INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

DNA viruses have developed numerous strategies to modulate or evade the immune system control of the host. Immune system modulators encoded within viral genomes include proteins that interrupt the complement cascade, act as cytokines or cytokine antagonists (34, 37, 40), inhibit the effector mechanisms mediated through antibodies, or interfere with antigen processing and presentation pathways (reviewed in references 15 and 28). Cytomegaloviruses (CMV) genomes encode several genes whose products modify the efficiency of host immune system control. Both human and mouse CMVs (HCMV and MCMV) contain genes which code for proteins that interfere with antigen processing and presentation in the major histocompatibility complex class I pathway and abolish the efficient recognition of CMV-infected cells by cytotoxic T lymphocytes and natural killer (NK) cells (2, 12, 17, 31, 39).

Viral proteins that directly interact with host immunoglobulins (Ig) have a potential to interfere with the humoral effector arm of the immune system and to modify the antibody response of the host. Viral Fc receptors (FcR) have been described for herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) (1, 10) and varicella-zoster virus (24). The binding of human IgG to HCMV-infected cells has been described previously (19), but the gene responsible for this phenomenon has not been identified. We have recently shown that a 88-kDa early glycoprotein of MCMV has FcR properties and have identified the corresponding gene, designated fcr-1 (38).

The biological role of herpesvirus FcRs is not clear. They are functional but not structural homologs of cellular FcRs. The HSV-1 glycoproteins E (gE) and I (gI) form an FcR complex, which protects infected cells from complement-mediated lysis and antibody-dependent cellular cytotoxicity in vitro (11). The mechanism is explained by bipolar bridging of specific IgG (13). However, protective effects mediated by the binding of the Ig Fc fragment have not yet been demonstrated in vivo. There is evidence for other in vivo functions of the HSV-1 gE-gI heterodimer, as well as its pseudorabies virus homolog. These functions are associated with direct cell-to-cell spread of the virus (3, 8, 9).

To investigate the biological role of the MCMV-encoded FcR, we constructed fcr-1 deletion mutants. The in vitro growth kinetics of the recombinant viruses was comparable to that of wild-type virus, indicating that the FcR is dispensable for viral growth in cell culture. After infection of experimental animals, FcR deletion mutants exhibited reduced growth in various organs. We analyzed whether this loss of virulence in vivo is caused by an increased virus sensitivity to specific antibody. Virus replication in antibody-deficient mice demonstrated the attenuating effect of the fcr-1 deletion is not linked to its IgG-specific function.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

Cells and virus. Mouse NIH 3T3 cells (ATCC CRL1658) were grown in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) newborn calf serum. A immortalized cell line of mouse fibroblasts (B12 cells) (7) was grown in minimum essential medium with 10% fetal calf serum. Third-passage mouse embryo fibroblasts, prepared from BALB/cJ mice and grown in minimum essential medium supplemented with 10% fetal calf serum, were used for virus infection. The Smith strain (ATCC VR-194) of MCMV was used as a tissue culture-grown virus.

Recombinant plasmids. Plasmid cloning was done by standard methods (26). To generate the recombination plasmid pJAR4, a lacZ cassette driven by the Rous sarcoma virus promoter from plasmid pATLacZ (a gift of G. Darai, University of Heidelberg, Heidelberg, Germany) was inserted between the BssHII and BglII sites of the phagemid pREG16. This plasmid contains a 2.4-kb BamHI-BamHI subfragment of the MCMV HindIII J fragment encompassing the fcr-1 gene (38).

Generation of a recombinase cre⁺ cell line (N2). A plasmid designated pneocre2 was generated by introducing a XhoI fragment containing the recombinase cre gene from pMC-Cre (16; a gift from K. Rajewsky, Cologne, Germany) into the XhoI site of the plasmid pUC21neo, which contains a neomycin resistance gene driven by the simian virus 40 enhancer-promoter. The cre recombinase-encoding plasmid pneocre2 (10 µg) was transfected into NIH 3T3 cells, and G418 resistant clones were isolated and tested for cre recombinase activity.

Construction of recombinant MCMV. The linearized recombination plasmid (30 fmol) and intact viral DNA (1.5 µg) were cotransfected into NIH 3T3 fibroblasts by the calcium phosphate precipitation technique (27). Recombinant viruses were isolated from the resulting virus progeny by at least five rounds of limiting dilution followed by 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) staining. The lacZ mutants were generated by a single passage through the recombinase cre⁺ cell line (N2) and purified by limiting dilution after X-Gal color screening.

DNA analysis. MCMV DNA was prepared from infected NIH 3T3 cells. Five days after infection, the cells were rinsed with phosphate-buffered saline, overlaid with lysis buffer (100 mM NaCl, 10 mM Tris [pH 8.0], 10 mM EDTA, 0.1% sodium dodecyl sulfate, 0.25 mg of proteinase K per ml), and incubated for 3 h at 37°C. Lysates were extracted twice with an equal volume of phenol and then once with phenol-chloroform-isoamyl alcohol (25:24:1) and chloroform-isoamyl alcohol (24:1). After addition of 2 volumes of isopropanol, the DNA was spooled on a glass rod, dried, and resuspended in 10 mM Tris-1 mM EDTA (pH 7.5).

Flow cytometry. For detection of FcR on the cell surface, MCMV-infected cells (16 h postinfection [p.i.]) were detached from the plastic with PBS-2 mM EDTA and incubated for 30 min with murine IgG (Dianova, Hamburg, Germany) at 100 µg/ml. Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG F(ab')₂ (Dianova) served as a secondary reagent.

Immunoprecipitation. B12 cells were infected either with wild-type MCMV or with FcR recombinants at a multiplicity of infection of 20. At 16 h p.i., the cells were pulse-labelled for 45 min with 150 µCi of [³⁵S]methionine (Amersham, Braunschweig, Germany) per ml in methionine-free minimal essential medium supplemented with 5% dialyzed fetal calf serum. Labelled cells were lysed in a buffer containing 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1% Nonidet P-40, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride. Cytoplasmic extracts were precleared twice with protein A-coupled Sepharose (Pharmacia, Uppsala, Sweden) and precipitated with murine IgG-coated protein A-Sepharose. Bound complexes were eluted by heating at 96°C for 5 min in reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis sample buffer and analyzed by polyacrylamide gel electrophoresis (9% polyacrylamide).

Animals and infection conditions. Newborn BALB/c mice (H-2^d), B-cell-deficient mice (µMT/µMT), and their heterozygous littermates (µMT/+) (H-2^b) (20) from the breeding colony at the Medical Faculty, University of Rijeka, were injected intraperitoneally with 10³ PFU of either wild-type or recombinant MCMV. Homozygous and heterozygous B-cell-deficient animals were selected by an enzyme-linked immunosorbent assay for the presence of the IgM in sera, as described previously (18).

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

Plasma membrane expression of the MCMV FcR. In previous experiments, the intracellular location of the FcR function was used to identify the gene (38). To fulfill an immunomodulatory function during infection, the FcR activity has to be present at the cell membrane. Therefore, we tested the surface expression of the viral FcR under various conditions. We found that trypsin addition to cells for removal of adherent cells destroyed the plasma membrane expression of the FcR activity. In the absence of trypsin, however, the binding of monomeric murine IgG can be demonstrated on the surface of infected fibroblasts (Fig. 1). Thus, the FcR activity is located at the site of potential immunological function.

View larger version (24K): [in this window] [in a new window]   FIG. 1.   Plasma membrane expression of the MCMV FcR function after infection. Mock-infected and MCMV-infected cells were incubated with murine IgG. Bound IgG was visualized with FITC-conjugated anti-mouse IgG F(ab')2. The dashed line indicates the FITC control in the absence of mouse IgG. The plasma membrane expression was assessed by fluorescence-activated cell sorter analysis.

Generation of recombinant MCMV. To analyze the role of the MCMV FcR in virus-host interactions, we constructed several FcR deletion mutants (illustrated in Fig. 2a). The recombinant virus MS94.4 was generated by homologous recombination with the recombination plasmid pJAR4. In this plasmid, the 1.35-kb BssHII-BglII fragment of the fcr-1 gene, including the promoter and the coding region for first 416 amino acids of the open reading frame, was deleted and replaced by the E. coli lacZ gene. The recombinant virus was isolated by five rounds of limiting dilution prior to generation of virus stocks.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Characterization of MCMV recombinants. (a) Schematic structure of recombinant MCMV. Shown is the HindIII cleavage map of the MCMV genome (top) and the expanded HindIII-J region of wild-type and recombinant viruses with HindIII (H) and XbaI (X) cleavage sites indicated (below). The position and orientation of the fcr-1 gene is indicated by an arrow. The open box depicts a 1.3-kb fragment of the fcr-1 gene deleted in the MS94.4, MC95.15, and MC95.16 recombinants, and the hatched boxes represent homologous regions used for recombination. The positions of the loxP sites are indicated by asterisks. The probe used for Southern blot analysis is represented by a bar. The expected sizes of the HindIII and XbaI restriction fragments are indicated. (b and c) Southern blot analysis of the recombinant viruses. DNA was isolated from infected NIH 3T3 cells and digested with HindIII (b) and XbaI (c). The sizes of the DNA fragments are indicated in kilobases.

Expression of MCMV FcR in wild-type, deletion mutant, and rescued virus. To verify that the recombinant viruses with the deletion of the fcr-1 gene do not express a protein with FcR properties, proteins from MCMV-infected and [³⁵S]methionine-labelled cells were precipitated with mouse IgG. In cells infected with wild-type MCMV and with fcr-1 rescued virus, proteins with molecular masses of 86 to 88 kDa and 105 kDa, corresponding to the MCMV FcR (38), were detected (Fig. 3). These proteins were absent in cells infected with MS94.4, MC95.15, and MC95.16, confirming the correct deletion of the FcR-encoding gene. In addition, it indicated the absence of any other proteins with FcR properties encoded by MCMV whose function could be disguised in the wild-type virus by the fcr-1 gene product.

View larger version (43K): [in this window] [in a new window]   FIG. 3.   Presence of glycoproteins with the FcR property in MCMV and mutants. B12 cells infected with wild-type MCMV and the different FcR recombinants were pulse-labelled for 45 min with 150 µCi of [35S]methionine per ml. Precipitation from cytoplasmic extracts was done with purified mouse IgG and protein A-Sepharose.

In vitro growth of MCMV mutants. To assess whether the fcr-1 gene affects the growth of virus in cell culture, single-step growth curves of recombinant and wild-type viruses were determined. After infection of NIH 3T3 fibroblasts at a multiplicity of infection of 0.1 PFU per cell, the replication kinetics of recombinants were indistinguishable from that of the wild-type MCMV (Fig. 4). Also, the size and morphology of viral plaques were identical in recombinant and parental viruses, indicating that the fcr-1 gene is dispensable for growth in cultured fibroblasts.

View larger version (16K): [in this window] [in a new window]   FIG. 4.   In vitro growth of MCMV recombinants. NIH 3T3 cells were infected with wild-type MCMV (open circles), MS94.4 (solid triangles), rMS95.9 (open diamonds), MC95.15 (solid circles), and MC95.16 recombinants (solid diamonds) at a multiplicity of infection of 0.1. Supernatants (A) and cells (B) were harvested at different time points p.i., and virus titers were determined. Standard deviations (not shown) were less than 15% of the mean values.

Reduced growth of fcr-1 deletion mutants in newborn mice. To investigate the influence of the fcr-1 gene deletion on in vivo replication, we infected 6-week-old BALB/c mice with wild-type and fcr-1 deletion mutant viruses. The tissues of mice infected with the fcr-1 deletion mutants contained very low virus titers that made quantitative comparisons difficult (data not shown). Therefore, we studied virus replication in the immunologically immature newborn mice, which are highly susceptible to MCMV infection (30). At 8 days p.i. the wild-type virus reached high titers in the organs of new-born mice (Fig. 5). At the same time, we observed a more than 100-fold-reduced replication of the fcr-1 deletion mutants in the lungs, liver, and spleen. Propagation of the rMS95.9 revertant virus was indistinguishable from that of the wild-type virus, confirming that the attenuation of the MS94.4 recombinant virus is entirely due to the site-specific mutation.

View larger version (26K): [in this window] [in a new window]   FIG. 5.   Reduced growth of fcr-1 deletion mutants in mice. Newborn BALB/c mice from timed pregnancies were inoculated intraperitoneally on day 0 with 103 PFU of wild-type (solid circles), MS94.4 (open circles), rMS95.9 (solid triangles), and MC95.15 (open triangles) recombinant MCMV. Virus titers were determined 8 days p.i. for individual mice (symbols), and median values (horizontal bars) were calculated. The solid line represents the detection limit.

Growth of the fcr-1 deletion mutant in B-cell-deficient mice. According to the hypothesis of bipolar bridging (13), the FcR should protect the virus and virus-infected cells from effects mediated through the Fc portion of Igs after binding to specific antigen. We reasoned that if the attenuated phenotype of the fcr-1 deletion mutants was due to the loss of an immunoevasive function mediated by antibody binding, the mutant virus should grow comparably to wild-type MCMV in mutant mice that lack B cells (20). Immunocompetent littermates heterozygous for the µ-chain mutation (µMT/+) severely restricted the growth of the MCMV mutant. The MCMV-specific antibody response of newborn heterozygous animals on day 21 p.i. was comparable to the response of adult mice (data not shown). Notably, the growth of the fcr-1 deletion mutant was inhibited to the same degree in µMT/µMT mice, which completely lack B cells and Igs (Fig. 6). Thus, the capacity to bind the Fc portion of Ig cannot represent the principle which dictates the attenuated phenotype of the fcr-1 deletion mutants.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   Growth of fcr-1 deletion mutants in B-cell-deficient mice. Newborn mice heterozygous (µMT/+) (circles) or homozygous (µMT/µMT) (triangles) for the µ exon mutation (20) were infected with 103 PFU of wild-type, MC95.15, and rMS95.9 MCMV. Virus titers were determined 3 weeks p.i. (symbols), and median values were calculated (horizontal bars).

Reduced growth of fcr-1 deletion mutants in T- and NK-cell-depleted mice. After depletion of T cells and NK cells, the C57BL/6 mice became increasingly susceptible to MCMV infection. Although the mutant virus showed marginal growth in immunodepleted juvenile mice, the significant difference between the growth of mutant and wild-type viruses and the growth of revertant virus remained evident. This suggested that the viral FcR does not interfere with the function of T cells and NK cells. Another explanation for reduced virulence could be the inability of the mutant virus to spread from the site of primary infection to the target organs. Therefore, we compared the virus titer that viruses reached in different organs after footpad and intravenous infection. The infection of the visceral organs and the salivary gland by the fcr-1 gene deletion mutant occurred at a low rate irrespective of the route of infection (Fig. 7). We therefore concluded that an inability of the mutant virus to spread hematogenously is not likely to explain the data.

View larger version (28K): [in this window] [in a new window]   FIG. 7.   Lack of effect of T cells, NK cells, and route of infection on the in vivo phenotype. C57BL/6 mice (6 weeks old) were depleted of CD4+, CD8+, and NK1.1+ cells and infected subcutaneously (footpad) or intravenously with 105 PFU of wild-type (solid circles), MS94.4 (open circles), and rMS95.9 (solid diamonds) MCMV. At 2 weeks p.i., virus titers were determined. Titers for individual mice (symbols) and median values (horizontal bars) are shown.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

We previously identified an MCMV protein with an FcR property that is highly selective for mouse Igs and subsequently identified and localized the corresponding gene (fcr-1) (38). Here, we analyzed its function during the in vivo infection. Due to the IgG binding property, this protein has a potential to modulate immune responses of the host that are triggered through the Fc fragment of the Ig. This potential could enable efficient replication and spread of the virus in the presence of an active immune system producing specific antibodies.

To investigate the biological role of the MCMV FcR, we generated MCMV mutants by deleting major parts of the open reading frame of the fcr-1 gene. By creating a revertant virus and a deletion mutant lacking the marker gene, we unambiguously connected the in vivo phenotype with the lack of the fcr-1 gene product expression. The fcr-1 gene is nonessential for replication in cell culture and has no phenotype in vitro. Herpesvirus functions have been shown to modulate virus-host interactions such as antigen presentation and immune system control (2, 4, 12, 15, 17, 31, 34, 37, 39-41), tissue tropism (3-5, 23, 27), virus spread (9, 25, 42), and the establishment of latency (33). Most of these functions are encoded by nonessential genes. In accordance with such an in vivo function, the growth of viruses that do not express the fcr-1 gene was significantly restricted in all organs tested. The differences were in the range between 1.5 and 3.5 log₁₀ PFU. This indicated that the fcr-1 gene product has an important function in vivo.

Considering the in vitro interaction of the viral FcR with the Fc fragment of mouse IgG (38), it was reasonable to presume that the reduced virulence of the fcr-1 deletion resulted from the more effective clearance through antibody-mediated mechanisms. Such protective effects have been suggested for FcRs in HSV-1 and varicella-zoster virus. Under in vitro conditions, they protect virus from neutralization and virus-infected cells from lysis mediated by antibody and complement and by antibody-dependent cellular cytotoxicity (10, 11, 13, 14). Our experiments showed, however, that the reduced replication capacity of fcr-1 deletion mutants is not resolved in the Ig-deficient host. Therefore, the interaction of MCMV FcR with the Ig Fc fragment either is irrelevant during in vivo infection or is minor in comparison with the effect of a strong second antibody-independent function. This mechanism is also T-cell and NK-cell independent, since depletion of these cell populations did not selectively improve the in vivo replication of the deletion mutant.

MCMV recombinants with deletions in the HindIII I and J fragments including the fcr-1 gene have been described recently (4), although a mutant with a singular deletion of the fcr-1 gene was not generated. In view of our results, the reduced in vivo virulence of these recombinants can now be explained at least in part by the lack of the fcr-1 gene. It has been shown that the deletion mutant missing both the fcr-1 gene and the m137 gene grew normally in the IC-21 macrophage cell line (4). This indicates that the FcR acts differently from the neighboring genes M140 and/or M141, to which macrophages growth deficiency was mapped.

An in vivo role of alphaherpesvirus proteins with FcR properties appears to be facilitation of infection through the direct cell-to-cell route (3, 9, 42). The HSV-1 gE and gI mutants fail in axonal spread due to the inefficient neuron-to-neuron transmission (9). They also form significantly smaller plaques than the wild type does (8, 42). Impaired cell-to-cell spread in polarized retinal epithelium cells is a characteristic of an HCMV US9 deletion mutant (25). Although the MCMV FcR shows no significant sequence homology to alphaherpesvirus FcRs or HCMV US9, it cannot yet be excluded that the MCMV FcR acts as a mediator of viral spread in certain tissues. The spread of the virus to several organs including the salivary gland is not inhibited after local injection, and also the clearance kinetics reflects, although at a lower level, that of the wild-type infection (data not shown). Thus, the phenotype of the infection in vivo does not yet provide a clue to the function of this glycoprotein, which clearly contributes to the virulence of the virus.

Although the deletion of the fcr-1 gene results in severely reduced viral growth in vivo, we could falsify the hypothesis concerning the underlying mechanism. Thus, potential immune system evasion principles, which appear obvious by studying isolated gene functions in vitro after high expression of the respective function, need to be confirmed by the analysis of appropriate mutants in vivo. The molecular basis for the reduced in vivo replication of fcr-1 deletion mutants is again unresolved. Attempts to identify the putative cellular ligand, perhaps a member of the Ig superfamily, are in progress.