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Essential role of hyperacetylated microtubules in innate immunity escape orchestrated by the EBV-encoded BHRF1 protein

['Damien Glon', 'Université Paris-Saclay', 'Cea', 'Cnrs', 'Institute For Integrative Biology Of The Cell', 'Gif-Sur-Yvette', 'Géraldine Vilmen', 'Crsa', 'Centre De Recherche Saint-Antoine', 'Umr-S']

Date: 2022-05

Innate immunity constitutes the first line of defense against viruses, in which mitochondria play an important role in the induction of the interferon (IFN) response. BHRF1, a multifunctional viral protein expressed during Epstein-Barr virus reactivation, modulates mitochondrial dynamics and disrupts the IFN signaling pathway. Mitochondria are mobile organelles that move through the cytoplasm thanks to the cytoskeleton and in particular the microtubule (MT) network. MTs undergo various post-translational modifications, among them tubulin acetylation. In this study, we demonstrated that BHRF1 induces MT hyperacetylation to escape innate immunity. Indeed, the expression of BHRF1 induces the clustering of shortened mitochondria next to the nucleus. This “mito-aggresome” is organized around the centrosome and its formation is MT-dependent. We also observed that the α-tubulin acetyltransferase ATAT1 interacts with BHRF1. Using ATAT1 knockdown or a non-acetylatable α-tubulin mutant, we demonstrated that this hyperacetylation is necessary for the mito-aggresome formation. Similar results were observed during EBV reactivation. We investigated the mechanism leading to the clustering of mitochondria, and we identified dyneins as motors that are required for mitochondrial clustering. Finally, we demonstrated that BHRF1 needs MT hyperacetylation to block the induction of the IFN response. Moreover, the loss of MT hyperacetylation blocks the localization of autophagosomes close to the mito-aggresome, impeding BHRF1 to initiate mitophagy, which is essential to inhibiting the signaling pathway. Therefore, our results reveal the role of the MT network, and its acetylation level, in the induction of a pro-viral mitophagy.

Viruses have developed numerous strategies to ensure their persistence in the host, notably by counteracting the innate immune system. The Epstein-Barr virus (EBV), which infects most humans worldwide, encodes a mitochondria and ER-localized protein named BHRF1, which participates in this viral persistence. Indeed, we have recently demonstrated that BHRF1, in addition to its well-described anti-apoptotic activity, has the ability to stimulate autophagy and to inhibit interferon (IFN) response. In this new study, we decipher the original mechanism used by BHRF1 to dampen antiviral immunity. We uncovered that a post-translational modification of the microtubule (MT) network induced by BHRF1 regulates IFN production. To do so, BHRF1 interacts with a cellular acetyltransferase called ATAT1 to increase the acetylation level of MT. One of the consequences of this MT modification is the clustering of autophagosomes and mitochondria in a juxtanuclear region. The sequestration of mitochondria inside the autophagosomes leads to the induction of mitophagy and blocks the signalling pathway triggering IFN production. Finally, we demonstrate that this mechanism, dependent on MT acetylation, occurs during EBV reactivation.

Funding: This work was supported by institutional funding from CNRS and Université Paris Saclay and by grants from DIM MALINF Région IDF to GV and from the Agence Nationale de la Recherche (ANR) to AE and ML (ANR-14-CE14-0022), and to GB (ANR-20-IDEES-0002). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2022 Glon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Our group recently unravelled the novel functions in innate immunity of the Epstein-Barr virus (EBV) BHRF1 protein, a viral Bcl-2 homolog [ 18 ], related to its mitochondrial localization [ 19 ]. We demonstrated that BHRF1 disturbs mitochondrial dynamics and subsequently stimulates mitophagy, a cellular process that can specifically sequester and degrade mitochondria by autophagy, leading to the inhibition of type I IFN response by EBV [ 19 ]. Here, we explored the role of the MT network in the innate immunity escape induced by BHRF1 and we revealed the contribution of the MT hyperacetylation on the BHRF1-induced mitochondrial clustering and on the BHRF1-mediated counteraction of the IFN responses.

MT functions are regulated by numerous post-translational modifications such as acetylation, detyrosination, phosphorylation, methylation or polyglutamylation [ 11 ]. Among them, the acetylation of α-tubulin, which mainly occurs on the Lysine in position 40 (K40), has emerged as an important regulator of numerous cell functions [ 12 ]. For example, α-tubulin acetylation, which is often linked to microtubule stabilization, modulates the binding of MAPs [ 13 – 15 ] and therefore may modulate organelle movement. Indeed, stable MTs are particularly involved in mitochondrial transport [ 16 ], and it has been reported that acetylation of α-tubulin mediates dynein-dependent transport of mitochondria along MTs [ 17 ].

The MT cytoskeleton is a highly dynamic polymer of α and β-tubulin heterodimers, which is involved in a variety of cellular functions, such as supporting cell structures, maintaining cell polarity, but also allowing the transport of organelles. Moreover, it has been shown that the MT network of virally infected cells could be used for intracellular transport of viral particle/genomic material to the sites of replication or assembly [ 6 ]. A broad range of MT-associated proteins (MAPs) contributes to regulating MT dynamic behavior. MAPs include kinesins and dyneins, which both allow the positioning of organelles and the long-distance transport of vesicles along MT tracks. Kinesins generally transport their cargos toward the plus-end of MTs at the cell periphery. Conversely, dyneins transport their cargos to the minus-end of MTs in the cell center [ 7 ]. The relationship between MTs and mitochondrial transport is not completely understood. Mitochondrial movement involves mitochondrial adaptors such as small mitochondrial Rho GTPases Miro1/2 and TRAK1/2 [ 8 ]. Indeed, Miro proteins can engage both kinesin and dynein to mediate the bidirectional movement of mitochondria along MT tracts [ 4 , 9 , 10 ].

Mitochondria carry out a crucial role in many cellular processes ranging from energy production to programmed cell death, from calcium homeostasis to cell immunity. They constitute a platform for signaling pathways involved in innate immunity thanks to the mitochondrial-resident protein MAVS (mitochondrial antiviral signaling protein), predominantly localized at the mitochondrial outer membrane surface [ 2 , 3 ]. RIG-I (retinoic acid-inducible gene) and MDA5 (melanoma differentiation-associated protein 5), two cytoplasmic PRRs that detect viral genomes, are notably translocated to the mitochondria to interact with MAVS that recruits and activates TBK1 (TANK-binding kinase 1). This kinase is required for the phosphorylation of the transcription factors IRF3 and IRF7 (interferon regulatory factors 3 and 7), leading to the subsequent activation of type I IFN promoter [ 2 , 3 ]. The functions of mitochondria depend on their morphology, which is in turn dependent upon mitochondrial dynamics, including fission, fusion, and motility. Moreover, mitochondria are actively recruited to specific cellular locations to respond to their functions. In eukaryotic cells, the cytoskeleton and notably microtubules (MTs) play a critical role in the distribution of mitochondria throughout the cytoplasm by facilitating their transport to areas with high metabolic demands [ 4 , 5 ].

The innate immune system provides the first line of defense against different invading pathogens. This process is based on the sensing of motifs from the foreign organism, called the pathogen-associated molecular pattern (PAMP), by different host pattern recognition receptors (PRR). In the case of a viral infection, their recognition notably leads to the induction of the interferon (IFN) response, which induces the expression of interferon-stimulated genes and the synthesis of cytokines known for their antiviral properties [ 1 ].

Results

BHRF1-induced mito-aggresome is organized around the centrosome The EBV-encoded BHRF1 localizes to mitochondria, and its expression in HeLa cells dramatically alters mitochondrial dynamics and distribution, [19]. By confocal microscopy, we observed abnormal mitochondrial clusters close to the nucleus in BHRF1-expressing cells, whereas in control cells (empty vector [EV] transfection), mitochondria remained distributed throughout the cytoplasm (Fig 1A). This mitochondrial phenotype, known as “mito-aggresome”, was in accordance with our previous observations. To quantify this phenotype, we arbitrarily considered that, when the compaction index (CI) of mitochondria is above 0.4, the cell presents a mito-aggresome, whereas when the CI value is below 0.4, mitochondria are distributed homogeneously throughout the cell cytoplasm [19,20]. The expression of BHRF1 significantly increased the CI values, which corresponds to the formation of a mito-aggresome in almost 80% of the cells (Fig 1B). To confirm the role of BHRF1 in the context of EBV infection, we analyzed the mitochondrial phenotype in EBV-positive Akata B cells, during latency or after treatment with anti-human IgG to induce EBV reactivation (S1 Fig). We clearly observed mito-aggresomes upon EBV reactivation, whereas the knockdown (KD) of BHRF1 completely blocked their formation. Since BHRF1 is associated with mitochondrial fragmentation (Fig 1A—insets), we precisely evaluated mitochondrial shape by calculation of the aspect ratio (AR) and the form factor (FF), two parameters that reflect the mitochondrial length and the branching of mitochondria, respectively. As calculated in Fig 1C, BHRF1 expression significantly reduced both AR and FF parameters, confirming the induction of mitochondrial fission. PPT PowerPoint slide

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TIFF original image Download: Fig 1. BHRF1 induces the formation of mito-aggresomes in a MT-dependent manner. (A-C) HeLa cells were transfected for 24 h with BHRF1-HA plasmid or with EV as a control. Mitochondria were labeled with MitoTracker and cells immunostained for BHRF1. Nuclei were stained with DAPI. (A) Confocal images with insets (3X) on mitochondrial phenotype. Values of mitochondrial CI are indicated on representative cells. Scale bars: 10 μm and 2 μm for insets. (B) Left, assessment of mitochondrial aggregation by calculation of CI. Right, percentage of cells presenting a mito-aggresome (n = 20 cells per condition). (C) Quantification of mitochondrial fission parameters, aspect ratio (AR; left panel) and form factor (FF, right panel). (D) Confocal images of HeLa cells transfected with BHRF1-HA plasmid (or EV) and immunostained for pericentrin and BHRF1 (upper panel) or mitochondria (lower panel). Nuclei were stained with DAPI. Scale bar: 20 μm. Arrowheads show the centrosomal localization of pericentrin. (E-F) MT network depolymerization assay. (E) Representative images where BHRF1 expression was visualized by HA immunostaining (gray), the MT network by α-tubulin immunostaining (green), and mitochondria are immunostained with an anti-TOM20 antibody (red). Values of mitochondrial CI are indicated on representative cells. Scale bars: 10 μm. (F) Percentage of BHRF1-HA-positive cells presenting a mito-aggresome (n = 30 cells per condition) in cells treated with nocodazole and after MT regrowth. Data represent the mean ± SEM of three independent experiments. ** P < 0.01; *** P < 0.001 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g001 As mitochondria localize near the nucleus upon BHRF1 expression, the involvement of the MT network in this mitochondrial redistribution was investigated. In mammalian cells, mitochondria move along cytoskeletal tracks to sites of high-energy demand in an MT-dependent manner [21]. At interphase, the MT network radiates from the centrosome, the main MT organizing center for directing the polarity and the orientation of MTs. By co-staining BHRF1-expressing cells for mitochondria and pericentrin, a classical centrosomal marker, we observed that mitochondria are concentrated around the centrosome to form a mito-aggresome (Fig 1D). Moreover, a 3D reconstruction confirmed the clustering of BHRF1 in the vicinity of the nucleus around the centrosome (S1 Movie).

The MT network is required for BHRF1-induced mito-aggresome formation To test whether MTs play a role in the mitochondrial redistribution induced by BHRF1 expression, the whole MT network was disassembled using a treatment mixing nocodazole, a classical MT-destabilizing drug, and exposure to cold [22]. As shown in Fig 1E—left panel this treatment disassembled MTs (fuzzy staining of α-tubulin). The removal of nocodazole followed by 1 hour in a warm complete medium at 37°C restored the MT network (Fig 1E—right panel). In cells without BHRF1 expression, mitochondria were homogeneously distributed in the cytoplasm, and the disorganization of the MT network had no impact on their distribution. Conversely, when MTs were depolymerized in BHRF1-positive cells, the mito-aggresome formation was abolished (Fig 1E—left panel) and mitochondria were then distributed throughout the cell (CI values mainly below 0.4). This phenomenon was reversible since the mitochondria rapidly re-aggregated to different extents within 1 hour after reassembly of the MT network (most of the CI values above 0.4) (Fig 1E—right panel). In contrast, mitochondria in non-transfected neighboring cells remained scattered within the cytoplasm after nocodazole removal. The quantification of mito-aggresomes in BHRF1-positive cells showed that whereas less than 20% of the cells presented mitochondrial clustering after depolymerization treatment (Fig 1F), this proportion went back to more than 50% after repolymerization of the MTs (compare to 80% without treatment). We concluded that the MT network is required for the mito-aggresome formation. The actin network is also known to be involved in mitochondrial transport and fission [8,23]. We thus checked if F-actin disorganization affects the mitochondrial phenotype induced by BHRF1. To do so, we treated HeLa cells with cytochalasin B to disrupt F-actin but we did not observe any changes in the mitochondrial phenotype in BHRF1-positive cells (S2A Fig). BHRF1 still induced mito-aggresome formation and fission at the same level as in the control (S2B Fig). Therefore, the actin network does not seem to be involved in the mitochondrial phenotype.

EBV reactivation induces BHRF1-dependent MT hyperacetylation We next explored whether BHRF1 could modify the MT network by assessing α-tubulin acetylation level. Interestingly, upon BHRF1 expression, whereas α-tubulin staining was unchanged, acetyl-α-tubulin was redistributed and colocalized with BHRF1 (Fig 2A). Moreover, we observed a brighter signal of acetyl-α-tubulin in BHRF1 expressing cells, suggesting an increase in α-tubulin acetylation. This was confirmed by western-blot analysis showing an increase of 60% under BHRF1 expression (Fig 2B). PPT PowerPoint slide

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TIFF original image Download: Fig 2. BHRF1 stimulates MT hyperacetylation. (A) Representative images of HeLa cells expressing BHRF1-HA and immunostained for HA and α-tubulin (upper panel) or acetyl-α-tubulin (lower panel). Nuclei were stained with DAPI. Scale bar: 20 μm. (B) Left, immunoblot analysis of acetyl and total α-tubulin in BHRF1-HA-transfected HeLa cells. Right, normalized ratios of acetyl-α-tubulin to total α-tubulin. (C) Immunoblot analysis of acetyl-α-tubulin, α-tubulin, Ea-D, ZEBRA and BHRF1 in the Akata and Ramos cells treated or not with anti-human IgG for 8 h or 24 h to induce EBV reactivation. (D) Immunoblot analysis of acetyl-α-tubulin, α-tubulin, Ea-D, ZEBRA and BHRF1 in Akata cells deficient for BHRF1 expression (sh-BHRF1). (E) Normalized ratios of acetyl-α-tubulin to α-tubulin in Akata cells. (F-G) EBV WT or EBV ΔBHRF1 were reactivated in HEK293 cells by transfection with ZEBRA and Rta plasmids for 24 h. (F) Left, immunoblot analysis of acetyl-α-tubulin, α-tubulin, Ea-D and BHRF1. Right, normalized ratios of acetyl-α-tubulin to α-tubulin. (G) Confocal images of HEK293/EBV+ cells immunostained for acetyl-α-tubulin and Ea-D. Nuclei were stained with DAPI. Scale bar: 20 μm. Stars indicate EBV-reactivated cells. Data represent the mean ± SEM of three independent experiments. ns = non-significant; ** P < 0.01 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g002 To confirm these results in the context of EBV infection, we analyzed the level of MT acetylation in EBV-positive Akata B cells (Fig 2C and 2E). We used, as a control, EBV-negative Ramos B cells similarly treated with anti-IgG. We observed an increase of MT acetylation only 24h after EBV reactivation in Akata cells (visualized by Ea-D and ZEBRA viral protein expression). This MT hyperacetylation coincided with BHRF1 expression. We confirmed that BHRF1 is required for this phenotype in the context of EBV infection of B cells, using an shRNA targeting BHRF1 (Fig 2D and 2E). In parallel, in HEK293/EBV+ epithelial cells, the EBV genomes WT and ΔBHRF1 were reactivated by co-transfection of plasmids encoding the trans-activator proteins ZEBRA and Rta (Fig 2F and 2G). We similarly observed a BHRF1-dependent MT hyperacetylation upon EBV reactivation in epithelial cells. Due to a low EBV reactivation rate in this model, the increase of MT hyperacetylation observed, although statistically significant, was less important than in Akata cells. However, by confocal microscopy, the increase of tubulin acetylation was clearly seen in WT HEK293/EBV+ and not in ΔBHRF1 (Fig 2G).

MT hyperacetylation is required for the mito-aggresome formation To determine whether this hyperacetylation could be involved in BHRF1-induced mitochondrial alterations, we expressed in HeLa cells a non-acetylatable α-tubulin mutant (mCherry-α-tubulin K40A) which prevents MT hyperacetylation [13]. Expression of this non-acetylatable α-tubulin mutant does not alter the MT network architecture and this protein is incorporated in the whole MT network [22]. However, the whole MT network hyperacetylation is prevented, since α-tubulin acetylation only occurs on polymers and the MT network is totally decorated with this non-acetylatable α-tubulin. We thus verified by immunoblot analysis that BHRF1 was unable to induce hyperacetylation of α-tubulin when the α-tubulin K40A mutant was expressed (S3A Fig). Then, the impact of α-tubulin K40A on the clustering of mitochondria was analyzed by confocal microscopy (Figs 3A, 3B and S3B). In cells co-expressing α-tubulin K40A and BHRF1, CI mean value was below 0.4 and mitochondria were homogeneously distributed throughout the cytoplasm, similarly to control cells. Thus, we concluded that BHRF1-induced mito-aggresome formation depends on MT hyperacetylation. This was confirmed in the context of EBV reactivation (Fig 3C and 3D). EBV reactivation was visualized in HEK293/EBV WT cells using the staining of Ea-D viral protein. PPT PowerPoint slide

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TIFF original image Download: Fig 3. MT hyperacetylation is required for BHRF1-induced mito-aggresomes formation. (A-B) HeLa cells were co-transfected with plasmids encoding BHRF1-HA and either mCherry-α-tubulin WT or a non-acetylatable form (K40A). (A) Confocal images with insets (3X) of cells immunostained for HA and TOM20. Nuclei were stained with DAPI. Scale bars: 10 μm and 4 μm for insets. Values of mitochondrial CI are indicated on representative cells. Images of control cells are presented in S3B Fig. (B) Quantification of CI, percentage of cells with a mito-aggresome and mitochondrial fission parameters (n = 20 cells per condition). (C-D) HEK293/EBV+ WT cells were co-transfected with plasmids encoding ZEBRA, Rta and either mCherry-α-tubulin WT or K40A. (C) Confocal images of cells immunostained for Ea-D and TOM20. Nuclei were stained with DAPI. Scale bar: 10 μm. Values of mitochondrial CI are indicated on representative cells. (D) Quantification of CI, percentage of cells with a mito-aggresome and mitochondrial fission parameters (n = 20 cells per condition). Data represent the mean ± SEM of three independent experiments. ns = non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g003 Interestingly, we observed that without α-tubulin hyperacetylation, BHRF1 expression or EBV reactivation were still able to promote mitochondrial fragmentation (Fig 3B and 3D). Confocal images (Fig 3A—insets) clearly showed that mitochondria were drastically smaller upon BHRF1 expression in both WT and K40A conditions. We previously reported that knockdown of Drp1 (dynamin-related protein 1), a master regulator of mitochondrial fission, prevents the BHRF1-induced mitochondrial phenotype [19]. We thus investigated the role of Drp1 in MT hyperacetylation and found that the loss of Drp1 inhibits MT hyperacetylation induced by BHRF1 expression (S4A Fig). In the same way, treatment of Akata cells with Mdivi-1, which inhibits mitochondrial fission, blocked MT hyperacetylation induced by EBV reactivation (S4B Fig). Taken together, these results demonstrated that the hyperacetylation of MTs requires mitochondrial fission and is essential for BHRF1-induced mito-aggresome formation.

BHRF1 requires acetyltransferase ATAT1 to modify MTs and form mito-aggresomes Thereafter, we wanted to characterize the mechanism of MT hyperacetylation induction by BHRF1. Tubulin acetylation results from the balance between the activities of tubulin acetyltransferases and deacetylases. Therefore, a hyperacetylation of the MTs can be the consequence of either the inhibition of deacetylases or the activation of an acetyltransferase. First of all, we explored a potential inhibitory role of BHRF1 on the two main α-tubulin deacetylases, HDAC6 (histone deacetylase 6) and SIRT2 (sirtuin type 2) [31,32]. Indeed, it has been observed that KD of SIRT2 results in an aberrant mitochondrial distribution in the perinuclear region [17], and another study showed that the depletion of SIRT2 results in nuclear envelope shape defects [33]. These observations are clearly similar to the effect of BHRF1 on mitochondria, as well as its distribution around the nuclear envelope and the modification of the nucleus shape (Fig 1A). We thus performed an in vitro tubulin deacetylation assay [14]. This test consists of mixing the highly acetylated porcine brain tubulin with lysates of BHRF1-expressing cells. Lysate of control EV-transfected cells reduced the acetylation of tubulin by approximately 50% compared to the condition without cell lysate (Fig 5A). The same deacetylase activity was observed with BHRF1-positive lysates, indicating that BHRF1 does not inhibit the deacetylases. PPT PowerPoint slide

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TIFF original image Download: Fig 5. MT hyperacetylation and mito-aggresome formation require ATAT1, which interacts with BHRF1. (A) Tubulin deacetylation assay. HeLa cells were transfected with BHRF1-HA plasmid (or EV) and lysed 48 h post-transfection. Cell lysates were incubated in vitro with porcine brain tubulin and NAD+. BHRF1-HA, acetyl-α-tubulin and α-tubulin were detected by immunoblot, and the histograms represent the normalized ratios of acetyl-α-tubulin to α-tubulin. (B-D) After knockdown of ATAT1 by siRNA transfection, HeLa cells were transfected with BHRF1-HA for 24 h. (B) Loss of ATAT1 activity was visualized by immunoblot analysis of acetyl-α-tubulin and α-tubulin. (C) Confocal images. Mitochondria were labeled with MitoTracker, and cells were immunostained for acetyl-α-tubulin and HA. Nuclei were stained with DAPI. Values of mitochondrial CI are indicated on representative cells. Scale bar: 20 μm. Images of EV-transfected cells are presented in S6 Fig. (D) Quantification of CI, percentage of cells with a mito-aggresome and mitochondrial fission parameters (n = 20 cells per condition). (E-F) HeLa cells were co-transfected with GFP-ATAT1 and BHRF1-HA plasmids for 24 h. (E) After immunoprecipitation of BHRF1 with an anti-HA antibody, proteins were detected by immunoblotting with anti-GFP and anti-HA antibodies. (F) After immunoprecipitation of GFP-ATAT1 with an anti-GFP antibody, proteins were detected by immunoblotting with anti-GFP and anti-HA antibodies.Data represent the mean ± SEM of three independent experiments. ns = non-significant; * P < 0.05; ** P < 0.01; *** P < 0.001 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g005 We subsequently investigated the role of ATAT1 (alpha-tubulin N-acetyltransferase 1) on BHRF1-induced MT hyperacetylation [34]. We knocked down ATAT1 expression by a siRNA approach and observed no more detectable acetylation activity under BHRF1 expression (Fig 5B), suggesting the involvement of ATAT1 in BHRF1-induced hyperacetylation. Then, we checked the mitochondrial network and observed that ATAT1 KD prevents mito-aggresome formation but not mitochondrial fission (Figs 5C, 5D and S6). These results led us to conclude that BHRF1 activates the acetyltransferase ATAT1, leading to MT hyperacetylation and subsequent mito-aggresome formation. To go further, we analyzed the distribution of GFP-tagged ATAT1 in presence of BHRF1 in HeLa cells (S7 Fig) and we observed a clear colocalization between GFP-ATAT1 and BHRF1 and, in all cells expressing GFP-ATAT1, a predictable increase in tubulin acetylation. Finally, co-immunoprecipitation assays revealed that BHRF1 interacts with ATAT1 in HeLa cells (Fig 5E and 5F). Our results suggest that the interaction between BHRF1 and ATAT1 leads to MT hyperacetylation.

Mito-aggresome formation requires retrograde transport of mitochondria via dyneins We observed in Fig 1E that after removal of nocodazole and regrowth of the MT network, mitochondria rapidly re-aggregated in BHRF1-expressing cells, suggesting that an MT-dependent motor activity is involved in the mito-aggresome formation. The hyperacetylation of MTs has been previously shown to facilitate the recruitment and binding of MAPs on the tubulin [13]. We therefore hypothesized that BHRF1-induced mitochondrial clustering next to the nucleus could use dyneins. To investigate this hypothesis, we inhibited dynein functions by overexpression of p50-dynamitin. The overexpression of one subunit of the dynactin complex (p50-dynamitin) disrupts it and results in the dissociation of dyneins from the MT network. We observed that the expression of this construct totally prevents mito-aggresome formation in BHRF1-expressing cells (Figs 6A, 6B and S8A). Moreover, the ability of BHRF1 to induce mitochondrial fission was decreased when dyneins were inhibited, as we observed a partial but significant restoration of mitochondrial fission parameters (AR and FF; Fig 6B). PPT PowerPoint slide

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TIFF original image Download: Fig 6. Dynein-based transport is required for BHRF1 to aggregate mitochondria next to the nucleus. (A-B) HeLa cells were co-transfected with plasmids encoding BHRF1-HA and p50-dynamitin-myc (or control) for 24 h. (A) Confocal images. Mitochondria were labeled with MitoTracker, and cells were immunostained for c-myc and HA. Nuclei were stained with DAPI. Values of mitochondrial CI are indicated on representative cells. Scale bar: 20 μm. Images of cells co-expressing EV and p50-dynamitin-myc are presented in S8A Fig. (B) Quantification of CI, percentage of cells with a mito-aggresome and mitochondrial fission parameters (n = 20 cells per condition). Data represent the mean ± SEM of three independent experiments. ns = non-significant; * P < 0.05, ** P < 0.01; *** P < 0.001 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g006 To confirm the importance of dynein motors in the mito-aggresome formation, we treated cells with ciliobrevin-D, a specific inhibitor of dyneins, which blocks their gliding along MTs and their ATPase activity. As with overexpression of p50-dynamitin, BHRF1 was not able to induce mito-aggresomes in cells treated with ciliobrevin-D (S8B and S8C Fig). However, BHRF1 still induced a slight mitochondrial fission phenomenon. Altogether, these results demonstrated that fragmented mitochondria are clustered next to the nucleus in a dynein-dependent manner.

MT hyperacetylation does not affect BHRF1-pro-autophagic activity, but is required for the first step of mitophagy induction Our group previously demonstrated that BHRF1 first stimulates autophagy and then induces mitophagy, the selective degradation of mitochondria [19]. Autophagy is a conserved cellular process allowing the removal and recycling of damaged or supernumerary cellular components. This process involves double-membrane vesicles, called autophagosomes, which fuse with lysosomes to degrade the content. Selective autophagy consists of the recognition of a specific cargo by a molecular receptor that links the cargo to the autophagosome for its removal [35]. Several studies demonstrated the role of MTs during the autophagic process, notably during the formation of the autophagosomes [22,36,37]. Thus, we investigated whether BHRF1 required MT hyperacetylation to induce autophagy. The autophagic flux was studied in HeLa cells co-expressing α-tubulin K40A and BHRF1. We first analyzed by immunofluorescence the accumulation of LC3 (microtubule-associated protein 1 light chain 3), a classical marker of autophagosomes in the presence or absence of CQ, an inhibitor of the autophagic flux. Expression of BHRF1 induced a clear accumulation of LC3 dots in the cytoplasm, and even more after CQ treatment, when non-acetylatable α-tubulin was expressed (Fig 7A). We quantified LC3 dots and we observed no difference when MTs were hyperacetylated or not (Figs 7B, S9A and S9B). We confirmed the result by analyzing the accumulation of lipidated LC3 (LC3-II) by immunoblot and we observed an increase in LC3-II levels in BHRF1-expressing cells, independently of MT hyperacetylation (Figs 7C and S9C). This increase is potentiated after CQ treatment. Therefore, we concluded that BHRF1 stimulates the biogenesis of autophagosomes and the autophagic flux independently of MT hyperacetylation. PPT PowerPoint slide

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TIFF original image Download: Fig 7. MT hyperacetylation is dispensable for BHRF1-pro-autophagic activity, but essential for mitophagy. HeLa cells were co-transfected for 24 h with plasmids encoding BHRF1-HA (or EV) and mCherry-α-tubulin K40A (or WT) and treated with chloroquine when indicated. (A) Confocal images. Cells were immunostained for BHRF1 and LC3 and nuclei stained with DAPI. Scale bar: 10 μm. Images of cells transfected with mCherry-α-tubulin WT are presented in S9A Fig. (B) Quantification of LC3 dots (n = 30 cells per condition). (C) Immunoblot analysis of LC3 and BHRF1-HA expression. β-actin was used as a loading control. (D) Measurement of the distance between autophagosomes and nucleus (n = 60 cells from three independent experiments). (E) Confocal images of cells immunostained for TOM20 and LC3. Nuclei were stained with DAPI. Insets (6X) show colocalization events (see arrows). Scale bars: 10 μm and 5 μm for insets. (F) Colocalization level (Manders coefficient) between mitochondria (TOM20) and autophagosomes (LC3) (n = 30 cells per condition). Data represent the mean ± SEM of three independent experiments. ns = non-significant; * P < 0.05, ** P < 0.01; *** P < 0.001 (Student’s t-test). https://doi.org/10.1371/journal.ppat.1010371.g007 Intriguingly, we noticed that the autophagosomes were localized close to the nucleus upon BHRF1 expression (Fig 7D). MT hyperacetylation is required for this juxtanuclear localization, suggesting its involvement in BHRF1-induced mitophagy. We therefore investigated the colocalization between mitochondria and autophagosomes in cells co-expressing α-tubulin K40A and BHRF1. When MT hyperacetylation was prevented, mitochondria did not colocalize anymore with autophagosomes (Fig 7E). The quantification of colocalization intensity, measured by Manders’ coefficient, confirmed that mitochondrial sequestration in autophagic vesicles did not occur when the mito-aggresome formation was impeded (Fig 7F). Altogether, this suggests that BHRF1-induced MT hyperacetylation triggers the localization of autophagosomes in the close vicinity of mitochondria.

[END]

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