Identification of common mechanisms by which human and mouse cytomegalovirus seven-transmembrane receptor homologues contribute to in vivo phenotypes in a mouse model

Abstract

The mouse cytomegalovirus chemokine receptor homologue (CKR) M33 is required for salivary gland tropism and efficient reactivation from latency, phenotypes partially rescued by the human cytomegalovirus CKR US28. Herein, we demonstrate that complementation of salivary gland tropism is mediated predominantly by G protein-dependent signaling conserved with that of M33; in contrast, both G protein-dependent and -independent pathways contribute to the latency phenotypes. A novel M33-dependent replication phenotype in cultured bone marrow macrophages is also described.

Fig 1

(A) Expression levels of HA-tagged US28 constructs detected by ELISA. COS-7 cells in 6-well dishes were transfected with 4 μg of the indicated HA-tagged US28 constructs or the pcDNA3 vector control using Lipofectamine 2000 (Invitrogen, Australia). At 6 h posttransfection, cells were trypsinized and seeded into replicate 96-well trays (six wells per construct per plate). The detection of the HA-tagged constructs in cells permeabilized with Triton X-100 and nonpermeabilized replicate cells was performed 24 h later as described previously (19). Receptor levels were measured as optical density (OD) readings at 450 nm and corrected for background via subtraction of mean control values (perm. or non-perm.). Data are presented as a cell surface index, i.e., the log₁₀ ratio of the cell surface (nonpermeabilized) to total (permeabilized) OD values. Bars indicate means and standard deviations (SD) (n = 6). Asterisks indicate significant differences between the wt and mutated US28 constructs (Kruskal-Wallis with Dunn's posttest, *, P < 0.05; ***, P < 0.001). (B) Quantitative assessment of endocytosis for wt US28 and US28 mutants. HeLa cells transfected with 4 μg of the indicated HA-tagged constructs using Lipofectamine 2000 were labeled with rabbit anti-HA (ab9110, 1:500; Abcam, Cambridge, United Kingdom) at 37°C for 1 h and then “chased” in the absence of antibody for a further 20 min. Endocytosis was stopped by incubating cells at 4°C and cell surface-retained receptors labeled with Alexa Fluor 594 (AF⁵⁹⁴) goat anti-rabbit IgG (1:1,000; A11037; Molecular Probes, Invitrogen, Australia) for 1 h at 4°C. The cells were fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100, and incubated with AF⁴⁸⁸ goat anti-rabbit IgG (A11034, 1:1,000; Molecular Probes, Invitrogen, Australia) for 1 h at room temperature. Images from random fields captured at a magnification of ×40 were converted to grayscale; the AF⁴⁸⁸ and AF⁵⁹⁴ channels were overlaid, and the ratio of AF⁴⁸⁸ (total) to AF⁵⁹⁴ (surface) staining of individual cells was determined using NIH ImageJ (http://rsb.info.nih.gov/ij/). The log₁₀ ratio provides an endocytosis index; this ratio increases as the proportion of endocytosed receptors increases. Data are plotted for individual cells, with bars indicating means and SD (50 cells). Asterisks indicate significant differences between the wt and other constructs (Kruskal-Wallis with Dunn's posttest, ***, P < 0.001). (C) Surface expression of wt US28 and US28 mutants determined by whole-cell binding of the chemokine ligand ¹²⁵I-CX3CL1 (fractalkine) as described previously (27). Whole-cell homologous competition binding was performed in MEF infected (MOI = 3) with either wt US28 (filled circles), US28ΔN[2-16] (open triangles), US28R129Q (open circles), US28ΔC54 (open diamonds), or US28RQ/ΔC54 (open squares). At 24 h p.i., cells were labeled with ¹²⁵I-CX3CL1 (fractalkine) and incubated with increasing amounts of cold CX3CL1 as indicated. Data are means ± SD from two experiments performed in triplicate.

Fig 2

Inositol phosphate turnover in transfected or MCMV-infected cells. (A) COS7 cells transfected 24 h previously with constructs expressing either wt US28 (black), US28ΔC54 (gray), US28R129Q (stippled), US28RQ/ΔC54 (striped), US28ΔN[2–16] (checked), or pcDNA3 (white). (B) MEF infected (MOI = 3) 24 h previously with MCMV recombinants expressing either wt or mutated US28 (shading as in panel A) or an M33-null mutant (white). In both panels, cells were incubated in the presence or absence of 10 nM human recombinant CX3CL1, prior to determination of accumulation of phosphorylated [³H]inositol according to methods described previously (28). Data are presented as means ± SD from 2 experiments performed in triplicate; asterisks indicate significant difference between the responses with and without fractalkine (Student's t test; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 3

HEK293 cells were transfected with 20 μg of the indicated HA-tagged US28 constructs or the pcDNA3 vector using calcium chloride precipitation. At 48 h after transfection, the cells were lysed on ice using radioimmunoprecipitation assay (RIPA) lysis buffer (Millipore). Samples were denatured at 92°C for 5 min and run on a 10% bis-Tris gel (Invitrogen) at 130 V for 1.5 h. The gel was blotted onto polyvinylidene difluoride (PVDF) membranes for 1.5 h and incubated with phosphospecific antibodies (Cell Signaling) against ERK (A) and JNK (B) simultaneously or p38 (C) overnight at 4°C. After incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies, the membranes were developed using Immobilon Western chemiluminescent (Millipore) and quantified using AlphaView software; blots were stripped and reprobed for β-actin as a loading control. For each blot, background signal (pcDNA3) was subtracted and results were expressed relative to the mean signal for wt US28 (100%). Results are expressed as means ± standard errors of the means (SEM) from five independent experiments, each performed with duplicate samples. Representative blots are shown above the quantification graphs in each case. Asterisks denote significant differences between wt US28 and US28 mutants (analysis of variance [ANOVA] with Bonferroni posttest; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 4

Induction of CREB-driven (A), NF-κB-driven (B), and NFAT-driven (C) luciferase expression in HEK cells transfected with either 50 ng of CREB-luciferase DNA (A), 50 ng NF-κB-luciferase DNA (B), or 50 ng NFAT-luciferase (C) together with increasing concentrations of either wt US28 (●), US28ΔC54 (♢), US28R129Q (○), or US28RQ/ΔC54 (□) expression plasmids. Experiments were performed according to previously reported methods (14). Baseline signaling measurements of HEK cells transfected with equivalent amounts of the pcDNA3 expression vector were set at 100%. Data are means ± SEM from two experiments performed in quadruplicate.

Fig 5

Multistep growth analysis of M33 MCMV recombinants (A) and US28 recombinants (B) in primary BMM cultures from adult C57BL/6 mice. BMM were harvested from femurs and tibias of mice and cultured in the presence of human recombinant colony-stimulating factor 1 (CSF-1; 10⁴ U/ml; a kind gift from M. Sweet, University of Queensland) for 5 to 9 days. BMM were infected at an MOI of 10 (relative to MEF; corresponds to an MOI of approximately 0.1 for BMM); supernatants were taken at the indicated times and assayed for infectious virus by plaque assay on MEF. (A) wt M33 (●), M33R131Q (○), M33ΔC38 (♢), and M33-null mutants (lacZ insertion [▲] and M33 premature stop [△]); (B) wt US28 (●), US28ΔC54 (♢), US28R129Q (○), US28RQ/ΔC54 (□) and M33-null mutants (lacZ insertion [▲] and M33 premature stop [△]). Data are mean titers (log₁₀ PFU/ml) ± SD from two experiments performed in triplicate. The origin of the y axis is adjusted to reflect the lower limit of virus detection.

Fig 6

(A) Replication of recombinant MCMV expressing wild-type or mutated US28 in BALB/c mice. Six-week-old mice were inoculated i.p. with 2 × 10⁶ PFU of either wt US28 recombinant MCMV (black) or the following US28 mutant MCMVs: US28ΔC54 (gray), US28R129Q (stippled), or US28RQ/ΔC54 (striped). Organs (n = 4) were harvested at 4 days p.i. (spleen and liver) or 17 days p.i. (salivary gland). Virus titers of homogenized organs (log₁₀ PFU/organ) were quantified on MEF by plaque assay. The origin of the y axis is adjusted to reflect the lower limit of virus detection. Comparisons between wt US28 and US28 mutant groups were made using ANOVA with a Bonferroni posttest (***, P < 0.001). (B) Cumulative reactivation curves of spleen explant cultures taken from latently infected adult BALB/c mice. Spleens (10 to 20 per group) were harvested 12 weeks following i.p. infection of 10⁵ PFU of either wt K181 (■), wt US28 (●), US28ΔC54 (♢), US28R129Q (○), US28RQ/ΔC54 (□), or M33R131Q (▲). Each spleen was finely dissected and distributed among 6 wells of a 24-well culture plate (i.e., 60 to 120 cultures in total). Supernatants (80 μl) were harvested twice during the first week and once per week for a period of 6 to 8 weeks and assayed for infectious virus on MEF by centrifugal enhancement. Cultures that were positive within the first 7 days (≤3% of total cultures) were likely to represent persistent shedding rather than a reactivation event and were excluded from the analysis. Comparisons of the proportion of reactivated to nonreactivated cultures were made using Fisher's exact test (**, P < 0.01).