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IRGM mediates GBF1 phosphorylation and associates with Arf1 at HCV replication sites. (A) Representative images of cells immunostained with antibodies against IRGM and GBF1 (magnification: 63×). DAPI (blue) staining marks the nucleus. The MOC represents observations from three independent experiments. (B) The effect of siIRGM on GBF1 and AMPKα phosphorylation was examined by immunoblotting. A merged image in the first row reveals overlap of signals (seen as orange) between phosphorylated (green) and total GBF1 (red) protein levels. The band corresponding to phospho-GBF1 (GBF1PT1337) is indicated by an arrow. GBF1PT1337 or AMPKαPT172 protein levels were normalized to total GBF1 or AMPKα protein levels. (C ) The effect of HCV infection on GBF1 and AMPKα phosphorylation were examined by immunoblotting as in B. (D and E) Representative images of cells immunostained with antibodies against IRGM and Arf1 (D) or NS5A and Arf1 (E). (Magnification: 63×.) DAPI staining (blue) marks the nucleus. The MCC quantification method was used because of the high level of IRGM or NS5A protein compared with the Arf1 protein level. Data shown are the mean ± SD of three independent experiments; *P < 0.05, paired t test. Med, uninfected hepatocytes.
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Without a vaccine or cost-effective antivirals against hepatitis C virus (HCV) there is a need to understand better the molecular mechanisms underlying the establishment of productive HCV infection and chronic liver disease. Recently, the Crohn's disease and tuberculosis risk factor immunity-related GTPase M (IRGM) was found to promote...
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... in uninfected or in HCV-infected cells, as assessed by Mander's coefficient analysis ( Fig. 2A and Fig. S5). Because this finding was in contrast with a report showing significant expression of IRGM at the mitochondria (14), we examined additional cell lines and similarly found only moderate overlap between IRGM and mito- chondrial staining (Fig. S5). The mouse IRGM homolog Irgm1 is recruited to phagosomes (11,12), and autophagic components may connect to lysosomal compartments during bacterial infection (33) and in vesicular trafficking (31). We found that antibodies to markers for early endosomes (EEA1) and late endosomes (LAMP1) did not appear to colocalize significantly with ...
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... localizes to the Golgi complex (38,39). To explore the relationship between IRGM and GBF1, we initially examined the colocalization of these proteins. We found that IRGM and GBF1 colocalized to a high extent in both uninfected (intact Golgi structure) and HCV-infected (fragmented Golgi) cells, as assessed by Mander's coefficient analysis (Fig. 5A). Next, we ex- amined if IRGM affected GBF1. It recently was found that in normal cells undergoing mitosis Golgi fragmentation is induced by AMPKα-dependent phosphorylation of GBF1 at T1337 (40, 41). Another study showed that IRGM stimulates AMPKα by stabilization of the kinase in its T172 phosphorylated form (16). Hence, we examined ...
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... phosphorylated form (16). Hence, we examined the effect of siRNA-mediated depletion of IRGM on HCV-triggered phosphorylation of GBF1T1337 and AMPKαT172, phosphorylation events that mediate the activation of AMPK and GBF1. We found that siRNA-mediated depletion of IRGM efficiently reduced HCV-triggered phosphorylation of GBF1T1337 and AMPKT172 (Fig. 5B), but depletion of IRGM did not influence the expression levels of total GBF1 or ...
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... to the Golgi apparatus (Fig. S9) in noninfected or in HCV-infected cells. Therefore, we suggest that the impact of IRGM on GBF1 is mediated by AMPKα activation rather than by affecting the Golgi residence of GBF1. Next, we examined kinetics of phosphorylation and found that HCV induced phosphorylation of GBF1T1337 starting between 3 and 4 d p.i. (Fig. 5C). We observed a strong increase in GBF1 phos- phorylation, and this increase correlated well with the kinet- ics of AMPKα phosphorylation, which was stimulated from day 3 p.i. (Fig. 5C). Throughout the time course, the total amount of GBF1 and AMPKα remained unchanged (Fig. 5C). Also, the presence of phosphorylated GBF1 at later times ...
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... the Golgi residence of GBF1. Next, we examined kinetics of phosphorylation and found that HCV induced phosphorylation of GBF1T1337 starting between 3 and 4 d p.i. (Fig. 5C). We observed a strong increase in GBF1 phos- phorylation, and this increase correlated well with the kinet- ics of AMPKα phosphorylation, which was stimulated from day 3 p.i. (Fig. 5C). Throughout the time course, the total amount of GBF1 and AMPKα remained unchanged (Fig. 5C). Also, the presence of phosphorylated GBF1 at later times in HCV infection correlated well with the increased amount of Golgi fragments at these times (Fig. 3B), supporting the role of phosphorylated GBF1 in Golgi dispersion by HCV. This ...
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... induced phosphorylation of GBF1T1337 starting between 3 and 4 d p.i. (Fig. 5C). We observed a strong increase in GBF1 phos- phorylation, and this increase correlated well with the kinet- ics of AMPKα phosphorylation, which was stimulated from day 3 p.i. (Fig. 5C). Throughout the time course, the total amount of GBF1 and AMPKα remained unchanged (Fig. 5C). Also, the presence of phosphorylated GBF1 at later times in HCV infection correlated well with the increased amount of Golgi fragments at these times (Fig. 3B), supporting the role of phosphorylated GBF1 in Golgi dispersion by HCV. This finding demonstrates that HCV stimulates the phosphorylation of GBF1. Overall, our results suggest ...
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... IRGM-AMPKα to induce the phos- phorylation of GBF1 at T1337, presumably as a signaling axis of HCV-triggered Golgi fragmentation. Notably, our imaging data showed that in HCV-infected cells GBF1 remains localized to fragmented Golgi structures, whereas our immunoblot analysis shows that GBF1 is phosphorylated at T1337 during HCV infec- tion ( Fig. 5 A and C). This result corroborates a previous study reporting that GBF1 colocalizes with Golgi markers both in con- trol cells and in cells treated with inducers of AMPK activation to cause Golgi fragmentation ...
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... together with IRGM- containing membranes. Arf1 is a small GTPase that exists in a GDP-bound cytosolic form and in a GTP-bound form that is mainly associated with the Golgi. Cells were infected with HCV and stained with antibodies against IRGM or Arf1 at 3 or 6 d p.i. In uninfected cells, both IRGM and Arf1 showed a ribbon- like Golgi pattern (Fig. 5D). In HCV-infected cells, IRGM and Arf1 displayed a dispersed, fragmented pattern. To assess the IRGM-Arf1 interplay in more detail, we quantified the fraction of IRGM that colocalizes with Arf1 and vice versa. To do so, we used Mander's colocalization coefficients (MCC) M1 and M2. MCC strictly measures the cooccurrence of proteins ...
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... with Arf1 and vice versa. To do so, we used Mander's colocalization coefficients (MCC) M1 and M2. MCC strictly measures the cooccurrence of proteins independently of signal proportionality and was useful because the IRGM signal was more intense than that of Arf1. We found that 20% of the total fraction of Arf1 colocalizes with IRGM (M2-Arf1 in Fig. 5D, Right). Interestingly, the fraction of Arf1 that colocalized with IRGM was markedly greater in HCV-infected cells than in un- infected cells, showing that around 80% of Arf1 was localized with IRGM at 6 d p.i. (Fig. 5D, Right). Hence, our results suggest that HCV infection induces an increased association of Arf1 with fragmented Golgi ...
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... IRGM signal was more intense than that of Arf1. We found that 20% of the total fraction of Arf1 colocalizes with IRGM (M2-Arf1 in Fig. 5D, Right). Interestingly, the fraction of Arf1 that colocalized with IRGM was markedly greater in HCV-infected cells than in un- infected cells, showing that around 80% of Arf1 was localized with IRGM at 6 d p.i. (Fig. 5D, Right). Hence, our results suggest that HCV infection induces an increased association of Arf1 with fragmented Golgi membranes and IRGM. (A and B) Cells were transfected with siRNAs against GBF1, Arf1, or control siRNA. GBF1 and Arf1 knockdown were controlled by immunoblotting. HCV protein levels after GBF1 or Arf1 knockdown were analyzed by ...
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... intensity compared with that of Arf1, the colocalization was quantified using MCCs M1, which measures the fraction of protein A in compartments containing protein B, and M2, which measures the fraction of protein B in compart- ments containing protein A. We found that up to 80% of Arf1 colocalized with NS5A, with a slight increase with time p.i. (Fig. 5E). This result could suggest that Arf1 is targeted to HCV replication sites. Therefore we speculate that HCV stim- ulates IRGM-dependent Golgi fragmentation and thereby fa- cilitates the association of Arf1-containing vesicles with HCV replication ...
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Citations
... Together with factors controlling vesicle shaping and trafficking, the different proteins interacting with APOL3 participate in secretion and exocytosis (Fig. 3). (IRGM1) triggers both Golgi fragmentation and autophagy through activation of the AMP-activated-protein-kinase (AMPK), which phosphorylates both the Golgi-specific-brefeldin-A-resistance-guanine-nucleotide-exchange-factor-1 (GBF1) and the Autophagy-1 kinase (ATG1) [42]. While GBF1 induces ARF1 dissociation from ADP-ribosylationfactor-GTPase-activating protein-1 (ARFGAP1) and activates ARF1 (GTP binding), ATG1 initiates autophagy at MERCS pre-autophagosomal structures, inducing the coalescence of Golgi-derived vesicles carrying the lipid scramblase autophagy-related-protein-9A (ATG9A). ...
The functions of human Apolipoproteins L (APOLs) are poorly understood, but involve diverse activities like lysis of bloodstream trypanosomes and intracellular bacteria, modulation of viral infection and induction of apoptosis, autophagy, and chronic kidney disease. Based on recent work, I propose that the basic function of APOLs is the control of membrane dynamics, at least in the Golgi and mitochondrion. Together with neuronal calcium sensor-1 (NCS1) and calneuron-1 (CALN1), APOL3 controls the activity of phosphatidylinositol-4-kinase-IIIB (PI4KB), involved in both Golgi and mitochondrion membrane fission. Whereas secreted APOL1 induces African trypanosome lysis through membrane permeabilization of the parasite mitochondrion, intracellular APOL1 conditions non-muscular myosin-2A (NM2A)-mediated transfer of PI4KB and APOL3 from the Golgi to the mitochondrion under conditions interfering with PI4KB-APOL3 interaction, such as APOL1 C-terminal variant expression or virus-induced inflammatory signalling. APOL3 controls mitophagy through complementary interactions with the membrane fission factor PI4KB and the membrane fusion factor vesicle-associated membrane protein-8 (VAMP8). In mice, the basic APOL1 and APOL3 activities could be exerted by mAPOL9 and mAPOL8, respectively. Perspectives regarding the mechanism and treatment of APOL1-related kidney disease are discussed, as well as speculations on additional APOLs functions, such as APOL6 involvement in adipocyte membrane dynamics through interaction with myosin-10 (MYH10).
... An increasing number of experiments show that virus-triggered GA fragmenta tion functions in various stages of viral replication cycle. Hepatitis C virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) promote self-replication by inducing GA fragmentation (53,54). Influenza A virus induces GA fragmentation to modulate vesicular transport, which facilitates intracellular transport of virus particles (55). ...
... Finally, we demonstrated that PRRSV triggered GA fragmentation-mediated autophagy to facilitate viral self-replication and overexpression of GRASP65 inhibited PRRSV replication when antagonizing GA fragmentation (Fig. 10). Earlier reports show that viruses induce GA fragmentation and utilize GA membrane fragments and vesicles for assembly of replication complexes (53,54,69). Consequently, we have provided a different manner of GA fragmentation for promoting virus replication via autophagy. ...
Porcine reproductive and respiratory syndrome virus (PRRSV) infection results in a serious swine disease affecting pig farming worldwide. Despite that numerous studies have shown that PRRSV triggers autophagy for its self-replication, how PRRSV induces autophagy is incompletely understood. Here, we identify that PRRSV Nsp2 degrades GRASP65 to induce GA fragmentation, which dissociates RAB2 from GM130 and activates RAB2-ULK1-mediated autophagy to enhance viral replication. This work expands our understanding of PRRSV-induced autophagy and PRRSV replication, which is beneficial for anti-viral drug development.
... SARS-CoV2 was recently shown to disrupt the Golgi apparatus via a currently undefined mechanism [15]. Hepatitis C virus was shown to activate the Golgi-localized immunity related GTPase M, which phosphorylates the guanine nucleotide exchange factor GBF1, leading to disruption of the Golgi [16]. Upon bacterial infection, host-cell invading Rickettsia rickettsii and Streptococcus pyogenes were previously shown to disrupt Golgi structure [17,18]. ...
Background:
Lung infections caused by Streptococcus pneumonia are a global leading cause of death. The reactive oxygen species H2O2 is one of the virulence factors of Streptococcus pneumoniae. The Golgi apparatus is essential for the inflammatory response of a eukaryotic cell. Golgi fragmentation was previously shown to be induced by bacterial pathogens and in response to H2O2 treatment. This led us to investigate whether the Golgi apparatus is actively involved and targeted in host-pathogen interactions during pneumococcal infections.
Methods:
Following in vitro infection of BEAS-2B bronchial epithelial cells with Streptococcus pneumoniae for 16 h, the structure of the Golgi apparatus was assessed by fluorescence staining of the Golgi-associated protein, Golgin-97. To investigate the effect of H2O2 production on Golgi structure, BEAS-2B cells were treated with H2O2 or the H2O2 degrading enzyme Catalase, prior to Golgi staining. Artificial disruption of the Golgi apparatus was induced by treatment of cells with the GBF1 inhibitor, Golgicide A. A proinflammatory cellular response was induced by treatment of cells with the bacterial cell wall component and TLR4 ligand lipoteichoic acid.
Results:
In vitro infection of bronchial epithelial cells with wild type Streptococcus pneumoniae led to a disruption of normal Golgi structure. Golgi fragmentation was not observed after deletion of the pneumococcal H2O2-producing gene, spxB, or neutralization of H2O2 by catalase treatment, but could be induced by H2O2 treatment. Streptococcus pneumoniae infection significantly reduced host cell protein glycosylation and artificial disruption of Golgi structure significantly reduced bacterial adherence, but increased bacterial counts in the supernatant. To understand if this effect depended on cell-contact or soluble factors, pneumococci were treated with cell-supernatant of cells treated with Golgicide A and/or lipoteichoic acid. This approach revealed that lipoteichoic acid conditioned medium inhibits bacterial replication in presence of host cells. In contrast, artificial Golgi fragmentation by Golgicide A treatment prior to lipoteichoic acid treatment rescued bacterial replication. This effect was associated with an increase of IL-6 and IL-8 in the supernatant of lipoteichoic acid treated cells. The increased cytokine release was abolished if cells were treated with Golgicide A prior to lipoteichoic acid treatment.
Conclusion:
Streptococcus pneumoniae disrupts the Golgi apparatus in an H2O2-dependent manner, thereby inhibiting paracrine anti-infective mechanisms. Video Abstract.
... For the Golgi morphology assessment, fragmented Golgi was defined as scattered dots (not connected) in the perinuclear region or multiple mini-golgi (isolated dots) dissociated from the major Golgi apparatus based on GM130 staining, a Golgi marker. Quantification of cells with fragmented Golgi per high power field (HPF) was performed using more than 300 cells per experiment with ImageJ software (NIH) as previous described [16,17,37]. ...
Sepsis is a serious clinical condition characterized by a systemic inflammatory response, a leading cause of acute liver and kidney injury, and is associated with a high morbidity and mortality. Understanding the molecular mechanisms underlying the acute liver and kidney injury is crucial for developing an effective therapy. Golgi apparatus plays important roles and has various substrates mediating cellular stress responses. Golgi phosphoprotein 3 (GOLPH3), linking Golgi membranes to the cytoskeleton, has been identified as an important oncogenic regulator; however, its role in endotoxemia-induced acute liver and kidney injury remains elusive. Here, we found that upregulation of GOLPH3 was associated with endotoxemia-induced acute liver and kidney injury. Lipopolysaccharide (LPS) treatment increased Golgi stress and fragmentation, and associated pro-inflammatory mediator (Tnfα, IL-6, and IL-1β) production in vivo and in vitro. Interestingly, the downregulation of GOLPH3 significantly decreased LPS-induced Golgi stress and pro-inflammatory mediators (Tnfα, IL-6, Mcp1, and Nos2), and reversed apoptotic cell deaths in LPS-treated hepatocytes and renal tubular cells. GOLPH3 knockdown also reduced inflammatory response in LPS-treated macrophages. The AKT/NF-kB signaling pathway was suppressed in GOLPH3 knockdown, which may be associated with a reduction of inflammatory response and apoptosis and the recovery of Golgi morphology and function. Taken together, GOLPH3 plays a crucial role in the development and progression of acute liver and kidney injury by promoting Golgi stress and increasing inflammatory response and apoptosis, suggesting GOLPH3 as a potential therapeutic target for endotoxemia-induced tissue injury.
... As an illustration, within the Flaviviridae family, Zika virus and dengue virus are known to induce the development of invaginated vesicles with negative membrane curvature, which are single-membrane structures that harbor dsRNA replication intermediates as well as viral replicase complex proteins (Cortese et al., 2017;Welsch et al., 2009), Whereas hepatitis C virus (HCV) induces the curvature of the endoplasmic reticulum (ER) membrane to create a DMVs web (Romero-Brey et al., 2012). Marianne D. Hansen et al. have demonstrated that in addition to DMVs, HCV also induces Golgi fragmentation and autophagosome formation to create complex MWs that facilitate replication (Hansen et al., 2017). The formation of DMVs has also been observed in other virus families including Picornaviridae, Arteriviridae and Coronaviridae Knoops et al., 2008Knoops et al., , 2012Miller & Krijnse-Locker, 2008). ...
Eukaryotic viruses are obligate intracellular parasites that rely on the host cell machinery to carry out their replication cycle. This complex process involves a series of steps, starting with virus entry, followed by genome replication, and ending with virion assembly and release. Negative strand RNA and some DNA viruses have evolved to alter the organization of the host cell interior to create a specialized environment for genome replication, known as IBs, which are precisely orchestrated to ensure efficient viral replication. The biogenesis of IBs requires the cooperation of both viral and host factors. These structures serve multiple functions during infection, including sequestering viral nucleic acids and proteins from innate immune responses, increasing the local concentration of viral and host factors, and spatially coordinating consecutive replication cycle steps. While ultrastructural and functional studies have improved our understanding of IBs, much remains to be learned about the precise mechanisms of IB formation and function. This review aims to summarize the current understanding of how IBs are formed, describe the morphology of these structures, and highlight the mechanism of their functions. Given that the formation of IBs involves complex interactions between the virus and the host cell, the role of both viral and cellular organelles in this process is also discussed.
... Since CPV-IIs originate from the medial/trans-Golgi [19], Golgi fragmentation may contribute to the formation of CPV-IIs [20]. Virus-induced Golgi fragmentation was previously reported for positive-sense RNA viruses such as picornaviruses and coronaviruses [93][94][95]. Interestingly, in the case of coronaviruses, Golgi fragmentation is induced by the accessory protein open reading frame 3a (ORF3a) which assembles into tetrameric ion channels [96][97][98]. ...
Many viruses encode ion channel proteins that oligomerize to form hydrophilic pores in membranes of virus-infected cells and the viral membrane in some enveloped viruses. Alphavirus 6K, human immunodeficiency virus type 1 Vpu (HIV-Vpu), influenza A virus M2 (IAV-M2), and hepatitis C virus P7 (HCV-P7) are transmembrane ion channel proteins that play essential roles in virus assembly, budding, and entry. While the oligomeric structures and mechanisms of ion channel activity are well-established for M2 and P7, these remain unknown for 6K. Here we investigated the functional role of the ion channel activity of 6K in alphavirus assembly by utilizing a series of Sindbis virus (SINV) ion channel chimeras expressing the ion channel helix from Vpu or M2 or substituting the entire 6K protein with full-length P7, in cis. We demonstrate that the Vpu helix efficiently complements 6K, whereas M2 and P7 are less efficient. Our results indicate that while SINV is primarily insensitive to the M2 ion channel inhibitor amantadine, the Vpu inhibitor 5-N, N-Hexamethylene amiloride (HMA), significantly reduces SINV release, suggesting that the ion channel activity of 6K is similar to Vpu, promotes virus budding. Using live-cell imaging of SINV with a miniSOG-tagged 6K and mCherry-tagged E2, we further demonstrate that 6K and E2 colocalize with the Golgi apparatus in the secretory pathway. To contextualize the localization of 6K in the Golgi, we analyzed cells infected with SINV and SINV-ion channel chimeras using transmission electron microscopy. Our results provide evidence for the first time for the functional role of 6K in type II cytopathic vacuoles (CPV-II) formation. We demonstrate that in the absence of 6K, CPV-II, which originates from the Golgi apparatus, is not detected in infected cells, with a concomitant reduction in the glycoprotein transport to the plasma membrane. Substituting a functional ion channel, M2 or Vpu localizing to Golgi, restores CPV-II production, whereas P7, retained in the ER, is inadequate to induce CPV-II formation. Altogether our results indicate that ion channel activity of 6K is required for the formation of CPV-II from the Golgi apparatus, promoting glycoprotein spike transport to the plasma membrane and efficient virus budding.
... During interphase, the mammalian GA is pericentrosomally anchored and exhibits a stacked structure comprised of a functionally defined set of cisternae (namely, cis-, medial-, and trans) (Klumperman, 2011;Yadav & Linstedt, 2011). However, at instances such as cell division, during certain neurodegenerative conditions and bacterial and viral infections, the GA loses its typical stacked architecture and its pericentrosomal localization (Gahmberg et al, 1986;Campadelli et al, 1993;Lavi et al, 1996;Nakagomi et al, 2008;Heuer et al, 2009;Quiner & Jackson, 2010;Corda et al, 2012;Thayer et al, 2013;Joshi et al, 2014;Kim & Satchell, 2016;Hansen et al, 2017;Wei & Seemann, 2017). Though critical to its function, a clear morphological understanding of the GA membrane remodeling pathways and their exact functional relevance is still lacking. ...
The Golgi apparatus (GA) in mammalian cells is pericentrosomally anchored and exhibits a stacked architecture. During infections by members of the alphavirus genus, the host cell GA is thought to give rise to distinct mobile pleomorphic vacuoles known as CPV-II (cytopathic vesicle-II) via unknown morphological steps. To dissect this, we adopted a phased electron tomography approach to image multiple overlapping volumes of a cell infected with Venezuelan equine encephalitis virus (VEEV) and complemented it with localization of a peroxidase-tagged Golgi marker. Analysis of the tomograms revealed a pattern of progressive cisternal bending into double-lamellar vesicles as a central process underpinning the biogenesis and the morphological complexity of this vacuolar system. Here, we propose a model for the conversion of the GA to CPV-II that reveals a unique pathway of intracellular virus envelopment. Our results have implications for alphavirus-induced displacement of Golgi cisternae to the plasma membrane to aid viral egress operating late in the infection cycle.
... We also screened the co-localization of ALSV proteins with the Golgi apparatus with the marker protein GM130, and found that no viral proteins was co-localize with the Golgi apparatus (Supplementary Figure S4). However, it cannot be ruled out that ALSV proteins interact with the Golgi apparatus by forming the newly synthesized virus particles which are transported from ER and Golgi apparatus to the cell surface (Hansen et al., 2017). ...
Alongshan virus (ALSV) in the Jingmenvirus group within the family Flaviviridae is a newly discovered tick-borne virus associated with human disease, whose genome includes four segments and encodes four structural proteins (VP1a, VP1b, VP2, VP3, and VP4) and two non-structural proteins (NSP1 and NSP2). Here, we characterized the subcellular distribution and potential function of ALSV proteins in host cells. We found that viral proteins exhibited diverse subcellular distribution in multiple tissue-deriving cells and induced various morphological changes in the endoplasmic reticulum (ER), and NSP2, VP1b, VP2, and VP4 were all co-localized in the ER. The nuclear transfer and co-localization of VP4 and calnexin (a marker protein of ER), which were independent of their interaction, were unique to HepG2 cells. Expression of NSP1 could significantly reduce mitochondria quantity by inducing mitophagy. These findings would contribute to better understanding of the pathogenesis of emerging segmented flaviviruses.
... Human IRGM has been extensively investigated as a xenophagy inducer during bacterial infection [12,47,48]. ...
... IRGM is also a fundamental negative regulator of type-1 IFN response against viral pathogens [21,55]. IRGM plays a key role in replication of HCV, an important risk factor for hepatocellular carcinoma, by regulating Golgi fragmentation and leading to co-localization of Golgi vesicles with replicating HCV [48]. Furthermore, autophagosome formation is stimulated in HIV-infected and HCV-infected HeLa cells, via IRGM interaction with HIV-NEF and HCV-NS3 [56]. ...
The human immunity-related GTPase M (IRGM) is a GTP-binding protein that regulates selective autophagy including xenophagy and mitophagy. IRGM impacts autophagy by (1) affecting mitochondrial fusion and fission, (2) promoting the co-assembly of ULK1 and Beclin 1, (3) enhancing Beclin 1 interacting partners (AMBRA1, ATG14L1, and UVRAG), (4) interacting with other key proteins (ATG16L1, p62, NOD2, cGAS, TLR3, and RIG-I), and (5) regulating lysosomal biogenesis. IRGM also negatively regulates NLRP3 inflammasome formation and therefore, maturation of the important pro-inflammatory cytokine IL-1β, impacting inflammation and pyroptosis. Ultimately, this affords protection against chronic inflammatory diseases. Importantly, ten IRGM polymorphisms (rs4859843, rs4859846, rs4958842, rs4958847, rs1000113, rs10051924, rs10065172, rs11747270, rs13361189, and rs72553867) have been associated with human inflammatory disorders including cancer, which suggests that these genetic variants are functionally relevant to the autophagic and inflammatory responses. The current review contextualizes IRGM, its modulation of autophagy, and inflammation, and emphasizes the role of IRGM as a cross point of immunity and tumorigenesis.
... Borders of each cell were drawn, and the ER area was then calculated and presented as a fraction of the total cell area [54]. Intracellular COL6 staining density measurements, such as corrected total cell fluorescence, were carried out with ImageJ [55]. For mCherry-EGFP-LC3B puncta analysis, Mks were outlined manually, and each channel was independently auto-thresholded using the same settings across all images from each channel. ...
Endoplasmic reticulum stress is an emerging significant player in the molecular pathology of connective tissue disorders. In response to endoplasmic reticulum stress, cells can upregulate macroautophagy/autophagy, a fundamental cellular homeostatic process used by cells to degrade and recycle proteins or remove damaged organelles. In these scenarios, autophagy activation can support cell survival. Here we demonstrated by in vitro and in vivo approaches that megakaryocytes derived from col6a1-⁄- (collagen, type VI, alpha 1) null mice display increased intracellular retention of COL6 polypeptides, endoplasmic reticulum stress and apoptosis. The unfolded protein response is activated in col6a1-⁄- megakaryocytes, as evidenced by the upregulation of molecular chaperones, by the increased splicing of Xbp1 mRNA and by the higher level of the pro-apoptotic regulator DDIT3/CHOP. Despite the endoplasmic reticulum stress, basal autophagy is impaired in col6a1-⁄- megakaryocytes, which show lower BECN1 levels and reduced autophagosome maturation. Starvation and rapamycin treatment rescue the autophagic flux in col6a1-⁄- megakaryocytes, leading to a decrease in intracellular COL6 polypeptide retention, endoplasmic reticulum stress and apoptosis. Furthermore, megakaryocytes cultured from peripheral blood hematopoietic progenitors of patients affected by Bethlem myopathy and Ullrich congenital muscular dystrophy, two COL6-related disorders, displayed increased apoptosis, endoplasmic reticulum stress and impaired autophagy. These data demonstrate that genetic disorders of collagens, endoplasmic reticulum stress and autophagy regulation in megakaryocytes may be interrelated.Abbreviations: 7-AAD: 7-amino-actinomycin D; ATF: activating transcriptional factor; BAX: BCL2 associated X protein; BCL2: B cell leukemia/lymphoma 2; BCL2L1/Bcl-xL: BCL2-like 1; BM: bone marrow; COL6: collagen, type VI; col6a1-⁄-: mice that are null for Col6a1; DDIT3/CHOP/GADD153: DNA-damage inducible transcript 3; EGFP: enhanced green fluorescent protein; ER: endoplasmic reticulum; reticulophagy: endoplasmic reticulum-selective autophagy; HSPA5/Bip: heat shock protein 5; HSP90B1/GRP94: heat shock protein 90, beta (Grp94), member 1; LAMP2: lysosomal associated membrane protein 2; MAP1LC3B/LC3B: microtubule-associated protein 1 light chain 3 beta; Mk: megakaryocytes; MTOR: mechanistic target of rapamycin kinase; NIMV: noninvasive mechanical ventilation; PI3K: phosphoinositide 3-kinase; PPP1R15A/GADD34: protein phosphatase 1, regulatory subunit 15A; RT-qPCR: reverse transcription-quantitative real-time PCR; ROS: reactive oxygen species; SERPINH1/HSP47: serine (or cysteine) peptidase inhibitor, clade H, member 1; sh-RNA: short hairpin RNA; SOCE: store operated calcium entry; UCMD: Ullrich congenital muscular dystrophy; UPR: unfolded protein response; WIPI2: WD repeat domain, phosphoinositide-interacting 2; WT: wild type; XBP1: X-box binding protein 1.
