AbstractVarious natural and synthetic polyanionic polymers with different chemical structures are known to exhibit potent antiviral activity in vitro toward a variety of enveloped viruses and may be considered as promising therapeutic agents. A water-soluble conjugate of 2,5-dihydroxybezoic acid (2,5-DHBA) with gelatin was synthesized by laccase-catalyzed oxidation of 2,5-DHBA in the presence of gelatin, and its antiviral activity against pseudorabies virus (PRV) and bovine herpesvirus type 1 (BoHV-1), two members of the Alphaherpesvirinae subfamily, was studied. The conjugate produced no direct cytotoxic effect on cells, and did not inhibit cell growth at concentrations up to 1000 µg/mL. It exhibited potent antiviral activity against PRV (IC50, 1.5–15 µg/mL for different virus strains) and BoHV-1 (IC50, 0.5–0.7 µg/mL). When present during virus adsorption, the conjugate strongly inhibited the attachment of PRV and BoHV-1 to cells. The 2,5-DHBA–gelatin conjugate had no direct virucidal effect on the viruses and did not influence their penetration into cells, cell-to-cell spread, production of infectious virus particles in cells, and expression of PRV glycoproteins E and B. The results indicated that the 2,5-DHBA–gelatin conjugate strongly inhibits the adsorption of alphaherpesviruses to cells and can be a promising synthetic polymer for the development of antiviral formulations against alphaherpesvirus infections. View Full-Text
Keywords: laccase; 2,5-dihydroxybezoic acid (2,5-DHBA); 2,5-DHBA–gelatin conjugate; pseudorabies virus; bovine herpesvirus type 1; antiviral activity; virus adsorptionlaccase; 2,5-dihydroxybezoic acid (2,5-DHBA); 2,5-DHBA–gelatin conjugate; pseudorabies virus; bovine herpesvirus type 1; antiviral activity; virus adsorption►▼ Figures
Derivation of bdh2 knockout mice. (A) Targeting strategy to delete exon 7 in the mouse bdh2 gene. TK, thymidine kinase. (B) Ribbon diagrams of wt and mutant mouse Bdh2 proteins with bound NAD+ in a stick model. Note that Tyr147 and Lys151, the critical residues for interaction with NAD+, are absent in mutant Bdh2. (C) Identification of positive ES clones by Southern blotting using both 5′ and 3′ external probes. (Left) Homologous recombination at the 5′ end. Two clones contained the expected 7.5-kb HindIII fragment with the 5′ external probe. (Right) Homologous recombination at the 3′ end. Two clones, which were targeted correctly at the 5′ end, were tested for targeting at the 3′ end. These clones contained the expected 22-kb EcoRI fragment with the 3′ external probe. (D) Genotype identification by PCR analysis. DNA extracted from tails of wt and bdh2 type II mice was subjected to PCR analysis. The diagnostic product for type II deletion is the 160-bp fragment amplified by the P1 and P4 primers. MM, molecular marker 1-kb DNA ladder.
Bdh2 null mice have no detectable 2,5-DHBA.We previously demonstrated that suppression of BDH2 results in depletion of 2,5-DHBA in cultured mammalian cells (18). We next determined 2,5-DHBA levels in bdh2 null mice. Salicylic acid derivatives such as 2,5-DHBA are easily detectable in urine specimens (29). Therefore, we analyzed 2,5-DHBA levels in urine samples collected from wt and bdh2 null mice by gas chromatography-mass spectrometry (GC-MS) analysis following derivatization with trimethylsilate (TMS) (18). We readily observed 2,5-DHBA in the urine samples obtained from wt mice, which was confirmed against a set of DHBA standards. In contrast, there was no detectable 2,5-DHBA in urine samples obtained from bdh2 null mice (22). Thus, these results not only confirm our previous findings (18) but also reinforce the notion that BDH2 catalyzes the biosynthesis of 2,5-DHBA.
Complete blood counts in 2,5-DHBA-deficient mice.Depletion of siderophore in developing zebrafish embryos results in anemia and heme deficiency (18). We next evaluated hematological parameters in our bdh2 null mice. Examination of complete blood counts (CBC) and red blood cell (RBC) indices in peripheral blood of bdh2 null mice (ages 3 to 14 months) revealed a few changes. We found that adult, but not geriatric, bdh2 null mice were mildly anemic, as noted by a decreased mean corpuscular volume (MCV) (Fig. 2C) and decreased mean corpuscular hemoglobin concentration (MCHC [Hgb/RBC]) (Fig. 2D). Additionally, analysis of peripheral blood smears indicated a mild hypochromia (Fig. 2G). Based on these results, we conclude that bdh2 null mice are microcytic and hypochromic. These observations are consistent with our previous findings that siderophore-depleted zebrafish embryos were hypochromic (17). Additionally, adult bdh2 null mice, but not geriatric mice, have a higher number of RBCs than wt controls (Fig. 2A). These results are consistent with the notion that mice with microcytic and hypochromic anemias have a higher number of RBCs (30–32). The increased number of RBCs with a decrease in size further suggests that the iron available for hemoglobin is restricted (30). Despite the presence of mild anemia in bdh2 null mice, we did not observe splenomegaly, although there was a trend toward increased erythropoiesis on histological examination (Fig. 2I) (unpublished data). No differences in white blood cell (WBC) counts or platelet numbers were detected in bdh2 null mice (Fig. 2H) (unpublished data). Thus, the results confirm our previous findings and suggest that siderophore depletion affects heme biogenesis.
Effect of BDH2 deficiency on hematological parameters. Complete blood counts were measured from whole blood of mice at the indicated ages. (A to F) RBC parameters were determined in female mice at 3 and 14 months of age. Data are presented as means ± SD. HCT, hematocrit; MCV, mean corpuscular volume; MCHC, mean corpuscular hemoglobin concentration; Hb, hemoglobin; RDW, red cell distribution width. (G) Wright-Giemsa-stained peripheral blood smears of mice at 3 months of age. bdh2 null RBCs are relatively hypochromic. (H) Platelet (PLT) levels in female mice at 3 and 14 months of age. Data are presented as means ± SD. (I) Increased extramedullary hematopoiesis in bdh2 null mice. Shown are H&E staining and Perls staining of histological sections of spleen from female mice at 14 months of age. WP, white pulp; RP, red pulp.
Mammalian siderophore 2,5-DHBA deficiency causes iron overload.Previously we demonstrated that depletion of 2,5-DHBA through inhibition of bdh2 expression alters cellular iron homeostasis (18). To assess the consequences of the BDH2 deficiency on iron homeostasis in various organs, we used both histological staining and quantitative determination of iron content (Fig. 3). At 3 months of age, bdh2 null mice showed a significant increase in iron content in the spleen (∼3-fold), which primarily reflects macrophage iron content recovered from the hemoglobin of senescent RBCs (Fig. 3B). We next examined sites of iron accumulation by staining histological sections for iron. We found enhanced iron accumulation in the red pulp region of the spleen (Fig. 3A). However, the histological appearance of the spleen remains unchanged compared to that of wt mice, as judged by H&E staining (Fig. 3A). Siderophore depletion elevates cytoplasmic iron and mitochondrial iron deficiency (18). Thus, the observed increase in splenic iron content may be secondary due to delayed iron utilization in the absence of siderophore.
Iron accumulation in bdh2 null mice. (A) Histological detection of tissue iron in liver and spleen sections from female mice at 14 months of age by Perls or H&E staining. Blue staining represents iron accumulation in cells. Representative images from specimens collected from at least 3 mice per genotype are shown. (B to E) Quantitative determination of tissue iron content (mg/g of tissue wet weight) in various organs was compared in female mice at 3 and 14 months of age. At least 3 mice per time point and genotype were analyzed. Data are presented as means ± SD. (F and G) Serum iron indices in female mice at 3 and 14 months of age. Iron and transferrin saturation were measured from nonhemolyzed serum of wt and bdh2 null mice. Data are presented as means ± SD.
We also examined the iron content of the liver in 3- and 14-month-old bdh2 null mice, which reflects total body iron stores. We observed a moderate increase in nonheme iron in liver (∼2-fold) in bdh2 null mice (Fig. 3C). The observed increase in iron content in liver is pronounced in parenchymal cells (Fig. 3A). However, the iron content in the pancreas, heart, and brain of bdh2 null mice is unremarkable (Fig. 3D and E). The increased iron content observed in spleen and liver samples from 3-month-old bdh2 null mice is not a permanent change, since analysis of iron content in samples from geriatric wt or bdh2 null mice (14 months of age) showed no significant difference in iron content (Fig. 3B and C). These findings indicate that bdh2 deficiency in mice leads to iron accumulation in spleen and, to a more limited extent, in liver.
Finally, analysis of serum iron in bdh2 null mice revealed a decrease in serum iron and transferrin saturation compared to the levels in wt mice (Fig. 3F and G).
Hepcidin and ferroportin levels are normal in bdh2 null mice.Hepcidin regulates systemic iron by controlling duodenal iron absorption and release of iron from splenic macrophages (33). Since bdh2 null mice specifically accumulate iron in the spleen, we reasoned that enhanced iron in spleen might be secondary to altered hepcidin levels. To examine this idea, we assessed levels of hepcidin in liver samples from wt and bdh2 null mice. Figure 4A shows that there is no significant difference in levels of hepcidin gene expression between wt and bdh2 null liver samples in aged mice. However, we did observe a trend toward increased hepcidin expression in liver samples from bdh2 null mice at 12 weeks of age, but the difference did not achieve statistical significance (P = 0.12).
Hepcidin and ferroportin levels are unaltered in bdh2 null mice. (A) Quantification of hepcidin mRNA levels in liver samples from control and bdh2 null mice. Shown is hepatic Hamp mRNA expression relative to β-actin mRNA expression in control and bdh2 null mice at 3 months of age. Data are presented as means ± SD. The P value is not significant compared with control mice. (B and C) Immunoblot (Western blot [WB]) analysis of ferroportin levels in liver and spleen samples from female mice 3 months of age. Protein samples were collected from mouse liver and spleen homogenates or 293T cells transfected with pCMV6 vector (Origene) or pCMV6 containing c-Myc-tagged full-length mouse FPN1 cDNA. (B) (Left) The blot was probed with anti c-Myc antibody. (Right) The blot was probed with antiferroportin antibody. (C) The blot was probed with antiferroportin antibody. The blot was later stripped and reprobed with an antiactin antibody to ensure equal loading. The positions and masses of molecular mass markers are indicated on the left.
Hepcidin binds to ferroportin on the plasma membrane of enterocytes, macrophages, hepatocytes, and other cells, promoting its internalization and eventually lysosomal degradation (34, 35). Ferroportin is the only exporter of inorganic iron in mammalian cells; therefore, inactivation of ferroportin by hepcidin leads to intracellular iron retention (34). We therefore assessed levels of ferroportin by performing an immunoblot of liver and spleen samples from control and bdh2 null mice. We found that ferroportin levels are similar in both sets of mice (Fig. 4C). Taken together, the results of Fig. 4A and C suggest that hepcidin and ferroportin levels are unaffected by BDH2 deficiency.
Mammalian siderophore 2,5-DHBA depletion confers mitochondrial iron deficiency.The ratio of erythrocyte zinc protoporphyrin IX to heme (ZnPP/H) is commonly used to determine whether iron scarcity affects heme biosynthesis (23, 31, 32). Chelation of iron to protoporphyrin by ferrochelatase is the final step in heme biogenesis. Zn is the alternative metal used by ferrochelatase when iron is not available (36). Therefore, iron deficiency resulting from excess hemolysis and defects in iron recycling contribute to an increase in the ZnPP/H ratio (31, 32). Yeast and zebrafish embryos deprived of siderophore display mitochondrial iron deficiency (18). Therefore, the ZnPP/H ratio was determined in whole-blood samples from wt and bdh2 null mice by hematofluorometry (23). The ZnPP/H ratio was increased in bdh2 null mice (Fig. 5A).
Mitochondrial iron deficiency in hematopoietic cells from bdh2 null mice. (A) Evaluation of ZnPP/H levels in RBC from control and bdh2 null mice at 3 months of age. Data are presented as means ± SD. (B) BDH2 deficiency impairs heme biosynthesis in hematopoietic cells. Control or bdh2 null immortalized hematopoietic cells were incubated with 2.5 μM 55Fe-Tf and 0.2 mM ALA for 8 h at 37°C. Radiolabeled heme was quantified by liquid scintillation following extraction from cell lysates. Data are presented as means ± SD. (C) Incorporation of 55Fe into reticulocytes from control or bdh2 null mice. Shown are uptake of 55Fe and the distribution of iron between heme and nonheme portions of cellular iron. Reticulocytes were incubated with 3.75 μM 55Fe-Tf at 37°C for 30 min. Time zero values were obtained by incubating ice-cold 55Fe-Tf with reticulocytes on ice. (D) Activity of mitochondrial aconitase (mAco; nmol/min/mg protein) was measured in mitochondria isolated from liver samples from female control and bdh2 null mice at 3 and 14 months of age. The activity of mAco was normalized to citrate synthase (CS) activity determined from the same samples. The percentage of mAco activity is indicated (mAco/CS × 100). Each value represents the mean ± SD of n = 5/genotype.
We next measured heme in immortalized hematopoietic cells by labeling them with 55Fe-transferrin and the heme precursor aminolevulinic acid (ALA) (7). As expected, wt cells efficiently incorporated 55Fe into heme; however, in contrast, incorporation of 55Fe into heme was significantly reduced in bdh2 null hematopoietic cells (Fig. 5B). Finally, we measured incorporation of iron into heme in isolated reticulocytes from wt and bdh2 null mice. Reticulocytes from serially phlebotomized mice were incubated with 55Fe-Tf (7), and the amount of intracellular iron incorporated into heme was measured. Iron incorporation into heme was lower in reticulocytes from bdh2 null mice (Fig. 5C).
To further explore mitochondrial iron deficiency, we measured the activity of mitochondrial aconitase (mAco), an iron-dependent enzyme, in liver samples from control and bdh2 null mice at 3 and 14 months of age (18). We found that mAco activity was substantially lower in liver from bdh2 null mice (Fig. 5D). In contrast, the activity of citrate synthase (CS), the activity of which is not dependent on iron, is unaltered in bdh2 null mice (Fig. 5D).
Mammalian siderophore 2,5-DHBA-deficient mice are sensitive to iron challenge.To investigate whether iron deposition in spleen was due to an intrinsic capacity of bdh2 null mice to load iron, 8-week-old control and bdh2 null mice were placed on iron-manipulated diets: a low-iron diet (0.0006% iron), a basal-iron diet (0.02% iron), and a high-iron diet (2% carbonyl iron) for 60 days. All control mice regardless of iron content in the diet survived for the duration of the experiment, whereas only 20% of bdh2 null mice were viable at 55 days post-high-iron-diet challenge (Fig. 6). In contrast, iron deficiency did not cause mortality in bdh2 null mice, which exhibited pale paws, extreme alopecia, and hypothermia (Fig. 6A). bdh2 null mice on the high-iron diet exhibited runted growth (Fig. 6A) and gained weight very slowly (Fig. 6). The observed delay in weight gain was unrelated to consumption of chow; both wt and bdh2 null mice consumed chow at comparable rates (unpublished data).
Iron challenge adversely affects the growth of bdh2 null mice. (A) Morphological features of mice on low-iron and high-iron diets. (B) Growth parameters of control and bdh2 null mice placed on a high-iron diet. (C) Survival analysis of control and bdh2 null mice on a high-iron diet.
To gain insight into the mechanism by which a high-iron diet kills bdh2 null mice, we systematically examined the biochemical and organ changes in these mice. We first evaluated the effect of dietary iron on hematological parameters in wt and bdh2 null mice. RBCs are elevated in both genotypes on a high-iron diet compared with RBCs in mice on the basal-iron diet or low-iron diet. However, the hemoglobin and MCH contents are significantly lower in bdh2 null mice, even on the high-iron diet, confirming that 2,5-DHBA deficiency results in heme deficiency (Table 1). Additionally, the MCV is also lower in bdh2 null mice, suggesting the development of microcytic hypochromic anemia, even in the face of excess iron (Table 1). In contrast, both wt and bdh2 null mice are anemic when maintained on a low-iron diet (Table 1).
We next examined tissue and serum iron levels in these mice. We found a significant increase in total serum iron and transferrin saturation in both wt and bdh2 null mice that were kept on a high-iron diet. However, serum iron levels in bdh2 null mice were much higher than those of wt mice on high-iron diets (Fig. 7A and B). In contrast to these findings, maintenance on a low-iron diet produced lower serum iron levels and decreased transferrin saturation in both groups of mice (Fig. 7A and B). Furthermore, the serum iron levels in mice on a low-iron diet were not significantly different between genotypes (Fig. 7A and B). Dietary carbonyl iron overload is associated with deposition of iron in periportal hepatocytes (37). Thus, we also found enhanced iron staining in periportal areas in control and bdh2 null mice (Fig. 7C). Additionally, iron accumulation in the liver of bdh2 null mice was significantly greater than that in liver samples from control mice (Fig. 7C). To confirm these findings, liver iron content was quantified by atomic absorption (AA) spectroscopy and colorimetry. Again, liver iron content was significantly higher in bdh2 null mice (Fig. 7D and E).
Iron parameters in control and bdh2 null mice fed with iron-manipulated diets. (A and B) Serum iron indices in control and bdh2 null mice placed on a low-iron diet, basal-iron diet, and high-iron diet. Serum iron and transferrin saturation were measured in nonhemolyzed sera of control and bdh2 null mice. Data are presented as means ± SD. (C) Perls Prussian blue staining to detect tissue iron in liver and spleen sections from control and bdh2 null mice fed with a low-iron diet, basal-iron diet, and high-iron diet. Nonheme iron stains blue. Representative images from specimens collected from at least 3 mice per genotype are shown. (D) Quantitative determination of liver iron content (μg/mg of wet tissue weight) of control and bdh2 null mice placed on a low-iron diet, basal-iron diet, and high-iron diet by colorimetry. At least 3 mice per time point and genotype were analyzed. Data are presented as means ± SD. (E) Atomic absorption spectroscopy analysis of liver iron content (μg/mg of tissue wet weight) in control and bdh2 null mice placed on a low-iron diet, basal-iron diet, and high-iron diet. At least 3 mice per time point and genotype were analyzed. Data are presented as means ± SD. (F) Quantitative determination of tissue iron content (μg/mg of wet tissue weight) in spleen from control and bdh2 null mice placed on a low-iron diet, basal-iron diet, and high-iron diet. At least 3 mice per time point and genotype were analyzed. Data are presented as means ± SD. (G) SDH levels in sera from control and bdh2 null mice fed with a low-iron diet, basal-iron diet, and high-iron diet. Enzyme levels are indicated in logarithmic scale. At least 3 mice per time point and genotype were analyzed. Data are presented as means ± SD.
We previously showed that BDH2 deficiency leads to abnormal accumulation of iron in the cytoplasm, leading to increased reactive oxygen species (ROS) production, which eventually contributes to premature cell death (18). Thus, it is possible that iron-loaded hepatocytes in bdh2 null mice undergo premature cell death. To investigate this possibility in vivo, we assessed hepatocyte cell injury by measuring serum levels of sorbitol dehydrogenase (SDH), a liver-specific intracellular enzyme that leaks into blood following hepatic damage (38). We found an ∼50-fold increase in serum SDH levels in bdh2 null mice placed on a high-iron diet over the level found in control mice on the same diet (Fig. 7G). In contrast to the findings observed with the high-iron diet, the low-iron diet significantly lowered liver iron content, as determined using histological and quantitative assays (Fig. 7D and E). Combined, these results suggest that the absence of bdh2 exacerbates iron accumulation when mice are challenged with excess iron.
Last, we evaluated the iron content in spleens of wt and bdh2 null mice that were fed a high-iron diet. Figure 3 demonstrates that bdh2 deficiency contributes to iron accumulation in the spleen. Iron accumulation in the spleen was further exacerbated when bdh2 null mice were placed on a high-iron diet (Fig. 7C). AA spectrometry and colorimetric determination of iron content confirmed the histological findings in both control and bdh2 null mice (Fig. 7F) (unpublished data). In contrast to these findings, a low-iron diet caused a decrease in iron content in the spleens of both wt and bdh2 null mice (Fig. 7C and F). Collectively, the iron imbalance observed in bdh2 null mice is likely to adversely affect organ function.
Hematologic parameters of wt and bdh2 null mice on normal and iron-manipulated diets
Supplementation with 2,5-DHBA alleviates iron overload.We previously showed that 2,5-DHBA protects cells from iron toxicity by sequestering iron (18). Additionally, bdh2 null mice display iron overload in spleen. If the basis for iron accumulation in spleen is indeed lack of 2,5-DHBA, then supplementation with 2,5-DHBA should correct this defect. To examine this hypothesis, we injected 12-week-old control or bdh2 null mice with 500 mg/kg 2,5-DHBA split into two doses, and each dose was injected intraperitoneally 24 h apart. Mice were sacrificed 24 h after last injection of 2,5-DHBA, and iron levels in the blood and tissues of these mice were determined. Injection with vehicle used to solubilize 2,5-DHBA had no effect on serum iron parameters in both control and bdh2 null mice (Fig. 8A and B). In contrast, injection of 2,5-DHBA lowered serum iron levels as well as serum Tf iron saturation in bdh2 null mice (Fig. 8A and B). Injection of 2,5-DHBA into control mice also lowered serum iron and serum Tf iron saturation (Fig. 8A and B).
Supplementation with 2,5-DHBA alleviates iron overload in bdh2 null mice. (A and B) Serum iron indices in control and bdh2 null mice in bdh2 null mice injected with vehicle or 2,5-DHBA. Serum iron and transferrin saturation were measured in nonhemolyzed sera of wt or bdh2 null mice injected with vehicle or 2,5-DHBA. Data are presented as means ± SD. (C) Quantitative determination of tissue iron content (μg/mg of tissue wet weight) in spleen of control or bdh2 null mice injected with vehicle or 2,5-DHBA. At least 3 mice per treatment condition were analyzed. Data are presented as means ± SD. (D) Histological detection of tissue iron in spleen sections from control or bdh2 null mice injected with vehicle control of 2,5-DHBA by Perls staining. Blue staining represents iron accumulation in cells. Representative images from specimens collected from at least 3 mice per treatment condition are shown.
Finally, we assessed iron levels in spleen samples from control or bdh2 null mice injected with 2,5-DHBA or vehicle. Iron content in the spleen of bdh2 null mice was higher than that in control mice (Fig. 8C and D). Injection of 2,5-DHBA reduced the iron content of the spleen in these mice as determined by histological and quantitative analyses (Fig. 8C and D). Combined, these results suggest that 2,5-DHBA supplementation reduces iron overload in the spleen.
Disruption of bdh2 does not alter ketone body metabolism.We next sought to determine the biological significance of BDH2 deficiency in vivo. An earlier study suggested a role for BDH2 in ketone body metabolism. Based on computational modeling, it was proposed that BDH2 binds to and oxidizes cytosolic ketone bodies (28, 39). However, whether BDH2 has a significant role in ketone body metabolism in vivo has not been demonstrated. Therefore, we evaluated levels of ketone bodies in plasma samples obtained from wt and bdh2 null mice, specifically d-β-OH butyric acid, the most abundant ketone in the body. As expected, wt mice displayed basal levels of d-β-OH butyric acid (Fig. 9A). Interestingly, measurement of d-β-OH butyrate in bdh2 null mice yielded levels comparable to that of wt mice (Fig. 9A) (22). Additionally, evaluation of acetoacetate also suggested no ketoacidosis in bdh2 null mice (Fig. 9B) (22). In mammals, ketone bodies are derived from the oxidation of fatty acids, which are then exported to other tissues to be used as fuel (40). Defects in ketone body oxidation result in ketosis and channeling of ketone bodies via coenzyme A (CoA) transferase to form fatty acids. Therefore, we next assessed cholesterol, free fatty acid (FFA), and triglyceride levels in plasma samples from wt and bdh2 null mice. Plasma fatty acid measurements in bdh2 null mice are similar to the levels found in wt mice (22). Thus, multiple lines of evidence suggest that BDH2 deficiency does not impair ketone body metabolism, counter to the computational model's prediction.
Bdh2 deficiency does not alter ketone body metabolism. (A and B) Measurement of d-β-OH butyric acid and acetoacetate levels in plasma of 8-week-old female wt and bdh2 null mice. Data are presented as means ± SD.