scholarly journals Bicaudal D1-Dependent Trafficking of Human Cytomegalovirus Tegument Protein pp150 in Virus-Infected Cells

2010 ◽  
Vol 84 (7) ◽  
pp. 3162-3177 ◽  
Author(s):  
Sabarish V. Indran ◽  
Mary E. Ballestas ◽  
William J. Britt
Keyword(s):  
Human Cytomegalovirus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Viral Assembly ◽  
Dominant Negative ◽  
Tegument Protein ◽  
Viral Glycoprotein ◽  
Infected Cells

ABSTRACT Human cytomegalovirus (HCMV) virion assembly takes place in the nucleus and cytoplasm of infected cells. The HCMV virion tegument protein pp150 (ppUL32) is an essential protein of HCMV and has been suggested to play a role in the cytoplasmic phase of HCMV assembly. To further define its role in viral assembly and to identify host cell proteins that interact with pp150 during viral assembly, we utilized yeast two-hybrid analyses to detect an interaction between pp150 and Bicaudal D1 (BicD1), a protein thought to play a role in trafficking within the secretory pathway. BicD1 is known to interact with the dynein motor complex and the Rab6 GTPase. The interaction between pp150 and BicD1 was confirmed by coimmunoprecipitation and fluorescence resonance energy transfer. Depletion of BicD1 with short hairpin RNA (shRNA) caused decreased virus yield and a defect in trafficking of pp150 to the cytoplasmic viral assembly compartment (AC), without altering trafficking to the AC of another essential tegument protein, pp28, or the viral glycoprotein complex gM/gN. The C terminus of BicD1 has been previously shown to interact with the GTPase Rab6, suggesting a potential role for Rab6-mediated vesicular trafficking in HCMV assembly. Finally, overexpression of the N terminus of truncated BicD1 acts in a dominant-negative manner and leads to disruption of the AC and a decrease in the assembly of infectious virus. This phenotype was similar to that observed following overexpression of dynamitin (p50) and provided additional evidence that morphogenesis of the AC and virus assembly were dynein dependent.

scholarly journals Human Cytomegalovirus pUL47 Modulates Tegumentation and Capsid Accumulation at the Viral Assembly Complex

2015 ◽  
Vol 89 (14) ◽  
pp. 7314-7328 ◽  
Author(s):  
Ilaria Cappadona ◽  
Clarissa Villinger ◽  
Gabi Schutzius ◽  
Thomas Mertens ◽  
Jens von Einem
Keyword(s):  
Human Cytomegalovirus ◽  
Nuclear Localization ◽  
Protein Interactions ◽  
Viral Assembly ◽  
Tegument Protein ◽  
Cell Periphery ◽  
Morphological Alterations ◽  
Tegument Proteins ◽  
Enveloped Virus ◽  
Infected Cells

ABSTRACTHuman cytomegalovirus (HCMV) tegument protein pUL47 is an interaction partner of pUL48 and highly conserved among herpesviruses. It is closely associated with the capsid and has an important function early in infection. Here, we report a specific role of pUL47 in the tegumentation of capsids in the cytoplasm. A newly generated mutant virus (TB-47stop), in which expression of pUL47 is blocked, exhibited a severe impairment in cell-to-cell spread and release of infectivity from infected cells. Ultrastructural analysis of TB-47stop-infected cells clearly showed cytoplasmic accumulations of nonenveloped capsids that were only partially tegumented, indicating that these capsids failed to complete tegumentation. Nevertheless, these accumulations were positive for HCMV inner tegument proteins pp150 and pUL48, suggesting that their attachment to capsids occurs independently of pUL47. Despite these morphological alterations, fully enveloped virus particles were found in the extracellular space and at the viral assembly complex (vAC) of TB-47stop-infected cells, indicating that pUL47 is not essential for the generation of virions. We confirmed findings that incorporation of pUL48 into virions is impaired in the absence of pUL47. Interestingly, pUL47 exhibited a strong nuclear localization in transfected cells, whereas it was found exclusively at the vAC in the context of virus infection. Colocalization of pUL47 and pUL48 at the vAC is consistent with their interaction. We also found a shift to a more nuclear localization of pUL47 when the expression of pUL48 was reduced. Summarizing our results, we hypothesize that pUL48 directs pUL47 to the vAC to promote tegumentation and secondary envelopment of capsids.IMPORTANCEGeneration of infectious HCMV particles requires an organized and multistep process involving the action of several viral and cellular proteins as well as protein-protein interactions. A better understanding of these processes is important for understanding the biology of HCMV and may help to identify targets for antiviral intervention. Here, we identified tegument protein pUL47 to function in tegumentation and proper trafficking of capsids during late phases of infection. Although pUL47 is not essential for the generation and release of infectious virions, its absence led to massive accumulations of partially tegumented capsids at the cell periphery. Detection of pUL48 at these accumulations indicated a pUL47-independent attachment of pUL48 to the capsid. On the other hand, localization of pUL47 to the vAC during infection appeared to be dependent on tegument protein pUL48, which suggests an intricate interplay of these proteins for normal generation of infectious virus progeny.

scholarly journals Human Cytomegalovirus pp28 (UL99) Localizes to a Cytoplasmic Compartment Which Overlaps the Endoplasmic Reticulum-Golgi-Intermediate Compartment

2000 ◽  
Vol 74 (8) ◽  
pp. 3842-3851 ◽  
Author(s):  
Veronica Sanchez ◽  
Elizabeth Sztul ◽  
William J. Britt
Keyword(s):  
Endoplasmic Reticulum ◽  
Human Cytomegalovirus ◽  
Protein Interactions ◽  
Secretory Pathway ◽  
Tegument Protein ◽  
Tegument Proteins ◽  
Intracellular Compartments ◽  
Infected Cells ◽  
Considerable Controversy ◽  
Intermediate Compartment

ABSTRACT Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for α- and β-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [3H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.

scholarly journals Cyclin-Dependent Kinase Activity Is Required for Efficient Expression and Posttranslational Modification of Human Cytomegalovirus Proteins and for Production of Extracellular Particles

2006 ◽  
Vol 80 (12) ◽  
pp. 5886-5896 ◽  
Author(s):  
Veronica Sanchez ◽  
Deborah H. Spector
Keyword(s):  
Steady State ◽  
Human Cytomegalovirus ◽  
Virus Titer ◽  
State Level ◽  
Early Gene ◽  
Tegument Protein ◽  
Cyclin Dependent Kinase ◽  
Viral Dna ◽  
Infected Cells ◽  
Steady State Levels

ABSTRACT We have previously shown that the addition of the cyclin-dependent kinase (cdk) inhibitor Roscovitine at the beginning of infection of cells with human cytomegalovirus (HCMV) significantly disrupts immediate-early gene expression and the progression of the infection. In the present study, we have examined the effects of cdk inhibition on late viral events by delaying addition of Roscovitine until 24 h postinfection. Although viral DNA replication was inhibited two- to threefold by treatment of infected cells with Roscovitine, the drop did not correspond to the 1- to 2-log-unit decrease in virus titer. Quantification of viral DNA in the supernatant from cells revealed that there was a significant reduction in the production or release of extracellular particles. We observed a lag in the expression of several viral proteins but there was a significant decrease in the steady-state levels of IE2-86. Likewise, the steady-state level of the essential tegument protein UL32 (pp150) was reduced. The levels of pp150 and IE2-86 mRNA were not greatly affected by treatment with Roscovitine and thus did not correlate with the reduced levels of protein. In contrast, the expression of the tegument protein ppUL69 was higher in drug-treated samples, and the protein accumulated in a hyperphosphorylated form. ppUL69 localized to intranuclear aggregates that did not overlap with viral replication centers in cells treated with Roscovitine. Taken together, these data indicate that cdk activity is required at multiple steps during HCMV infection, including the expression, modification, and localization of virus-encoded proteins.

scholarly journals Interaction of Human Cytomegalovirus Tegument Proteins ppUL35 and ppUL35A with Sorting Nexin 5 Regulates Glycoprotein B (gpUL55) Localization

2018 ◽  
Vol 92 (9) ◽  
Author(s):  
Gregor Maschkowitz ◽  
Sabine Gärtner ◽  
Heike Hofmann-Winkler ◽  
Helmut Fickenscher ◽  
Michael Winkler
Keyword(s):  
Human Cytomegalovirus ◽  
Retrograde Transport ◽  
Viral Assembly ◽  
Glycoprotein B ◽  
Tegument Protein ◽  
Immunocompromised Patients ◽  
Structural Protein ◽  
Transport Pathway ◽  
Sorting Nexin ◽  
Sorting Nexin 5

ABSTRACTHuman cytomegalovirus (HCMV) is a widespread human pathogen that causes asymptomatic infection in healthy individuals but poses a serious threat to immunocompromised patients. During the late phase of HCMV infection, the viral capsid is transported to the cytoplasmic viral assembly center (cVAC), where it is enclosed by the tegument protein layer and the viral envelope. The cVAC consists of circularly arranged vesicles from thetrans-Golgi and endosomal networks. The HCMV gene UL35 encodes ppUL35 and its shorter form, ppUL35A. We have previously shown that the UL35 gene is involved in HCMV assembly, but it is unknown how UL35 proteins regulate viral assembly. Here we show that sorting nexin 5 (SNX5), a component of the retromer and part of the retrograde transport pathway, interacts with UL35 proteins. Expression of wild-type proteins but not mutants defective in SNX5 binding resulted in the cellular redistribution of the cation-independent mannose-6-phosphate receptor (CI-M6PR), indicating that UL35 proteins bind and negatively regulate SNX5 to modulate cellular transport pathways. Furthermore, binding of UL35 proteins to SNX5 was required for efficient viral replication and for transport of the most abundant HCMV glycoprotein B (gB; gpUL55) to the cVAC. These results indicate that ppUL35 and ppUL35A control the localization of the essential gB through the regulation of a retrograde transport pathway. Thus, this work is the first to define a molecular interaction between a tegument protein and a vesicular transport factor to regulate glycoprotein localization.IMPORTANCEHuman cytomegalovirus is ubiquitously present in the healthy population, but reactivation or reinfection can cause serious, life-threatening infections in immunocompromised patients. For completion of its lytic cycle, human cytomegalovirus induces formation of an assembly center where mature virus particles are formed from multiple viral proteins. Viral glycoproteins use separate vesicular pathways for transport to the assembly center, which are incompletely understood. Our research identified a viral structural protein which affects the localization of one of the major glycoproteins. We could link this change in glycoprotein localization to an interaction of the structural protein with a cellular protein involved in regulation of vesicle transport. This increases our understanding of how the virus intersects into cellular regulatory pathways to enhance its own replication.

scholarly journals PKC-α mediates flow-stimulated superoxide production in thick ascending limbs

2010 ◽  
Vol 298 (4) ◽  
pp. F885-F891 ◽  
Author(s):  
Nancy J. Hong ◽  
Guillermo B. Silva ◽  
Jeffrey L. Garvin
Keyword(s):  
Nadph Oxidase ◽  
Oxidase Activity ◽  
Mesangial Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Oxidase Inhibitor ◽  
Dominant Negative ◽  
Aortic Endothelial Cells ◽  
Nadph Oxidase Activity ◽  
Measured Flow

We showed that luminal flow increases net superoxide (O2−) production via NADPH oxidase in thick ascending limbs. Protein kinase C (PKC) activates NADPH oxidase activity in phagocytes, cardiomyocytes, aortic endothelial cells, vascular smooth muscle cells, and renal mesangial cells. However, the flow-activated pathway that induces NADPH oxidase activity in thick ascending limbs is unclear. We hypothesized that PKC mediates flow-stimulated net O2− production by thick ascending limbs. Initiation of flow (20 nl/min) increased net O2− production from 4 ± 1 to 61 ± 12 AU/s ( P < 0.007; n = 5). The NADPH oxidase inhibitor apocynin completely blocked the flow-induced increase in net O2− production (2 ± 1 vs. 1 ± 1 AU/s; P > 0.05; n = 5). Flow-stimulated O2− was also blocked in p47phox-deficient mice. We measured flow-stimulated PKC activity with a fluorescence resonance energy transfer (FRET)-based membrane-targeted PKC activity reporter and found that the FRET ratio increased from 0.87 ± 0.02 to 0.96 ± 0.04 AU ( P < 0.05; n = 6). In the absence of flow, the PKC activator phorbol 12-myristate 13-acetate (200 nM) enhanced net O2− production from 5 ± 2 to 92 ± 6 AU/s ( P < 0.001; n = 6). The PKC-α- and βI-selective inhibitor Gö 6976 (100 nM) decreased flow-stimulated net O2− production from 54 ± 15 to 2 ± 1 AU/s ( P < 0.04; n = 5). Flow-induced net O2− production was inhibited in thick ascending limbs transduced with dominant-negative (dn)PKC-α but not dnPKCβI or LacZ (Δ = 11 ± 3 AU/s for dnPKCα, 55 ± 7 AU/s for dnPKCβI, and 63 ± 7 AU/s for LacZ; P < 0.001; n = 6). We concluded that flow stimulates net O2− production in thick ascending limbs via PKC-α-mediated activation of NADPH oxidase.

scholarly journals Activation of the NF-κB Pathway in Human Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major Immediate-Early Promoter

2004 ◽  
Vol 78 (9) ◽  
pp. 4498-4507 ◽  
Author(s):  
Ian B. DeMeritt ◽  
Liesl E. Milford ◽  
Andrew D. Yurochko
Keyword(s):  
Human Cytomegalovirus ◽  
Time Course ◽  
Hcmv Infection ◽  
Dominant Negative ◽  
Immediate Early ◽  
Primary Role ◽  
Early Promoter ◽  
Ikk Complex ◽  
Infected Cells ◽  
Major Immediate Early Promoter

ABSTRACT We previously demonstrated that human cytomegalovirus (HCMV) infection induced the activation of the cellular transcription factor NF-κB. Here, we investigate the mechanism for the HCMV-induced NF-κB activation and the role that the induced NF-κB plays in transactivation of the major immediate-early promoter (MIEP) and production of immediate-early (IE) proteins. Using a dominant-negative inhibitor of NF-κB, the IκB-superrepressor, we demonstrated that active NF-κB is critical for transactivation of the HCMV MIEP. Investigation of the mechanisms of NF-κB activation following HCMV infection showed a rapid and sustained decrease in the inhibitors of NF-κB, IκBα and IκBβ. Because the IκB kinases (IKKs) regulate the degradation of the IκBs, virus-mediated changes in the IKKs were examined next. Using dominant-negative forms of the IKKs, we showed significant decreases in transactivation of the MIEP in the presence of these mutants. In addition, protein levels of members of the IKK complex and IKK kinase activity were upregulated throughout the time course of infection. Lastly, the role NF-κB plays in HCMV IE mRNA and protein production during infection was examined. Using aspirin and MG-132, we demonstrated that production of IE protein and mRNA was significantly decreased and delayed in infected cells treated with these drugs. Together, the results of these studies suggest that virus-mediated NF-κB activation, through the dysregulation of the IKK complex, plays a primary role in the initiation of the HCMV gene cascade in fibroblasts and may provide new targets for therapeutic intervention.

scholarly journals The Src Family Kinase c-Yes Is Required for Maturation of West Nile Virus Particles

2005 ◽  
Vol 79 (18) ◽  
pp. 11943-11951 ◽  
Author(s):  
Alec J. Hirsch ◽  
Guruprasad R. Medigeshi ◽  
Heather L. Meyers ◽  
Victor DeFilippis ◽  
Klaus Früh ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Secretory Pathway ◽  
Virus Assembly ◽  
Cellular Protein ◽  
Viral Envelope ◽  
Src Family Kinase ◽  
West Nile ◽  
Virus Particles ◽  
Cellular Genes ◽  
Infected Cells

ABSTRACT The role of cellular genes in West Nile virus (WNV) replication is not well understood. Examination of cellular transcripts upregulated during WNV infection revealed an increase in the expression of the src family kinase (SFK) c-Yes. WNV-infected cell lines treated with the SFK inhibitor PP2 demonstrated a 2- to 4-log decrease in viral titers, suggesting that SFK activity is required for completion of the viral replication cycle. RNA interference mediated knock-down of c-Yes, but not c-Src, and similarly reduced virus yield, specifically implicating c-Yes in WNV production. Interestingly, PP2 treatment did not reduce intracellular levels of either viral RNA or protein, suggesting that the drug does not act on the early stages of replication. However, endoglycosidase H (endoH) digestion of the viral envelope (E) glycoprotein revealed that the acquisition of endoH-resistant glycans by E, but not endogenous major histocompatibility complex class I, was reduced in PP2-treated cells, demonstrating that E specifically does not traffic beyond the endoplasmic reticulum in the absence of SFK activity. Electron microscopy further revealed that PP2-treated WNV-infected cells accumulated an increased number of virions in the ER compared to untreated cells. Therefore, we conclude that inhibition of SFK activity did not interfere with virus assembly but prevented transit of virions through the secretory pathway. These results identify c-Yes as a cellular protein that is involved in WNV assembly and egress.

scholarly journals Sequence Requirements for Localization of Human Cytomegalovirus Tegument Protein pp28 to the Virus Assembly Compartment and for Assembly of Infectious Virus

2006 ◽  
Vol 80 (11) ◽  
pp. 5611-5626 ◽  
Author(s):  
Jun-Young Seo ◽  
William J. Britt
Keyword(s):  
Amino Acids ◽  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Virus Assembly ◽  
Tegument Protein ◽  
Reading Frame ◽  
Transfected Cells ◽  
Recombinant Viruses ◽  
Assembly Compartment ◽  
Sequence Requirements

ABSTRACT The human cytomegalovirus UL99 open reading frame encodes a 190-amino-acid (aa) tegument protein, pp28, that is myristoylated and phosphorylated. pp28 is essential for assembly of infectious virus, and nonenveloped virions accumulate in the cytoplasm of cells infected with recombinant viruses with a UL99 deletion. pp28 is localized to the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) in transfected cells, while in infected cells, it is localized together with other virion proteins in a juxtanuclear compartment termed the assembly compartment (AC). We investigated the sequence requirements for pp28 trafficking to the AC and assembly of infectious virus. Our studies indicated that the first 30 to 35 aa were required for localization of pp28 to the ERGIC in transfected cells. Mutant forms of pp28 containing only the first 35 aa localized with other virion structural proteins to cytoplasmic compartments early in infection, but localization to the AC at late times required a minimum of 50 aa. In agreement with previous reports, we demonstrated that the deletion of a cluster of acidic amino acids (aa 44 to 59) prevented wild-type trafficking of pp28 and recovery of infectious virus. A recombinant virus expressing only the first 50 aa was replication competent, and this mutant, pp28, localized to the AC in cells infected with this virus. These findings argued that localization of pp28 to the AC was essential for assembly of infectious virus and raised the possibility that amino acids in the amino terminus of pp28 have additional roles in the envelopment and assembly of the virion other than simply localizing pp28 to the AC.

scholarly journals IL-2 receptors preassemble and signal in the ER/Golgi causing resistance to antiproliferative anti–IL-2Rα therapies

2019 ◽  
Vol 116 (42) ◽  
pp. 21120-21130 ◽  
Author(s):  
Julianna Volkó ◽  
Ádám Kenesei ◽  
Meili Zhang ◽  
Péter Várnai ◽  
Gábor Mocsár ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Surface ◽  
T Cell Activation ◽  
Cell Activation ◽  
Secretory Pathway ◽  
Interleukin 2 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Autocrine Signaling

Interleukin-2 (IL-2) and IL-15 play pivotal roles in T cell activation, apoptosis, and survival, and are implicated in leukemias and autoimmune diseases. Their heterotrimeric receptors share their β- and γc-chains, but have distinct α-chains. Anti–IL-2Rα (daclizumab) therapy targeting cell surface-expressed receptor subunits to inhibit T cell proliferation has only brought limited success in adult T cell leukemia/lymphoma (ATL) and in multiple sclerosis. We asked whether IL-2R subunits could already preassemble and signal efficiently in the endoplasmic reticulum (ER) and the Golgi. A combination of daclizumab and anti–IL-2 efficiently blocked IL-2–induced proliferation of IL-2–dependent wild-type (WT) ATL cells but not cells transfected with IL-2, suggesting that in IL-2–producing cells signaling may already take place before receptors reach the cell surface. In the Golgi fraction isolated from IL-2–producing ATL cells, we detected by Western blot phosphorylated Jak1, Jak3, and a phosphotyrosine signal attributed to the γc-chain, which occurred at much lower levels in the Golgi of WT ATL cells. We expressed EGFP- and mCherry-tagged receptor chains in HeLa cells to study their assembly along the secretory pathway. Confocal microscopy, Förster resonance energy transfer, and imaging fluorescence cross-correlation spectroscopy analysis revealed partial colocalization and molecular association of IL-2 (and IL-15) receptor chains in the ER/Golgi, which became more complete in the plasma membrane, further confirming our hypothesis. Our results define a paradigm of intracellular autocrine signaling and may explain resistance to antagonistic antibody therapies targeting receptors at the cell surface.

scholarly journals Retrieval of Human Cytomegalovirus Glycoprotein B from Cell Surface Is Not Required for Virus Envelopment in Astrocytoma Cells

2002 ◽  
Vol 76 (10) ◽  
pp. 5147-5155 ◽  
Author(s):  
Michael A. Jarvis ◽  
Kenneth N. Fish ◽  
Cecilia Söderberg-Naucler ◽  
Daniel N. Streblow ◽  
Heather L. Meyers ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Infectious Virus ◽  
Critical Role ◽  
Dominant Negative ◽  
Glycoprotein B ◽  
Virion Assembly ◽  
Virion Envelope ◽  
Infected Cells ◽  
Dynamin I ◽  
Golgi Network

ABSTRACT Human cytomegalovirus (HCMV) is a prototypic member of the betaherpesvirus family. The HCMV virion is composed of a large DNA genome encapsidated within a nucleocapsid, which is wrapped within an inner proteinaceous tegument and an outer lipid envelope containing viral glycoproteins. Although genome encapsidation clearly occurs in the nucleus, the subsequent steps in the virion assembly process are unclear. HCMV glycoprotein B (gB) is a major component of the virion envelope that plays a critical role in virus entry and is essential for the production of infectious virus progeny. The aim of our present study was to identify the secretory compartment to which HCMV gB was localized and to investigate the role of endocytosis in mediating gB localization and HCMV biogenesis. We show that HCMV gB is localized to the trans-Golgi network (TGN) in HCMV-infected cells and that gB contains all of the trafficking information necessary for TGN localization. Endocytosis of gB was shown to play a role in mediating TGN localization of gB and in targeting of the protein to the site of virus envelopment. However, inhibition of endocytosis with a dominant-negative dynamin I molecule did not affect the production of infectious virus. These observations indicate that, although endocytosis is involved in the trafficking of gB to the site of glycoprotein accumulation in the TGN, endocytosis of gB is not required for the production of infectious HCMV.

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