Ligand induction of retinoic acid receptors alters an acute infection by murine cytomegalovirus

Abstract

Here we report that administration of retinoids can alter the outcome of an acute murine cytomegalovirus (MCMV) infection. We show that a crucial viral control element, the major immediate-early enhancer, can be activated by retinoic acid (RA) via multiple RA-responsive elements (DR2) that bind retinoid X receptor-retinoic acid receptor (RAR) heterodimers with apparent dissociation constants ranging from 15 to 33 nM. Viral growth is dramatically increased upon RA treatment of infected tissue culture cells. Using synthetic retinoid receptor-specific agonists and antagonists, we provide evidence that RAR activation in cells is required for mediating the response of MCMV to RA. Oral administration of RA to infected immunocompetent mice selectively exacerbates an infection by MCMV, while cotreatment with an RAR antagonist protects against the adverse effects of RA on MCMV infection. In conclusion, these chemical genetic experiments provide evidence that an RAR-mediated pathway can modulate in vitro and in vivo infections by MCMV.

FIG. 1

RAREs in the MCMV enhancer. (A) Schematic representation of the MCMV MIEP sequence from position −2000 to +50 present in the reporter construct pON405. The locations of the seven potential RAREs are marked by open boxes. The AGGTCA-related motifs of the two types of elements, named MDR2a and MDR2b, are indicated. The numbers refer to the nucleotide position relative to the transcription start site. A schematic of the MCMV MIEP deletion mutant present in the reporter construct pON407 in which sequences from position −146 to −2000 are abolished is shown below. One copy each of MDR2a and MDR2b was independently transferred to the heterologous promoter of the CAT gene expression vector tkCAT to generate the reporter plasmids MDR2a and MDR2b shown in the lower portion. (B) NT-2/D1 cells were transfected with 5 μg of either the pON405 or pON407 reporter plasmid and incubated for 36 h with 10⁻⁵ M ATRA (+) or the vehicle (−). Transfection efficiency was standardized by cotransfection of 5 μg of tkLuc. The fold induction of β-galactosidase activity was calculated for each construct by taking the activity in the absence of ATRA as 1. Data shown represent the means and standard deviations of triplicate determinations. (C) Five micrograms of the reporter plasmids MDR2a and MDR2b was cotransfected with 5 μg of the β-galactosidase pRSVβgal internal control expression vector into NT-2/D1 cells and then treated for 36 h with 10⁻⁵ M ATRA (lanes +) or with vehicle (lanes −). The figure shows the result of a representative assay for CAT enzyme activity from cell lysates. Similar results were obtained in three independent experiments. (D) The response of MDR2a and MDR2b to RA (b+) is plotted as the normalized activity observed in these experiments, calculated for each construct by taking the activity in the absence of ATRA (−) as 1.

FIG. 2

Binding of RAR-RXR heterodimers to the MCMV enhancer RAREs. (A) Baculovirus-derived preparations of RARβ or RXRβ were incubated either independently or in combination with ³²P-labeled probes representing the MDR2a and MDR2b elements and analyzed in a gel mobility retardation assay. The arrow indicates the major specific nucleoprotein complex detected. (B) The apparent dissociation constants (K_D) were determined by using double-reciprocal plots (see Materials and Methods for details). The figure shows the results of a representative experiment, and the apparent K_D data represent average values from three separate determinations.

FIG. 3

Activation of the MCMV enhancer by RA in the context of the infection. (A) A schematic representation of the HindIII map of the MCMV genome is shown on the top line. The HindIII-J, -K, and -L fragments have been expanded to show the region containing the MIE genes (ie1, ie2, and ie3) and the sgg1 gene. The hatched box depicts the murine MIEP-enhancer (Enh.). The recombinant viruses RM408, RM427, and RM461, which carry an insertion of the lacZ reporter gene, are shown in the lower portion. The position and orientation of the insertion for each virus are shown. The black box depicts the HCMV promoter-enhancer sequences from position −219 to −19. The empty box depicts position −146 to +50 from the MCMV promoter-enhancer. Wt, wild type. (B) NIH 3T3 cells were exposed to 9-cis-RA (10⁻⁵ M) (+) or the vehicle (−), infected with MCMV recombinant RM408, RM427, or RM461 at an MOI of 0.1 PFU/cell, and assayed 48 h later for β-galactosidase activity. Shown is the normalized β-galactosidase activity observed in these experiments, calculated in each case by taking the activity in the absence of RA as 1. Error bars indicate standard deviations.

FIG. 4

Effect of RA on MCMV growth. (A) NIH 3T3 cells were exposed to 9-cis-RA at 10⁻⁵ M (+RA) or vehicle (−RA) for 4 h. Subsequently, the cells were infected with MCMV (Smith strain) at an MOI of 0.01 PFU/cell and reexposed to 9-cis-RA at 10⁻⁵ M (+RA) or vehicle (−RA). At the different times after infection indicated, the presence of extracellular virus in the cultures was determined. Each data point represents the average and standard deviation for three separate cultures. Similar results were obtained with 10⁻⁵ M ATRA (data not shown). dpi, days postinfection. (B) Same as panel A except that MCMV infections were carried out at an MOI of 10. hpi, hours postinfection. (C) Same as panel A except that NIH 3T3 cells were infected with vaccinia virus at an MOI of 0.01 PFU/cell. In this case, the presence of intracellular virus in the cultures was determined. (D) Same as panel A except that NIH 3T3 cells were infected with herpes simplex virus type 1 at an MOI of 0.01 PFU/cell. Growth of herpes simplex virus type 2 in NIH 3T3 cells was also unaffected by treatment with 9-cis-RA at 10⁻⁵ M (data not shown).

FIG. 5

RAR activation in cells is required for mediating the induction of MCMV by RA. (A) NT-2/D1 cells were transfected with 5 μg of pON405 and incubated for 36 h with the vehicle (−RA), 9-cis-RA (9cRA) at 10⁻⁵ M, ATRA at 10⁻⁵ M, TTNPB at 10⁻⁷ M, LG100069 at 10⁻⁷ M, or a combination of TTNPB and LG100069 at 10⁻⁷ M each. Transfection efficiency was standardized by cotransfection of 5 μg of tkLuc. The response of pON405 to the different retinoids is plotted as the fold induction of β-galactosidase activity observed in these experiments, calculated for each construct by taking the activity in the absence of RA as 1. Each bar represents the average and standard deviation of triplicate determinations. (B) NIH 3T3 cells were exposed to 9-cis-RA at 10⁻⁵ M, ATRA at 10⁻⁵ M, TTNPB at 10⁻⁷ M, LG100069 at 10⁻⁷ M, a combination of TTNPB and LG100069 at 10⁻⁷ M each, or the vehicle (−RA) for 4 h. Subsequently, the cells were infected with MCMV (RM461) at an MOI of 0.01 PFU/cell and reexposed to the different retinoids or the vehicle (−RA). On day 5 after infection, the presence of extracellular virus in the cultures was determined. The fold enhancement of MCMV replication was calculated in each case by taking the amount of virus in the absence of RA as 1. Each bar represents the average and standard deviation for three separate cultures. (C) Structure of the RAR antagonist. The 50% inhibitory concentrations of AGN 193109 are 9 (±1) nM for RARα, 7 (±3) nM for RARβ, and 5 (±1) nM for RARγ. The 50% inhibitory concentrations were determined by performing transfection assays on CV-1 cells with a reporter construct, MTV-4(R5G)-LUC, containing four copies of the DR-5 RARE R5G and expression plasmids for either RARα, -β, or -γ and using a 10⁻⁸ M dose of ATRA (25). (D) NT-2/D1 cells were cotransfected with 5 μg of pON405 and incubated for 36 h with the vehicle or 10⁻⁶ M 9-cis-RA in the presence or absence of the indicated increasing concentrations of the retinoid antagonist AGN 193109 or the vehicle. Transfection efficiency was standardized by cotransfection of 5 μg of tkLuc. β-Galactosidase activity is expressed as the normalized response, which is the β-galactosidase activity divided by the luciferase activity. Activation obtained with 9-cis-RA treatment alone was considered 100%. The data presented are representative of results of triplicate experiments. (E) NIH 3T3 cells were exposed to the vehicle or 9-cis-RA at 5 × 10⁻⁵ M in the presence or absence of the indicated concentrations of the retinoid antagonist AGN 193109 or the vehicle. Subsequently, cells were infected with MCMV (RM461) at an MOI of 0.01 PFU/cell and reexposed to the different retinoids. On day 5 after infection, the presence of extracellular virus was determined. The amount of virus obtained with 9-cis-RA treatment alone was considered 100%. Each data point represents the average and standard deviation for three separate cultures.

FIG. 6

Oral administration of ATRA selectively increases the susceptibility of mice to MCMV infection. BALB/c.ByJ mice (six per group) were pretreated by intragastric intubation with vehicle (corn oil) (−RA) or 50 mg of ATRA per kg in vehicle (+RA) 2 h before intraperitoneal infection with 1 × 10⁵ PFU of MCMV (Smith strain) (A) or 2 × 10⁷ PFU of vaccinia virus (VV) (B). Treatment with vehicle or ATRA in vehicle was repeated on days 2, 4, 7, 9, 11, and 14 after infection. Mice were monitored daily for survival up to and including day 15. Survival curves were statistically analyzed by the log rank test and showed that RA treatment significantly influenced the rate of mortality from MCMV (P value of 0.01) but not from vaccinia virus (P value of 0.64).

FIG. 7

Levels of lethality of MCMV in mice treated with ATRA and those treated with solvent alone. BALB/c.ByJ mice were pretreated by intragastric intubation with vehicle (corn oil) (−RA) or 50 mg of ATRA per kg in vehicle (+RA) 2 h before intraperitoneal infection with different doses of MCMV (Smith strain). Treatment with vehicle or ATRA in vehicle was repeated on days 2, 4, 7, 9, 11, and 14 after infection. Mice were monitored daily for survival up to and including day 15. Logit regression curves relating survival to the log (to the base 10) dose of virus were fit to each group (deviance = 2.10 and P = 0.95 for the control treated mice; deviance = 6.80 and P = 0.56 for the mice treated with RA). The points indicate the observed survival at each dose, with the integers above the points denoting the numbers of animals tested at that dose. The LD₅₀ for each group was estimated from the fitted logit regression line as 6.27 × 10⁴ for the group of mice treated with RA and 2.34 × 10⁵ for the control group. The bars along the x axis represent approximate 95% confidence intervals for the LD₅₀s. A formal test of the equivalence of the LD₅₀s for the two groups would be rejected at an alpha level of less than 0.001.

FIG. 8

Oral administration of ATRA increases the severity of an MCMV infection in the spleens of infected mice. BALB/c.ByJ mice (five per group) were pretreated by intragastric intubation with vehicle (−RA) or 50 mg of ATRA per kg (+RA) 2 h before intraperitoneal infection of 5 × 10³ PFU of MCMV (Smith strain). Treatment with vehicle or ATRA was repeated on day 2 after infection. All mice were sacrificed on day 4 after infection for determination of virus titers (A) and spleen weight (B). There was a trend in higher viral titers in the spleens of the ATRA-treated animals in comparison with control treated mice (P value of ∼0.05) as analyzed by the Mann-Whitney test (two tailed). Spleen weights were also significantly different (P < 0.05) as determined by Student’s t test (two-tailed). Error bars indicate standard deviations.

FIG. 9

Oral administration of ATRA enhances MCMV damage in the spleens of infected mice. Splenic sections from BALB/c.ByJ mice treated by intragastric intubation with vehicle (−RA) or with 50 mg of ATRA per kg (+RA) and infected with 5 × 10³ PFU of MCMV for 4 days are shown. Magnification, ×100. Some of the cytomegalic cells present are indicated by arrowheads, and the areas of necrosis are indicated by arrows. The presence of selected hematopoietic cells, such as megakaryocytes (M), is shown. The red pulp (RP) and white pulp (WP) are indicated.