MG-132 interferes with iron cellular homeostasis and alters virulence of bovine herpesvirus 1
Filomena Fiorito a,*, 1, Carlo Irace b, 1, Francesca Paola Nocera a, Marialuisa Piccolo b, Maria Grazia Ferraro b, Roberto Ciampaglia b, Gian Carlo Tenore b, Rita Santamaria b,*,
Luisa De Martino a
a Department of Veterinary Medicine and Animal Production, University of Naples Federico II, Naples, Italy
b Department of Pharmacy, University of Naples Federico II, Naples, Italy
A R T I C L E I N F O
Keywords: BoHV-1 MG-132
Ferritin TfR-1
Total iron content Virus yield
A B S T R A C T
Bovine herpesvirus 1 (BoHV-1) requires an iron-replete cell host to replicate efficiently. BoHV-1 infection pro- vokes an increase in ferritin levels and a decrease of transferrin receptor 1 (TfR-1) expression, ultimately lowering iron pool extent. Thus, cells try to limit iron availability for virus spread. It has been demonstrated that MG-132, a proteasome inhibitor, reduces BoHV-1 release. Since ferritin, the major iron storage protein in mammalian cells, undergoes proteasome-mediated degradation, herein, the influence of MG-132 on iron metabolism during BoHV-1 infection was examined.
Following infection in bovine cells (MDBK), MG-132 reduced cell death and viral yield. Western blot analysis showed a significant ferritin accumulation, likely due to the inhibition of its proteasome-mediated degradation pathway. In addition, the concomitant down-regulation of TfR-1 expression, observed during infection, was counteracted by proteasome inhibitor. This trend may be explained by enhanced acidic vesicular organelles, detected by acridine orange staining, determining a reduction of intracellular pH, that promotes new synthesis of
TfR-1 degraded in a recycling pathway. In addition, MG-132 influences cellular iron distribution during BoHV-1 infection, as revealed by Perls’ Prussian blue staining. However, cellular iron content, evaluated by Atomic Absorption Spectrophotometry, resulted essentially unaltered.
These findings reveal that MG-132 may contribute to limit cellular iron availability for virus replication thereby enhancing cell survival.
⦁ Introduction
Bovine herpesvirus 1 (BoHV-1), a member of the alphaherpesvirus subfamily, is an important pathogen responsible for significant economic losses to the cattle industry (Muylkens et al., 2007; Jones, 2019). It can cause infectious bovine rhinotracheitis (IBR), conjunctivitis, abortions or complicated polymicrobial infections due to its immunosuppressive properties, leading to pneumonia and occasionally to death. In acute disease, BoHV-1 initiates infection in mucosal surfaces and causes high amount of programmed cell death. Then, by cell-cell spread, BoHV-1 establishes latency in sensory neurons of the infected host. Reac- tivation from latency can be stimulated by increased levels of cortico- steroids resulting in virus shedding and spread to susceptible hosts
(Muylkens et al., 2007; Jones, 2019).
Infection in permissive cells (Madin Darby bovine kidney, MDBK) with BoHV-1 provokes cell death, in part due to apoptosis. It takes place during the late stages of infection as a result of activation of caspases, via modulation of Bcl-2 family members (Devireddy and Jones, 1999; Fiorito et al., 2008a, 2014, 2017b, 2017c).
Previous studies have established that BoHV-1, a double-stranded DNA virus, requires iron-replete host to replicate efficiently, as this metal plays a critical role in the catalytic center of viral ribonucleotide reductase (Lamarche et al., 1996). Iron metabolism is an important area for virus/host interaction and that viral infection is potentially influ- enced by the iron status of the host (Wessling-Resnick, 2018). Though this metal is essential for all living cells, excess of free iron can be toxic
* Corresponding authors.
E-mail addresses: [email protected] (F. Fiorito), [email protected] (R. Santamaria).
1 Equally contributed first authors.
https://doi.org/10.1016/j.rvsc.2021.04.023
Received 27 January 2021; Received in revised form 2 April 2021; Accepted 19 April 2021
Available online 21 April 2021
0034-5288/© 2021 Elsevier Ltd. All rights reserved.
due to oxidative stress and the generation of free radicals that in turn can damage lipids, proteins, DNA, and other cellular components. On the contrary, iron deficiency abrogates the activity of iron-dependent pro- teins and impairs some cellular processes, triggering cellular growth arrest and death (Muckenthaler et al., 2017). As both iron deficiency and iron overload are harmful to the cell, iron homeostasis must be tightly controlled by modulating the expression of proteins involved in cellular iron uptake, utilization, storage and export. Specifically, iron is trans- ported bound to serum transferrin, taken up by cells through the transferrin binding to the transferrin receptor 1 (TfR1), stored in the ferritin (H/L-Fer), a protein that accumulates the intracellular iron in a redox-inactive form, and exported via the transmembrane protein fer- roportin. All these proteins are pH-dependent and regulated mainly at the post-transcriptional level by Iron Regulatory Proteins (IRP1 and IRP2). IRPs activity is in turn dependent on the chelatable or labile iron pool (LIP) (Muckenthaler et al., 2017). As both host and pathogen require iron, host innate immune response carefully orchestrates iron metabolism control to limit its availability to fight infection by invading microorganisms (Wessling-Resnick, 2018). Indeed, BoHV-1 infection in MDBK cells, an epithelial cell line used routinely for generating virus stocks, leads to an increase in ferritin levels accompanied by reduction of TfR-1 expression, finally lowering the levels of LIP (Maffettone et al., 2008; Fiorito et al., 2013). Therefore, by the regulating LIP, mammalian cells could limit iron availability required for spread of virus. Indeed, infection in the presence of ferric ammonium citrate induced an enhancement in BoHV-1 replication and in expression of bICP0, the major regulatory protein in lytic cycle of BoHV-1 (Fiorito et al., 2013). Viruses utilize several cellular signaling pathways in the course of their replication. The ubiquitin-proteasome system seems to be a cellular pathway that herpesviruses use for their own benefit. Indeed, it has been shown that proteasome inhibitors can alter herpesviruses replication by playing significant roles in replication cycle. Certainly, proteasome in- hibitors decrease immediate early and late proteins expression in HSV-1 (La Frazia et al., 2006), play a role in post-entry stages of HSV-1 infec- tion (Everett et al., 1998; Eom and Lehman, 2003; Burch and Weller, 2004; Hagglund and Roizman, 2004; Dai-Ju et al., 2006) and facilitate the entry of HSV-1 at a post-penetration stage (Delboy et al., 2008). Recently, it has been proven that BoHV-1 requires proteasome-mediated proteolysis for successful entry and infection in MDBK cells (Pastenkos et al., 2018). Following BoHV-1 infection in MDBK cells, MG-132, a peptide aldehyde that competitively prevents the degradative activity of the proteasome, inhibits virus-induced apoptosis and stimulates auto- phagy (Fiorito et al., 2017a). In this contest, bICP0 acts as a ubiquitin ligase and its activating effect was attenuated by MG-132 (Diao et al., 2005). In addition, bICP0, which is constitutively expressed during infection (Inman et al., 2001; Fiorito et al., 2010), is completely inhibited by MG-132 (Fiorito et al., 2017a). Intriguingly, the efficient virus release triggered by apoptosis is significantly lowered by protea- some inhibitor, suggesting that MG-132 might counteract BoHV-1 pro- ductive infection (Fiorito et al., 2017a). Hence, given that the cellular iron homeostasis represents a critical point for virus-host interaction, in this study we investigated the regulation of iron metabolism in bovine cells during BoHV-1 infection in the presence of MG-132, focusing on its
possible impact on infection worsening.
⦁ Materials and methods
⦁ Cell cultures and virus infection
MDBK cells (American Type Culture Collection, CCL22) were cultured in Dulbecco’s modified Eagle’s minimal essential medium (DMEM), supplemented with 2% foetal calf serum (FCS), 1% penicillin/
streptomycin, 1% L-glutamine, 0.2% sodium pyruvate and 0.1% tylosin. Cells were incubated at 37 ◦C (in 5% CO2/95% air). MDBK cell line was
maintained free of mycoplasma and bovine viral diarrhoea virus, as previously described (Fiorito et al., 2011). BoHV-1, Cooper strain, was
used throughout the study. Virus stocks were grown on MDBK cells. In addition, MDBK cells were also used for virus titer analysis (Fiorito et al., 2008b, 2020).
The proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132) (Calbiochem, 474,790), dissolved in dimethyl sulfoxide, was
added to the medium to a final concentration of 1 μM, as described
(Fiorito et al., 2017a). Confluent monolayers of MDBK cells, washed with DMEM, were infected or not with BoHV-1, at multiplicity of infection (MOI) of 5, in the presence or not of MG-132. So, we had four
groups: uninfected or infected cells, MG-132 treated infected and un- infected cells. After 1 h of adsorption at 37 ◦C, MDBK cells were incu-
bated and processed at 0, 1, 4, 8, 12, 24 and 48 h post infection (p.i.). The virus was in culture medium through the course of the experiment.
⦁ Cell viability
×
±
Cell viability was assessed by TB exclusion test, as described (Fiorito et al., 2008a). Briefly, after the indicated times of infection, cells were collected by trypsinization. Then, cell suspension was mixed with 0.2% Trypan-blue (Sigma) in 1 phosphate-buffered saline (PBS) (1:1) for 10 min. Then, cells were counted by TC20 automated cell counter (Bio- Rad). Cell viability was calculated as percentage of live cells over total cells number. Obtained results are the mean S.E.M. of three inde- pendent experiments carried out in duplicate.
⦁ Evaluation of cellular acidic vesicular organelles
In order to identify the acidic vesicular organelles (AVOs) acridine orange staining was performed (Fiorito et al., 2017a). Confluent monolayers of MDBK cells, grown on glass coverslip, were washed with DMEM, infected or not with BoHV-1 at MOI of 5, in the presence or in
×
absence of MG-132 and incubated at 37 ◦C. At 4, 24 and 48 h p.i., cells
were washed with PBS, then stained with acridine orange (Sigma, A6014) for 15 min. After that, stained cells placed on a microscope slide, were observed by fluorescence microscopy (Nikon).
⦁ Protein extraction and Western blot analysis
Protein extraction and Western blot analysis were performed as previously reported (Fiorito et al., 2008a; Santamaria et al., 2011). After 48 h of BoHV-1 infection, in the presence or not of MG-132, cells were
collected by scraping and low-speed centrifugation. Then, cell pellets were lysed at 4 ◦C for 30 min in a buffer containing 20 mM Tris-HCl, pH
× —
7.4, 5 mM EDTA, 150 mM NaCl, 10 mM NP-40, 5% (v/v) glycerol and protease inhibitor tablets (Roche). The supernatant fraction, obtained by centrifugation at 15,000 g for 10 min at 4 ◦C, was stored at 80 ◦C.
Protein concentration was measured by the Bio-Rad protein assay (Bio- Rad, Milan, Italy). Samples, containing 50 μg of proteins, were dena- tured, separated on a 10% SDS-polyacrylamide gel and then electro-
transferred onto a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, Buckinghamshire, UK) by using a Bio-Rad Transblot (Bio-Rad). Then, proteins were visualized on the membranes by reversible staining with Ponceau-S solution and de-stained in PBS. After that, the membranes were blocked at room temperature in milk buffer
×
[1 PBS, 0.2% (v/v) Tween-20, 5–10% (w/v) non-fat dry milk] and incubated at 4 ◦C overnight with anti-bICP0 polyclonal rabbit (a.a.
663–676) serum (1:800), kindly provided by Prof. M. Schwyzer (Uni- versity of Zurich, Switzerland), rabbit polyclonal antibody to human ferritin (Dako Cytomation, Glostrup, Denmark) (1:1000), and with
+
mouse monoclonal antibody to human transferrin receptor 1 (Zymed Laboratories Inc., CA) (1:1000). Afterward, the membranes were incu- bated for 90 min at room temperature with peroxidase-conjugated goat anti-rabbit IgG, or peroxidase-conjugated goat antimouse IgG IgM (Jackson ImmunoResearch Laboratories, Baltimore Pike, West Grove, PA). The developed immunocomplexes were visualized by using chemiluminescent Western blotting detection reagents (ECL, Amersham
Biosciences), then examined by an imaging system (ImageQuantTM
400, GE Healthcare Life Sciences). Densitometric analysis was per- formed by using a GS-800 imaging densitometer (Bio-Rad). β-actin (Sigma–Aldrich) was used as internal control to normalize the results.
⦁ Perls’ Prussian blue iron staining
Non-heme iron (predominantly in the Fe3+ form) can be visualized in
cells by the classic Perl’s iron stain, in which soluble ferrocyanide reacts with the tissue Fe3+ to form crystals making an insoluble Prussian blue
dye (Meguro et al., 2007). MDBK cells, exposed or not to MG-132, were infected with BoHV-1, and after 48 h of infection, cells were washed twice with PBS, fixed with 95% ethanol, drained and dried. Afterward,
cells were stained with Prussian blue Perls’ staining (Bio-Optica, 04–180,807/L), according to the manufacturer’s instructions and as
previously reported (Meli et al., 2013).
⦁ Determination of cellular iron content
×
MDBK cells (1.5 106) exposed or not to MG-132, were infected with BoHV-1, and after 48 h of infection, cell pellets were collected by centrifugation. For digestion, samples were treated with ultra-pure concentrated nitric acid and 30% hydrogen peroxide, and subjected to digestion in a microwave digestion apparatus (MW-AD, Ethos EZ mi- crowave digester, Mileston, Shelton, CT, USA). Samples were trans- ferred to TFM®PTFE vessels, and 6 mL of concentrated HNO3 (14.33
mol/L) and 1 mL of 30% H2O2 were added. The heating program for digestion was: 160 ◦C for 5 min using 80% of microwave power; 190 ◦C
for 10 min using 90% of microwave power; 50 ◦C for 11 min. Final so- lutions were diluted up to 25 mL with water. Analysis for iron deter- mination was performed by Atomic Absorption Spectrophotometry according to method 900.02 (AOAC International, 1995) by mineral-
izing the sample at 550 ◦C for 4 h in muffle furnace (Heraeus K1251F,
Thermo Scientific, Southend-on-Sea, UK). The analysis was performed by using an AA-6300 spectrophotometer (Shimadzu, Columbia, MD, USA) equipped with an ASC-6100 autosampler (Shimadzu, Columbia, MD, USA) and a GFA-EX7i graphite furnace atomizer (Shimadzu, Columbia, MD, USA). The working conditions were the following: wavelength, 248.3 nm; slit width, 0.5 nm; lamp current, 5 mA; Gas, Argon.
⦁ Virus production
MDBK cells were infected with BoHV-1 at MOI 5, in the presence or not of MG-132, and incubated at 37 ◦C. At 0, 1, 4, 8, 12, 24 and 48 h of
infection, virus titration and viral cytopathic effects (CPE) were per-
formed. For virus titration, cell extracts, obtained by three cycles of freezing and thawing, were collected and stored in aliquots at —80 ◦C. Virus titration was assayed by TCID50 method, according to Reed and
Muench (1938), as described (Fiorito et al., 2020). To evaluate CPE, at indicated times of infection, cells were examined under light microscope.
⦁ Statistical analysis
±
Data are presented as mean S.E.M. One-way ANOVA with Tukey’s post-test was carried out by using GraphPad InStat Version 3.00 for Windows 95 (GraphPad Software, San Diego, CA). P value <0.05 was considered statistically significant.
⦁ Results
⦁ MG-132 decreases cell death as well as virus yield during BoHV-1 infection and inhibits bICP0 expression
In order to explore the biological effect of MG-132 during BoHV-1
infection in MDBK cells, we first assessed cell viability by Trypan Blue
(TB) exclusion test. From 4 h to 48 h p.i., MG-132, at the non-toxic concentration of 1 μM (Fiorito et al., 2017a), significantly decreased the cell death in MDBK cells during BoHV-1 infection (Fig. 1A).
Following BoHV-1 infection in MDBK cells, we analyzed virus titra- tion and viral cytopathic effect (CPE) to confirm the MG-132 biological effect on virus production. For this reason, MDBK cells were infected with BoHV-1, in the presence or not of MG-132, and processed. Analysis of viral growth curves showed similar kinetics, and the levels of viral yield in MG-132 treated cells were considerably lower than in MG-132 untreated cells (Fig. 1B). Indeed, from 24 to 48 h after infection, in
MG-132 treated infected cells a significant (P < 0.05) decrease in viral
yield was detected, and the reduction was substantial as the difference in virus titer, expressed in Log, was about 1/10 in viral particles. In addi- tion, after 48 h of infection, CPE, evaluated by ample syncytia formation along with elimination of cellular sheet, was marked in infected cells, whereas in MG-132 treated infected cells an appreciable reduction of CPE was detected (see Fig. 5). Moreover, we confirmed that MG-132 completely inhibited protein expression of bICP0 (Fig. 1C).
⦁ MG-132 increases the acidity of vesicular organelles during BoHV-1 infection
To evaluate the effect of MG-132 on acidity compartments during BoHV-1 infection, uninfected or infected cells, MG-132 treated infected and uninfected cells were cultured for 48 h and then stained by using lysosomotropic agent acridine orange for the detection of acidic vesic- ular organelles (AVOs). Specifically, acridine orange allows to distin- guish possible acidic vesicles with different internal acidity. Indeed, acidity is an important factor in accumulation of AVOs, due to dye protonation. Briefly, the vesicles with higher acidity stimulate AVOs protonation. Consequently, the intensely acidic AVOs may display or- ange or red fluorescence, whereas the weakly acidic AVOs should appear green or yellow (Pierzyn´ska-Mach et al., 2014). As shown in Fig. 2, both cytoplasm and nucleolus of uninfected cells fluoresced bright green and dim red by acridine orange staining. The same fluorescent pattern also occurs for MG-132 treated uninfected cells, as well as for infected cells. In contrast, incubation with MG-132 of infected cells significantly enhanced acidic compartments which exhibited orange luminescence due to increased AVOs protonation.
⦁ MG-132 changes the transferrin receptor (TfR-1) levels during BoHV-1 infection
To evaluate the expression of the key proteins involved in the regulation of cellular iron metabolism, we examined the levels of TfR-1 by immunoblot analysis on lysates obtained from uninfected or infected cells, MG-132 treated infected and uninfected cells cultured for 48 h. As depicted in Fig. 3, TfR-1 protein was clearly expressed in uninfected cells, whereas MG-132 treatment significantly reduced the protein levels.
During BoHV-1 infection a down-regulation of TfR-1 has been demonstrated. Interestingly, TfR-1 levels were significantly (P < 0.01) enhanced in MG-132 treated infected cells, showing an increase of about
two-fold with respect to infected cells (Fig. 3).
⦁ MG-132 up-regulates the ferritin levels during BoHV-1 infection
We then examined the expression of ferritin by Western blot analysis. As shown in Fig. 4, the H/L ferritin levels in MDBK cells, were well represented.
A noticeable (P < 0.001) increase of ferritin content was observed in
MG-132 treated cells. Viral infection determined a strong increase in ferritin expression, further increased by proteasome inhibition (Fig. 4).
Fig. 1. MG-132 decreases cell death and virus yield during BoHV-1 infection and inhibits bICP0 expression. (A) Uninfected (CC) or infected cells (BoHV-1), MG-132 treated infected (BoHV-1 + MG-132) and MG-132 treated uninfected cells (MG-132) at 0, 4, 24 or 48 h of treatment cells were stained with Trypan-blue and scored with a Burker chamber under a light microscope; (B) For viral growth curves, infected cells (BoHV-1) and MG-132 treated infected (BoHV-1 + MG-132) at different times p.i., cells were frozen. Virus titer was assayed by TCID50 method and reported as Log TCID50/mL. Data are presented as mean ± S.E.M. of three independent
experiments performed in duplicate. Significant differences between infected untreated and infected MG-132-treated groups are indicated by probability P. *P < 0.05, **P < 0.01 and ***P < 0.001. (C) Cell lysates were prepared from uninfected (Control) or infected cells (BoHV-1), MG-132 treated infected and uninfected cells at the indicated times, Western blot analysis was performed by using an antibody which specifically recognized bICP0 or β-actin. Results are the mean of three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. MG-132 stimulates the formation of acidic vesicular organelles in MDBK cells during BoHV-1 infection. Photomicrographs showing untreated uninfected (Control), or infected cells (BoHV-1), MG-132 treated infected (BoHV-1 + MG-132) and uninfected cells (MG-132), stained with acridine orange. After 48 of infection, compared to the control cells, MG-132 stimulated the formation of acidic vesicular organelles, (magnification×1000).
Fig. 3. MG-132 changes TfR-1 levels during infection in MDBK cells. (A)Cell lysate was prepared from uninfected (Control) or infected cells (BoHV-1), MG- 132 treated infected (BoHV-1 + MG-132) and uninfected cells (MG-132) at 48 h
p.i., Western blot analysis was performed with an antibody which specifically
recognized TfR-1. β-actin was used as an internal loading control. Blots are representative of three separate experiments. (B). Densitometry analysis. Re- sults are expressed as the mean ± S.E.M. of three separate experiments. Sig- nificant differences between groups are indicated by probability P. Comparing
Control group to all infected groups (untreated and treated with MG-132) ***P
< 0.001. Comparing MG-132 treated infected groups to untreated infected groups **P < 0.01.
⦁ MG-132 influences cellular iron distribution during BoHV-1 infection
To assess the distribution of intracellular iron, we performed cyto- chemical analysis in MDBK cells by Prussian blue Perls’ staining, revealing “non-heme” iron, including soluble free iron, and iron-protein deposits, such as ferritin and hemosiderin (Meguro et al., 2007). To this
aim, uninfected or infected cells, MG-132 treated infected and unin- fected cells were incubated for 48 h and processed. As shown in Fig. 5, during BoHV-1 infection in uninfected cells, evidences of iron accumu- lation were substantial, probably as ferritin and hemosiderin deposits.
MG-132 treatment during infection further enhances iron accumu-
×
lation. These outcomes were substantially in accordance with ferritin expression. In addition, we evaluated total iron content following treatments in about 1.5 106 cells (total cell number, TCN, as mean
±
values standard deviation of three separate experiments) by using
atomic absorption spectrophotometry analysis. Surprisingly, total cellular iron amount essentially remained unaltered (P > 0.05) in infected and in MG-132 treated infected cells, compared to uninfected
and MG-132 treated cells (see Table 1).
⦁ Discussion
The ubiquitin-proteasome system, recognized as the major pathway of intracellular protein degradation in eukaryotes, seems to be a cellular pathway that viruses use for their own needs. In this frame, BoHV-1 requires proteasome-mediated proteolysis for a successful entry and infection of MDBK cells (Pastenkos et al., 2018). Recently, it has been shown that proteasome inhibitor MG-132 may influence BoHV-1 repli- cation by playing a significant role in replication cycle (Fiorito et al.,
Fig. 4. MG-132 up-regulates ferritin (H/L) levels during infection in MDBK cells. (A) Cell lysate was prepared from uninfected (Control) or infected cells
(BoHV-1), MG-132 treated infected (BoHV-1 + MG-132) and uninfected cells
(MG-132), and, at 48 h p.i., Western blot analysis was performed with an antibody which specifically recognized H/L ferritin. β-actin was used as an internal loading control. Blots are representative of three separate experiments. (B). Densitometry analysis. Results are expressed as the mean ± S.E.M. of three separate experiments. Significant differences between groups are indicated by
probability P. Control group and all infected groups (untreated and treated with MG-132) ***P < 0.001. No significant differences were detected comparing MG-132-treated infected groups to untreated infected groups.
2017a). Herein, we explored the influence of MG-132 on iron meta- bolism during BoHV-1 infection. In our in vitro experimental model, MG- 132 significantly reduced BoHV-1-dependent cell death as previously reported (Fiorito et al., 2017a). Furthermore, we observed a reduction of virus replication in MG-132 treated infected cells, through a decrease in CPE, as well as a significant decline in virus yield. These results, accompanied by complete inhibition of bICP0 expression, indicated that the virus release was significantly decreased by proteasome inhibitor MG-132.
Proteasome system is involved in the catabolic regulation of proteins implicated in the maintenance of cellular iron homeostasis (Rudeck et al., 2000; Tachiyama et al., 2011). Recent studies indicate mamma- lian cells strategically operate to limit iron availability during the innate immune response to counteract infection (Wessling-Resnick, 2018). Alterations in iron homeostasis may have a role in the pathogenesis of several virus infections, as well as, changes in host iron status could influence the virulence of many microorganisms (Wessling-Resnick, 2018). Emerging evidence suggests that many viruses use the TfR-1, which represents the main gate for cellular iron import, to enter cells. For example, feline panleukopenia virus and canine parvovirus are able to bind TfR-1 (Parker et al., 2001; Palermo et al., 2003; Lee et al., 2019). Herein, our results showed that MG-132 decreased the levels of TfR-1 protein at the cell surface. Similarly, BoHV-1 infection induced a sig- nificant down-regulation of TfR-1 expression, as previously described (Maffettone et al., 2008; Fiorito et al., 2013). Interestingly, in MG-132 treated infected cells, the levels of TfR-1, albeit reduced, were
Fig. 5. MG-132 influences cellular iron distribution during infection in MDBK cells. Representative mi- crophotographs of uninfected (Control) or infected
cells (BoHV-1), MG-132 treated infected (BoHV-1 +
MG-132) and uninfected cells (MG-132), stained with Perls’ Prussian Blue after 48 h of infection. Iron de- posits are indicated by arrows. Slides were visualized
at phase contrast microscope (Leica) (magnification x 150). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 1
Determination of total iron content by atomic absorption spectrophotometry.
Control MG-132 BoHV-1 BoHV-1 + MG-
132
degradation of TfR in lysosomes (Tachiyama et al., 2011). We thereby presume that the ferritin up-regulation could reduce both iron pool extent and iron-induced degradation of TfR-1. This may explain the enhanced levels of TfR-1 observed during infection in the presence of MG-132, compared to those detected in the absence of the proteasome
28.48 ng/TNC* ±
0.98
27.59 ng/TNC ±
2.37
27.68 ng/TNC ±
2.11
29.47 ng/TNC ±
1.04
inhibitor.
It is known that ubiquitination of membrane proteins triggers their
Footnote: Mean values ± standard deviation obtained by Atomic Absorption Spectrophotometry analysis. *Total cell number (1.5 × 106).
significantly doubled compared to those in MG-132 untreated infected cells.
Ferritin is a cytosolic protein storing iron in a redox-inactive form but at the same time rapidly bioavailable, thereby protecting cells from iron toxicity. It is well known that ferritin can be degraded by the ubiquitin- proteasome pathway. The regulation of this catabolic machinery may thereby have a deep impact on virus cycle requiring iron for productive infection in mammalian cells (Wessling-Resnick, 2018). Here, we found ferritin was up-regulated by MG-132 proteasome inhibitor, and a sig- nificant increase of ferritin was observed after infection, as previously described (Maffettone et al., 2008; Fiorito et al., 2013). In addition, an enhancement in ferritin expression was detected in MG-132 treated infected cells. Interestingly, cellular iron content, as evaluated by Atomic Absorption Spectrophotometry, was unaltered in all studied groups compared to control cells, as indicated in Table 1. This outcome could be due to simultaneous induction of ferritin expression and TfR-1 level reduction. Thus, the cell, in response to viral infection and/or proteasome inhibition, maintained iron balance. In addition, in line with enhanced levels of the ferritin, changes in cellular distribution of iron
were observed during BoHV-1 infection. Indeed, Perls’ staining revealed
iron accumulation, probably due to ferritin and hemosiderin, in infected cells, especially when exposed to MG-132. This finding is consistent with proteasome-mediated ferritin degradation (Rudeck et al., 2000). The use
of proteasome inhibitors results in ferritin‑iron accumulation, compa-
rable with what we found after treatment with MG-132. In these con- ditions, ferritin‑iron accumulation enhances both ubiquitination and
endocytosis and subsequent lysosomal degradation. However, while the recycling route and iron-regulated expression of TfR-1 are well charac- terized, the mechanism regulating the TfR-1 degradation remains largely unknown. Indeed, transferrin and TfR-1 are involved in delivery and uptake of iron into the cell, respectively. After binding of transferrin, TfR-1 is internalized and transported to early endosomes, where iron is released, then TfR-1 is transported to recycling endosomes and finally to the plasma membrane (recycling pathway). Since TfR-1 protein could be damaged during recycling process, for example by acidic pH in early/ recycling endosomes, TfR-1 is degraded and newly synthesized (Max- field and McGraw, 2004). This process could further clarify the increased levels of TfR-1 detected in MG-132 treated infected cells compared to MG-132 untreated infected cells. Moreover, we hypothe- size that, during infection, MG-132 could induce an increase of intra- cellular acidity. Indeed, in eukaryotic cells, organelles contribute to regulate intracellular pH (Casey et al., 2010). In acridine orange-stained cells, the cytoplasm and nucleolus fluoresce bright green and dim red, whereas acidic compartments fluoresce bright red (Traganos and Dar- zynkiewicz, 1994; Longo et al., 2009; De Martino et al., 2010). The in- tensity of orange or red fluorescence is proportional to the acidity degree of the cellular acidic compartment (Pierzyn´ska-Mach et al., 2014) and MG-132 enhanced acidic compartments.
Taken together, our results demonstrated that the inhibition of proteasome pathway by MG-132 could further limit iron availability for BoHV-1 replication in MDBK cells, as illustrated in the Diagram (Fig. 6).
⦁ Conclusions
Our findings reveal that proteasome inhibitor MG-132 reduces
Fig. 6. Schematic diagram illustrating the hypothesized mechanisms as to how MG-132 exerts its effects on the iron metabolism during BoHV-1 infection, resulting in decreased virus yield in MDBK cells.
BoHV-1 release in bovine cells and provokes ferritin accumulation during infection. Moreover, down-regulation of transferrin receptor 1, detected during BoHV-1 infection, is counteracted by MG-132. In addition, during infection MG-132 can influence cellular iron distribu- tion without altering intracellular iron amount.
Taken together, our findings reveal that proteasome inhibitor MG-
132 could contribute to limit cellular iron availability for virus repli-
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cation thereby enhancing cell survival. Thus, proteasome pathway
https://doi.org/10.1002/jcb.22700.
`
modulation represents a potential target to be further explored for the development of novel antiviral strategies.
Further investigations are needed to clarify the mechanism by which the proteasome is involved in the regulation of iron metabolism in BoHV-1 infection.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Authors’ contributions
The study was conceived, designed and critically revised by FF, CI, RS and LDM. Data collection, study execution, data analysis and the drafted manuscript were done by FF, CI, FPN, GCT, RC, MP, MGF and RS. All authors have read and approved the final manuscript.
Declaration of competing interest
None.
Acknowledgments
The authors would like to thank Gaetano Vitagliano for technical support and Prof. Martin Schwyzer (Institute of Virology, Faculty of Veterinary Medicine, University of Zürich, Switzerland) for polyclonal rabbit anti-bICP0 serum.
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