicates with similar efficiency as wt RCMV in spleen and liver at day 5 p.i., pR33-EGFP could not be detected by immunohistochemistry using anti-EGFP antibodies in spleen or liver samples from RCMV33G-infected rats. In contrast, pR33-EGFP is expressed markedly in bone marrow cells from these rats. Based on these observations, it could be hypothesized that R33 is expressed specifically in bone marrow cells at early times of infection in vivo. Whether or not this expression reflects the expression of native R33 by the wt virus is currently unknown. Our future studies will, therefore, be directed at (i) the generation of anti-pR33 antibodies, which has hitherto been unsuccessful; and (ii) the construction of RCMV strains expressing GPCR homologues with alternative, smaller protein tags, such as FLAG. In these studies, an important aim will be to address how pR33 determines the salivary gland tropism of RCMV and how this relates to RCMV virulence and putative expression of the protein in bone marrow.
The recombinant RCMV strain expressing pR78-EGFP was found to be attenuated, albeit moderately, both in vitro and in vivo. In cell culture, RCMV78G produced virus titres 3- to 4-fold lower than wt RCMV in the culture medium. In comparison, an R78-deleted virus strain, RCMVR78a, produced virus titres approximately 50 times lower than wt RCMV (this study; Beisser et al., 1999
). Similarly, an MCMV strain with a deletion of the MCMV homologue of R78, M78, was reported to be at least 50 times less effective in producing infectious virus (Oliveira & Shenk, 2001
). Taken together, these results indicate that the expression of pR78-EGFP by strain RCMV78G can complement only partly the absence of the native R78 gene. The strong similarity between wt RCMV and RCMV78G in transcription of R78(-EGFP) and neighbouring genes in cell culture indicates that the differences between these strains in virus replication are not caused by differences in transcription but rather by dissimilarities in other processes, such as translation, subcellular distribution, proteinprotein interactions and/or post-translational modification of pR78(-EGFP).
The ability of pR78-EGFP to only partially functionally replace the native pR78 protein was also observed in vivo. The most dramatic difference between wt RCMV and RCMV78G was seen at day 5 p.i. in the spleen of infected rats. While infectious virus was readily detectable in the spleen of all wt RCMV-infected rats, virus was not detected in the spleen of any of the RCMV78G-infected rats. Additionally, infectious virus was not found in the spleens of RCMVR78a-infected animals. Significant differences between RCMV and RCMV78G in the production of infectious virus were not seen in any of the other investigated organs. Although we studied previously the differences between wt RCMV and RCMV
R78a in virus dissemination (Beisser et al., 1999
), the replication defect of RCMV
R78a in the spleen has hitherto remained unnoticed. Since similar viral DNA levels were found in the spleen of wt RCMV-, RCMV
R78a- and RCMV78G-infected animals, we concluded that (i) R78 plays a crucial role in the production of infectious RCMV in the spleen; and (ii) R78-EGFP does not function (as efficiently) as R78 during infection in the spleen. We hypothesize that R78 does not play a role in virus dissemination or viral DNA replication but rather functions in intracellular processes following DNA synthesis, such as virion assembly or egress. Previously, we reported that a different cell morphology was seen in cultures infected with RCMV
R78a as compared to cultures infected with wt virus (Beisser et al., 1999
). Taken together, our findings indicate that pR78 may play a role in stabilizing intra- and/or intercellular structures required for optimal release of virions from infected cells. It is interesting to note that our data on virus dissemination differ from those reported by Oliveira & Shenk (2001)
in a study of an M78-deleted MCMV strain. This strain was shown to replicate less efficiently than wt MCMV not only in spleen but also in salivary glands and liver. We do not currently have an explanation for this discrepancy, although it is clear that pR78 and the M78 gene product may show differences in function as a consequence of differences between their predicted amino acid sequences (Vink et al., 2000
). Challenges for future studies will be (i) to elucidate the putative intracellular pathways by which pM78, and maybe also pR78, mediate the accumulation of IE transcripts; (ii) to characterize how the activities of these proteins stimulate the production of infectious virus in vitro; and (iii) to identify the processes that determine the R78-dependent spleen tropism of RCMV.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 13 March 2003;
accepted 15 May 2003.