Virus attenuation after deletion of the cytomegalovirus Fc receptor gene is not due to antibody control

Abstract

The murine cytomegalovirus (MCMV) fcr-1 gene codes for a glycoprotein located at the surface of infected cells which strongly binds the Fc fragment of murine immunoglobulin G. To determine the biological significance of the fcr-1 gene during viral infection, we constructed MCMV fcr-1 deletion mutants and revertants. The fcr-1 gene was disrupted by insertion of the Escherichia coli lacZ gene. In another mutant, the marker gene was also deleted, by recombinase cre. As expected for its hypothetical role in immunoevasion, the infection of mice with fcr-1 deletion mutants resulted in significantly restricted replication in comparison with wild-type MCMV and revertant virus. In mutant mice lacking antibodies, however, the fcr-1 deletion mutants also replicated poorly. This demonstrated that the cell surface-expressed viral glycoprotein with FcR activity strongly modulates the virus-host interaction but that this biological function is not caused by the immunoglobulin binding property.

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.

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 ΔMS94.4, ΔMC95.15, and ΔMC95.16 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.

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.

FIG. 4

In vitro growth of MCMV recombinants. NIH 3T3 cells were infected with wild-type MCMV (open circles), ΔMS94.4 (solid triangles), rMS95.9 (open diamonds), ΔMC95.15 (solid circles), and ΔMC95.16 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.

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), ΔMS94.4 (open circles), rMS95.9 (solid triangles), and ΔMC95.15 (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.

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, ΔMC95.15, and rMS95.9 MCMV. Virus titers were determined 3 weeks p.i. (symbols), and median values were calculated (horizontal bars).

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), ΔMS94.4 (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.