Expression of iron-related proteins during infection by bovine herpes virus type-1

Bovine herpesvirus 1 (BHV-1), a dsDNA animal virus, is an economically important pathogen of cattle and the aetiological agent of many types of disease. The efficient replication of a DNA virus is strictly dependent on iron since this metal plays a crucial role in the catalytic center of viral ribonucleotide reductase. Consequently, iron metabolism is an important area for virus/host interaction and a large body of evidence suggests that viral infection is potentially influenced by the iron status of the host. The aim of the present study was to address the effects of BHV-1 on iron metabolism in Madin-Darby bovine kidney (MDBK) cells at different times of post-infection. For this purpose, cell viability, iron regulatory proteins (IRPs) activity and levels, transferrin receptor 1 (TfR-1), ferritin expression and LIP were evaluated. Our data demonstrate that a productive BHV-1 infection in MDBK cells determines an overall decrease of IRPs RNA-binding activity without affecting their expression. As consequence of this modulation, an increased ferritin mRNA translation and a decreased TfR-1 mRNA translation were also observed. Moreover, the LIP level was decreased following viral infection. These results are consistent with the hypothesis that by reducing the iron up-take and by enhancing the sequestration of free iron, animal cells will limit the iron availability for virus proliferation. Therefore, the results presented herein support the view that iron metabolism could be critical for the interaction between DNA viruses, such as BHV-1, and mammalian cells. Delineation of the interplay among pathogen and host may provide new antimicrobial agents.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes.     

2,3,7,8-Tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin regulates Bovine Herpesvirus type 1 induced apoptosis by modulating Bcl-2 family members

Exposure to environmental contaminants, like 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), leads to an increased susceptibility to infectious agents. Infection of bovine cells (MDBK) with Bovine Herpesvirus 1 (BHV-1) anticipates virus-induced apoptosis, suggesting an involvement of TCDD in virus infection. Herein we analyzed the effects of TCDD on apoptotic pathway in MDBK cells infected with BHV-1. After 12 h of infection, TCDD induced a significant increase in apoptotic cells. TCDD caused a dose-dependent up-regulation and anticipated activation of caspases 3, 8 and 9, with respect to unexposed groups. TCDD anticipated cleavage of PARP, compared to controls. Furthermore TCDD increased Bax and Bid levels, and decreased Bcl-2 and Bcl-XL levels. Such events took place earlier in exposed than unexposed cells. These results showed that TCDD influences BHV-1 induced apoptosis through members of Bcl-2 family and up-regulating activation of caspases.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells

Cellular iron metabolism is essentially controlled by the binding of cytosolic iron regulatory proteins (IRP1 or IRP2) to iron-responsive elements (IREs) located on mRNAs coding for proteins involved in iron acquisition, utilization and storage. The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is one of the most potent toxins of current interest that occurs as poisonous chemical in the environment. TCDD exposure has been reported to induce a broad spectrum of toxic and biological responses, including significant changes in gene expression for heme and iron metabolism associated with liver injury. Here, we have investigated the molecular effects of TCDD on the iron metabolism providing the first evidence that administration of the toxin TCDD to mammalian cells affects the maintenance of iron homeostasis. We found that exposure of Madin-Darby Bovine Kidney cell to TCDD caused a divergent modulation of IRP1 and IRP2 RNA-binding capacity. Interestingly, we observed a concomitant IRP1 down-regulation and IRP2 up-regulation thus determining a marked enhancement of transferrin receptor 1 (TfR-1) expression and a biphasic response in ferritin content. The changed ferritin content coupled to TfR-1 induction after TCDD exposure impairs the cellular iron homeostasis, ultimately leading to significant changes in the labile iron pool (LIP) extent. Since important iron requirement changes occur during the regulation of cell growth, it is not surprising that the dioxin-dependent iron metabolism dysregulation herein described may be linked to cell-fate decision, supporting the hypothesis of a central connection among exposure to dioxins and the regulation of critical cellular processes. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

2,3,7,8-Tetrachlorodibenzo-p-dioxin increases Bovine Herpesvirus type-1 (BHV-1) replication in Madin-Darby Bovine Kidney (MDBK) cells in vitro

Dioxin-2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental toxin of current interest. In the last years, higher levels of TCDD than those permitted in UE [European Commission. 2002. European Commission Recommendation 2002/201/CE. Official Gazette, L 67/69] were detected in milk samples from cow, water buffalo, goat, and sheep raised on some areas of Campania Region (South Italy). Dioxin often causes immunosuppression and might render the animal liable to viral infections. In addition, viral infections are able to alter the pattern of dioxin distribution in different organs of the exposed animals. Bovine Herpesvirus type-1 (BHV-1) is a widespread pathogen, which causes infectious rhinotracheitis and infectious pustular vulvovaginitis in cattle. Herein, we have studied the effects of TCDD and BHV-1 infection, in Madin-Darby Bovine Kidney (MDBK) cells, alone as well as in association, so as cellular proliferation, apoptosis, and virus replication. We have observed an increase in cell viability of confluent monolayers at low TCDD concentrations. TCDD treated cells demonstrated increased viability compared to controls as evaluated by MTT test. TCDD exposure increased cell proliferation but induced no changes on apoptosis. Cells exposed to TCDD along with BHV-1 showed a dose-dependent increase in cytopathy, represented by ample syncytia formation with the elimination of the cellular sheets and increased viral titer. These results suggest that TCDD increases viral replication in MDBK cells while BHV-1 further decreases viability of TCDD exposed cells. Since very low concentrations (0.01 pg/ml) are sufficient to augment BHV-1 titer, TCDD may contribute to reactivate BHV-1 from latency, leading to recurrent disease and increase virus transmission.     

Transactivator protein BICP0 of bovine herpesvirus 1 (BHV-1) is blocked by prostaglandin D2 (PGD2), which points to a mechanism for PGD2-mediated inhibition of BHV-1 replication

The immediate-early protein, BICP0, of bovine herpesvirus 1 (BHV-1) transactivates a variety of viral and cellular genes. In a yeast two-hybrid cDNA library screening, we found that lipocalin-type prostaglandin D synthase, which catalyzes the production of prostaglandin D(2) (PGD(2)), is a cellular target of BICP0. We observed that, during wild-type BHV-1 infection, PGD(2) levels were increased intracellularly and decreased in the medium. These effects were absent upon infection with recombinant BHV-1 expressing beta-galactosidase instead of BICP0 (A2G2). Transient-expression assays showed that BICP0 alone caused a significant increase in PGD(2) levels in the cell. PGD(2) repressed BHV-1 replication in cultured cells. Antiviral activities of prostaglandins have been documented long ago, but their mode of action remains to be clarified. Here we provide evidence that PGD(2) impairs the transactivation ability of BICP0 that is necessary for efficient virus replication.     

Hepatitis C Virus Infection Causes Iron Deficiency in Huh7.5.1 Cells

Patients with chronic hepatitis C virus (HCV) infection frequently develop systemic iron overload, which exacerbates morbidity. Nevertheless, iron inhibits HCV replication in cell culture models and thereby exerts antiviral activity. We hypothesized that the cellular iron status is crucial for the establishment of HCV infection. We show that HCV infection of permissive Huh7.5.1 hepatoma cells promotes an iron deficient phenotype. Thus, HCV leads to increased iron regulatory protein (IRP) activity, accumulation of IRP2 and suppression of transferrin receptor 1 (TfR1) and divalent metal transporter 1 (DMT1) in the host. These data suggest that HCV regulates cellular iron levels to bypass iron-mediated inhibition in viral replication.     

Bovine herpesvirus 1 tegument protein VP22 interacts with histones, and the carboxyl terminus of VP22 is required for nuclear localization

The bovine herpesvirus 1 (BHV-1) UL49 gene encodes a viral tegument protein termed VP22. UL49 homologs are conserved among alphaherpesviruses. Interestingly, the BHV-1 VP22 deletion mutant virus is asymptomatic and avirulent in infected cattle but produces only a slight reduction in titer in vitro. Attenuation of the BHV-1 VP22 deletion mutant virus in vivo suggests that VP22 plays a functional role in BHV-1 replication. In herpes simplex virus type 1, the VP22 homolog was previously shown to interact with another tegument protein,VP16, the alpha-transinducing factor in vitro. In this report, we show that (i) the nuclear targeting of VP22 is independent of other viral factors, (ii) the carboxyl terminus of VP22 is required for its nuclear localization, (iii) VP22 associates with histones and nucleosomes, (iv) an antihistone monoclonal antibody cross-reacts with VP22, and (v) acetylation of histone H4 is decreased in VP22-expressing cells as well as virus-infected cells. Our data suggest that VP22 may have a modulatory function during BHV-1 infection.     

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.     

Bovine cells expressing bovine herpesvirus 1 (BHV-1) glycoprotein IV resist infection by BHV-1, herpes simplex virus, and pseudorabies virus.

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.     

Serologic survey of white-tailed deer on Anticosti Island, Quebec for bovine herpesvirus 1, bovine viral diarrhea, and parainfluenza 3.

In 1985 unusual mortality was observed among the 3- to 4-yr-old white-tailed deer (Odocoileus virginianus) on Anticosti Island, Québec (Canada). A viral pathogen was suspected to be the cause of the deaths. Thus, a serologic survey for bovine herpesvirus 1 (BHV-1), bovine viral diarrhea (BVD) virus and parainfluenza-3 (PI-3) virus was conducted. We examined 396 deer sera from 1985. Results indicated that the high mortality mainly afflicted 3- to 4-yr-old deer. In 1985, 57% of deer sampled were seropositive for viral neutralizing antibodies against BHV-1. Prevalences decreased over the next 2 yr of the survey. Prevalence of antibodies against PI-3 virus, determined by hemagglutination inhibition test, remained high (82% to 84%) for the 3 yr period. No deer were seropositive for neutralizing antibodies against BVD virus during the survey period. Analysis of antibodies against BHV-1 and PI-3 viruses according to sex, age and antibody titers revealed that an epizootic BHV-1 infection occurred in 1985; PI-3 infection appears to be enzootic in Anticosti deer.     

Interstrain differences in the progression of nonalcoholic steatohepatitis to fibrosis in mice are associated with altered hepatic iron metabolism

Nonalcoholic fatty liver disease (NAFLD) is a major health problem worldwide. Currently, there is a lack of conclusive information to clarify the molecular events and mechanisms responsible for the progression of NAFLD to fibrosis and cirrhosis and, more importantly, for differences in interindividual disease severity. The aim of this study was to investigate a role of interindividual differences in iron metabolism among inbred mouse strains in the pathogenesis and severity of fibrosis in a model of NAFLD. Feeding male A/J, 129S1/SvImJ and WSB/EiJ mice a choline- and folate-deficient diet caused NAFLD-associated liver injury and iron metabolism abnormalities, especially in WSB/EiJ mice. NAFLD-associated fibrogenesis was correlated with a marked strain- and injury-dependent increase in the expression of iron metabolism genes, especially transferrin receptor (Tfrc), ferritin heavy chain (Fth1), and solute carrier family 40 (iron-regulated transporter), member 1 (Slc40a1, Fpn1) and their related proteins, and pronounced down-regulation of the iron regulatory protein 1 (IRP1), with the magnitude being A/J<129S1/SvImJ<WSB/EiJ. Mechanistically, down-regulation of IRP1 was linked to an increased expression of microRNAs miR-200a and miR-223, which was negatively correlated with IRP1. The results of this study demonstrate that the interstrain variability in the extent of fibrogenesis was associated with a strain-dependent deregulation of hepatic iron homeostasis.