Autophagic response in the Rabbit Hemorrhagic Disease, an animal model of virally-induced fulminant hepatic failure
© Vallejo et al.; licensee BioMed Central Ltd. 2014
Received: 4 November 2013
Accepted: 22 January 2014
Published: 4 February 2014
The Rabbit Hemorrhagic Disease Virus (RHDV) induces a severe disease that fulfils many requirements of an animal model of fulminant hepatic failure. However, a better knowledge of molecular mechanisms contributing to liver damage is required, and it is unknown whether the RHDV induces liver autophagy and how it relates to apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize their role in the context of RHDV pathogenesis. Rabbits were infected with 2 × 104 hemmaglutination units of a RHDV isolate. The autophagic response was measured as presence of autophagic vesicles, LC3 staining, conversion of LC3-I to autophagosome-associated LC3-II and changes in expression of beclin-1, UVRAG, Atg5, Atg12, Atg16L1 and p62/SQSTM1. RHDV-triggered autophagy reached a maximum at 24 hours post-infection (hpi) and declined at 30 and 36 hpi. Phosphorylation of mTOR also augmented in early periods of infection and there was an increase in the expression of the endoplasmic reticulum chaperones BiP/GRP78, CHOP and GRP94. Apoptosis, measured as caspase-3 activity and expression of PARP-1, increased significantly at 30 and 36 hpi in parallel to the maximal expression of the RHDV capsid protein VP60. These data indicate that RHDV infection initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from downregulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, cells begin to exhibit apoptosis.
The Rabbit Hemorrhagic Disease Virus (RHDV) is a positive-strand RNA virus, member of the Caliciviridae family, that causes in wild and domestic rabbits an acute highly fatal disease first reported three decades ago . Hepatic damage plays a central pathogenic role and is histologically similar to fatal viral hepatitis causing fulminant hepatic failure (FHF) in humans . We have shown by data on animal survival, clinical features, histopathological findings, changes in blood chemistry and intracranial pressure monitoring that the RHD fulfils many of the requirements of an animal model of FHF . Moreover, loss of the oxidant/antioxidant balance , presence of apoptosis and endoplasmic reticulum (ER) stress [5–7], and lack of regeneration [8, 9] are constant features in rabbits experimentally infected with the RHDV. This model could therefore be useful to improve our insight into the pathophysiology of viral FHF and to facilitate the development and evaluation of new therapeutic modalities.
Macroautophagy (thereafter referred to as autophagy) pathway is a bulk degradation system which controls the clearance and recycling of intracellular constituents for the maintenance of cellular survival  and can participate in the host response to infection . Autophagy starts with the formation of a doubled-membrane-bound vacuole, known as the autophagosome, that engulfs fractions of the cytoplasm in an either unselective or selective manner via the activity of the autophagy adaptors, such as sequestrosome 1 (p62/SQSTM1), that forms a bridge between the target and the growing autophagosome membrane . After being formed, most autophagosomes receive input from the endocytic vesicles to form an amphisome, in which the autophagic cargo is degraded and delivered into the lysosomal lumen . The first step in the initiation of autophagy is the activation of a molecular complex containing the serine/threonine kinase ULK1. The activation of this complex is down-regulated by mammalian target of rapamycin (mTOR), which integrates multiple signalling pathways sensitive to the availability of amino acids, ATP, growth factors, or level of reactive oxygen species . The nucleation of the autophagosomal membrane is controlled by another molecular complex containing Bcl-2-interacting protein (beclin)-1, which allows the production of phosphatidylinositol 3-phosphate (PI3P) to occur . Several interacting proteins which participate in this complex include positive factors such as UV radiation resistance-associated gene (UVRAG) . In the elongation step, two distinct ubiquitin-like conjugation systems are involved. The ubiquitin-like autophagy-related (Atg)12 is conjugated to Atg5 and then forms a complex with Atg16L1, which is required in the elongation of the autophagosome membrane and determines its curvature. The main player in the second conjugation system is microtubule-associated protein 1 light chain (LC)3, which is cleaved to generate the LC3-I form. LC3-I conjugates with phosphatidylethanolamine to LC3-II, which localizes to the autophagosomal membrane and is suited to serving as an autophagy-specific marker .
Autophagy primarily fulfills a pro-survival role during adaptation to unfavourable growth conditions or following cellular stress. In addition, autophagy contributes to innate immunity by degrading intracellular pathogens, and its inhibition results in an increased replication of virulence of different viruses such as herpex simplex virus 1 (HSV1) or vesicular stomatitis virus (VSV) . However, many viruses, including hepatitis C virus (HCV), Dengue virus, or human immunodeficiency virus (HIV)-1, have evolved mechanisms to evade autophagy and in some cases manage to be even more subversive, using the autophagic response for enhanced replication and viral release [18, 19]. ER stress, which is one of the typical stress responses initiated in cells after viral infection, is important in maintaining the physiology of healthy cells and functions to down-regulate protein synthesis through the unfolded protein response (UPR) . It has been reported that autophagy is activated upon ER stress as a defensive mechanism for survival , and it is known that some viruses stimulate signalling pathways from UPR to autophagy . Interference of the autophagic response with cell death mechanisms plays an important role in determining the fate of infected cells, and recent data suggest the existence of a cross talk between autophagic and apoptotic pathways . For example some studies have demonstrated that the autophagy process can contribute to the death of virus-infected cells through apoptotic mechanisms . However, the autophagy-dependent modulation of cell death is a complex phenomenon and it has also been reported that autophagy can prolong survival of virus-infected cells by counteracting the apoptotic response [25, 26].
We have previously reported that RHDV leads to the activation of the different branches of the UPR  and induces apoptotic death in the last stages of the disease [5, 6]. However it is unknown whether the RHDV induces autophagy in the liver of infected rabbits and how it relates to ER stress and apoptosis. In this study, we attempted to explore which signalling pathways were involved in the autophagic response induced by the RHDV and to characterize the role of autophagy in the context of RHDV pathogenesis.
Materials and methods
Virus and experimental model
Nine-week-old male New Zealand white rabbits were kept in the animal facility of the University of León with 12-h light cycle at 21–22 °C and 50% relative humidity. They were given a standard dry rabbit food and water ad libitum. Rabbits were injected intramuscularly with 2 × 104 hemagglutination units of the RHDV isolate AST/89 [3, 4] at 21 h pm. We have previously reported that during experimental RHDV-infection biochemical data and expression of genes involved in injury, apoptosis, ER stress and regeneration change remarkably at 36–48 hpi, with a 10-15% survival rate by 48 hpi [6–8]. So, we decided to study the effects of infection on the mechanisms of autophagy in rabbits that were infected with the RHDV and sacrificed at 12, 18, 24, 30 and 36 hpi (n = 6 each). The study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and was specifically approved by the Ethics Committee of the University of León.
Transmission electron microscopy
For transmission electron microscopy (TEM) analysis, liver tissues were dissected into 1-mm3 pieces for good penetration of the fixative, and then immersed in a modified Karnovsky fixative (2% glutaraldehyde + 4% buffered formalin (0.1 mol/L phosphate buffer)) overnight. The samples were post-fixed in 2% osmium tetroxide for 2 h at 4 °C and dehydrated with ascending grades of alcohol. The tissue block was then infiltrated and embedded in epon resin at 60 °C for 72 h. Ultrathin sections (70 nm) were cut with an automatic ultra-microtome (Reichert Ultracut E, Vienna, Austria) using a diamond knife. The sections were collected on copper grids (200 meshes) and stained with uranyl acetate and lead citrate solutions. TEM images were observed under a transmission electron microscope (JEOL Ltd, Tokyo, Japan) operating at an accelerating voltage of 80 kV.
Primers used in this study
Sense primer (5′-3′)
Antisense primer (5′-3′)
Western blot analysis
For Western blot analysis liver tissue (25 mg) was homogenised in 1 mL from RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche Diagnostics GmbH). Further disrupt and homogenize tissue with a manual homogenizer, maintaining temperature at 4 °C throughout all procedures. Then the homogenate was incubated on ice for 30 min and finally the samples were centrifuged at 13 000 g for 30 min at 4 °C . The supernatant fraction was recollected and stored at −80 ° C in aliquots until use. Protein concentration was measured by Bradford assay. Equal amounts of protein extracts (30 μg) were separated by 7-12% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and transferred electrically to polyvinyllidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were then blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 30 min at 3 ° C and probed overnight at 4 ° C with polyclonal anti-p62/SQSTM1, PARP-1, Bcl-2, Bcl-xL (Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3I/II, and phospho-mTOR (Abcam, Cambridge, UK) antibodies at 1:200–1:1000 dilution with PBST containing 2.5% non-fat dry milk. Equal loading of protein was demonstrated by probing the membranes with a rabbit anti-GAPDH polyclonal antibody (1:2000 Sigma, St Louis, MO, USA). After washing with TBST, the membranes were incubated for 1 h at room temperature with secondary HRP conjugated antibody (Dako, Glostrup, Denmark, 1:5000), and visualized using ECL detection kit (Amersham Pharmacia, Uppsala, Sweden) . The density of the specific bands was quantified with an imaging densitometer (Scion Image J Software 1.46a, Bethesda, MD, USA).
Tissue samples were recovered, fixed in 10% buffered formalin and embedded in paraffin. Sections (4 μm) were dewaxed and hydrated through graded ethanol, cooked in 25 mM citrate buffer, pH 6.0, in a pressure cooker for 10 min, transferred into boiling deionized water and let to cool for 20 min. Tissue sections were then treated with 3% hydrogen peroxide to inactivate endogenous peroxidase activity. The slides were incubated with rabbit anti-VP60 and anti-LC3 antibodies (Ingenasa, Madrid, Spain and Abcam, respectively) overnight at 4 °C. Subsequently, the sections were incubated for 30 min using the EnVision + system and developed with a solution of 3-3-diaminobenzidine (DAB) (Vector Lab, Burlingame, CA, USA). The slides were stained with hematoxylin for 10 s and mounted. The specificity of the technique was evaluated by negative controls (omitting the incubation with the primary antibody and incubating it with non-immune sera). Pathological findings were assessed by one of the authors blinded to the group allocations.
Lysates were prepared by homogenizing liver tissue in 0.25 mM sucrose, 1 mM EDTA, 10 mM Tris and a protease inhibitor cocktail (Roche Diagnostics GmbH). The lysates were then centrifuged at 14 000 g for 10 min at 4 °C, and supernatants (50 μg protein) were incubated for 1 h at 37 °C in HEPES buffer containing 100 μM concentrations of the specific fluorogenic substrate (7-amino-4-methylcoumarin N-acetyl-L-aspartyl-L-glutamyl-L-valyl-l-aspartic acid amide, Ac-DEVD-AMC). Cleavage of the caspase substrate was monitored using a spectrofluorimeter (Hitachi F-2000 fluorimeter, Hitachi LTD, Tokyo, Japan) at excitation/emission wavelengths of 380/460 nm. Activity was expressed as fluorescence units per milligram of protein per min of incubation.
Results are expressed as mean values ± standard error of the mean (SEM). Data were compared by analysis of variance (ANOVA); when the analysis indicated the presence of a significant difference, the means were compared with the Newman-Keul’s test. Significance was accepted when p was less than 0.05. Values were analyzed using the statistical package SPSS 19.0 (IBM Corporation, Armonk, NY, USA).
Expression of the capsid protein VP60
Autophagy vesicles were detected in RHDV-infected hepatocytes by transmision electron microscopy
RHDV infection induced autophagy molecular signalling
Monitoring static levels of autophagosomes is not sufficient to elucidate effects of RHDV on autophagy, because accumulation of autophagosomes may result either from an increased in their formation or a decrease in their fusion with lysosomes . Thus, to examine autophagy in RHDV-infected rabbits and to avoid misinterpretation, in this research we combined the TEM study with immunohistochemical analysis, Western blot and RT-PCR of different autophagy markers.
Effects of RHDV infection on pathways regulating autophagy induction
One of the major pathways regulating autophagy involves mTOR. It is known that activation of mTOR in nutrient-proficient cells acts as a negative regulator of autophagy, while repression of mTOR by nutrient deprivation or rapamycin treatment induces autophagy . However, the cross talk between mTOR pathway and autophagy induction during viral infection is complex, and it has been reported that some viruses activate mTOR signalling [23, 34]. We analyzed the hepatic expression of phospho-mTOR by Western blot at different RHDV-infection periods (Figure 3C-D). A progressive increased hepatic expression of phospho-mTOR was observed at 12, 18, and 24 hpi in RHDV-infected animals. However, at 30 hpi phospho-mTOR hepatic level decreased to values below the control group, and it was undetectable at 36 hpi.
Effect of Rabbit Hemorrhagic Disease virus (RHDV) infection on mRNA levels of genes related to autophagy
100 ± 11
118 ± 8
203 ± 16*
253 ± 36*
105 ± 6
87 ± 7
100 ± 6
104 ± 8
227 ± 19*
131 ± 15
81 ± 9*
99 ± 5
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125 ± 11*
163 ± 19*
139 ± 7*
87 ± 7
63 ± 5
100 ± 9
173 ± 12*
296 ± 41*
177 ± 9*
104 ± 8
82 ± 7
100 ± 10
106 ± 9
223 ± 6*
140 ± 12*
130 ± 7*
106 ± 10
100 ± 4
145 ± 6*
192 ± 8*
202 ± 8*
86 ± 6
69 ± 4*
100 ± 7
118 ± 17
177 ± 41*
304 ± 29*
241 ± 39*
184 ± 18*
100 ± 8
169 ± 10*
211 ± 19*
540 ± 79*
158 ± 9*
161 ± 13*
100 ± 4
80 ± 4*
163 ± 5*
412 ± 54*
314 ± 13*
226 ± 21*
Apoptotic death in RHDV-infected liver cells
This research examined the occurrence of autophagy during experimental infection by the RHDV. Similarly to other studies conducted with viruses that promote autophagy, TEM analysis showed that number and content of autophagy vesicles increased in RHDV-infected livers. We further analyzed the impact of RHDV infection of several proteins that regulate distinct molecular events leading to autophagy vesicle formation, including its initiation (beclin-1), and maturation by the Atg12 and LC3 conjugation systems. Data obtained demonstrate an early increased expression of the Atg16L1 complex components, together with enhanced LC3 immunostaining and conversion of soluble cytosolic LC3-I to its lipidated, autophagosome-associated form LC3-II, which unequivocally demonstrates that the autophagy was induced at an early stage in rabbits infected with the RHDV. Real-time PCR confirmed that the key autophagy gene beclin-1 was also activated, a fact which suggests a crucial role for this protein in the induction of the autophagic response by the RHDV. Although beclin-1 up-regulation is a frequent finding following viral infection , there are data of beclin-1-independent autophagy induction by enterovirus 71  and it has been reported a late and rather limited increase in the expression of this proautophagic protein by HSV-1 .
In our experiments, p62/SQSTM1 expression increased from 12 hpi and remained elevated until 24 hpi. p62/SQSTM1 is a multifunctional protein, involved in the delivery of ubiquitin-bound cargo to the autophagosome, that interacts with LC3 and is specifically degraded by the autophagic-lysosome pathway, being commonly measured to detect autophagic flux . Viral infection with different herpes viruses has been reported to result in a decrease of p62/SQSTM1 in parallel to increase in the protein LC3-II [39, 40]. However, upregulated expression of both p62/SQSTM1 and LC3 has been shown to exist in different types of tumours, whose growth is significantly inhibited by p62/SQSTM1 down-regulation . Moreover, the expression of p62/SQSTM1 and LC3-II also increases in livers from patients with primary biliary cirrhosis and cultured biliary epithelial cells treated with hydrogen peroxide, with an accumulation of p62-positive aggregates . In Huh 7.5 cells it has been reported that after the transfection of the HCV RNA there is a continuous increase of p62/SQSTM1 which indicates that HCV does not enhance autophagic protein degradation . Results from the present research suggest a similar response to RHDV infection, with an upregulation of p62/SQSTM1 which may reflect a dysfunctional process in which the capacity of autophagy is not much enough to process the damaged proteins bound to p62/SQSTM1.
mTOR is an important signalling molecule which in nutrient-proficient cells acts as a negative regulator of autophagy . When the expression of phospho-mTOR was monitored by Western blot assay we observed an increased expression between 12 and 24 hpi, showing that infection with the RHDV stimulates the mTOR signalling pathway in parallel to the development of the autophagic process. A similar unexpected result has been previously reported in HCV-infected human hepatocytes , in U251 glioma cells after infection with the Newcastle virus , and in bovine kidney cells infected with the bovine herpesvirus type-4 . Our data demonstrate that mTOR is not a negative regulator during RHDV-induced autophagy, and could indicate that induction of autophagy occurs upstream of mTOR signalling or that both processes act concurrently. In HCV-infected hepatocytes it has been suggested that mTOR activation is necessary for cell growth through regulation of phospho-eukaryotic translation initiation factor 4E-binding protein (EBP)1 . Further work would be necessary to identify if there is a similar requirement following infection with the RHDV.
Autophagy is also triggered in response to ER stress through the induction of the UPR . In mammalian cells, knockdown of the upstream UPR regulator BiP inhibits autophagosome formation, but does not affect the conversion of LC3-I to LC3-II, suggesting that ER stress induction is an obligatory factor for autophagy and may function at the phagophore expansion rather than induction step . Previous studies have shown that induction of autophagosomes by the HCV virus depends on the UPR , and the three branches of the UPR contribute to regulate HCV replication via modulation of autophagy . It has also been reported that the tobacco mosaic virus RNA induces ER stress-related autophagy in HeLa cells , and it is known that autophagosome formation during varicella-zoster virus infection follows ER stress and the UPR . In a previous work, our research group, using the RHDV model of FHF, reported that ER stress was induced in RHDV infected rabbits through a modulation of the three branches of the UPR . Here, it is shown that mRNA levels of the molecular chaperones CHOP, BiP and GRP94 reached a peak at 24 hpi, in parallel to the increase of the expression of beclin-1 and the components of the two ubiquitin-like conjugation systems Atg12-Atg5-Atg16L1 and LC3. Our data suggest that autophagy could be provoked at least in part upon ER stress. This hypothesis is further supported by the RHDV-induced increase in the upregulation of beclin-1, whose expression is required for ER-stress induced autophagy .
The interplay between autophagy and programmed cell death is complex. Autophagy is a cytoprotective mechanism which enables cells to survive unfavourable growth conditions and can prevent cell death by apoptosis. However, some studies have demonstrated that autophagy may have an active contribution to cell death in virus infected cells. Thus, it has been reported that pharmacological inhibition of autophagy efficiently suppresses apoptosis induced by human adenovirus type 5 Delta-24-RGD mutant in mouse fibroblast or human U251 glioma cells . Blocking of autophagy also attenuates cell death caused by the avian influenza A H5N1 virus both in vitro and in vivo , and it is known that knockdown of beclin-1 or Atg5 protects human rhabdomyosarcoma cells from enterovirus 71-induced apoptotic death . We and others have previously reported that RHDV infection induces in rabbits a marked apoptotic response at 36–48 hpi with increased caspase-3 activity and immunoexpression and a marked proteolysis of PARP-1 [5, 6, 8, 51]. Results from the present study indicate that apoptosis is present in the late stages of the disease, with no significant increase in caspase-3 activity and PARP-1 degradation or decreased expression of the antiapoptotic proteins Bcl-2 or Bcl-xL occurring in early periods. The fact that autophagy develops in hepatocytes at early stage and cells begin to exhibit apoptosis in parallel to the decline of the autophagy response, suggests that autophagy play a beneficial role in an attempt to protect cells from the impending noxious effects of the virus. It has been recently shown that cardiomyocites exposed to angiotensin II exhibit a similar behavior, with autophagy occurring at early stages whereas apoptosis occurs late . A number of studies have also demonstrated the ability of virally-induced autophagy to prevent or delay death of infected cells. For example, apoptotic death of hepatoma cells expressing the oncogenic HBV X protein increases when autophagy is blocked , and the infection with Japanese encephalitis virus increases caspase activation and cell death in beclin-1 or Atg5-deficient cells . It has also been shown that in HSV-1-infected U251 glioma cells the autophagic response markedly delayed caspase activation and other hallmarks of apoptotic cell death . Data here obtained also point to a role of virally-induced autophagy to support survival of the infected cells and suggest that autophagy might contribute to limit the pathological consequences associated with cell death triggered by the RHDV infection. Confirmation of the connection between autophagy and RHDV pathogenecity should require the use of cell culture systems, that are unavailable at present . An additional interesting finding concerns the increased expression of p62/SQSTM1 observed in liver cells. This reflects inhibition of autophagosome-lysosomal function and dysfunctional autophagy, which may contribute to altered signal transduction pathway and liver damage . In fact, it is known that upregulation of p62/SQSTM1 positively controls apoptosis by polyubiquitination and aggregation of the key initiator caspase 8 [56, 57], thus playing a potential role in the cross-regulation between autophagy and apoptosis.
In summary, experiments here reported were aimed to enhance our understanding of the interplay between the RHDV and the host liver cells. The most important finding is that RHDV infection in vivo initiates a rapid autophagic response, perhaps in an attempt to protect liver, which associates to ER stress development and is independent from down-regulation of the major autophagy suppressor mTOR. As the infection continues and the autophagic response declines, the process of apoptosis dominates. Although it is necessary to be cautious, considering that autophagy is also involved in modulation of viral replication and recognition/presentation of viral antigens , therapeutic potential of autophagy modulation in controlling RHDV-induced cell death is worthy to be explored, considering the importance of RHDV infection as a model of human FHF of viral origin. Findings from the present study could contribute to the search for new pharmacological strategies to protect livers from FHF injury.
CIBERehd is funded by Instituto de Salud Carlos III, Spain.
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