The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Elizabeth Sztul - One of the best experts on this subject based on the ideXlab platform.
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modeling the dynamic behaviors of the copi vesicle formation regulators the small gtpase arf1 and its activating sec7 guanine nucleotide exchange factor GBF1 on golgi membranes
bioRxiv, 2020Co-Authors: Garrett Sager, John F Presley, Ryoichi Kawai, Elizabeth SztulAbstract:ABSTRACT The components and subprocesses underlying the formation of COPI-coated vesicles at the Golgi are well understood. The coating cascade is initiated after the small GTPase Arf1 is activated by the Sec7 domain-containing guanine nucleotide exchange factor GBF1. This causes a conformational shift within Arf1 that facilitates stable association of Arf1 with the membrane, a process required for subsequent recruitment of the COPI coat. Although we have an atomic level knowledge of Arf1 activation by Sec7 domain-containing GEFs, our understanding of the biophysical parameters that regulate Arf1 and GBF1 association with Golgi membranes and with each other is limited. We used Fluorescence Recovery After Photobleaching (FRAP) data and kinetic Monte Carlo simulation based on continuous-time random walk to assess behavior of Arf1 and GBF1 during COPI vesicle formation in live cells. Our analyses support a model in which Arf1 and GBF1 associate with Golgi membranes independently, with an excess of GBF1 relative to Arf1, and in which Arf1 activation is much faster than GBF1 cycling on the membrane. Interestingly, modeling the behavior of the GBF1/E794K mutant stabilized on the membrane is inconsistent with the formation of a stable complex between it and an endogenous Arf1, and suggests that its prolonged association with the membrane occurs independently of complex formation.
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regulating the regulators role of phosphorylation in modulating the function of the GBF1 big family of sec7 arf gefs
FEBS Letters, 2020Co-Authors: Kendall Walton, Andre Leier, Elizabeth SztulAbstract:Membrane traffic between secretory and endosomal compartments is vesicle-mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF-GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the Golgi-specific brefeldin A-resistance factor 1 (GBF1)/brefeldin A-inhibited guanine nucleotide exchange protein (BIG) family of ARF-GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N-terminal domains of the GBF1/BIG ARF-GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C-terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the nonconserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2.
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a redundant mechanism of recruitment underlies the remarkable plasticity of the requirement of poliovirus replication for the cellular arfgef GBF1
Journal of Virology, 2019Co-Authors: Ekaterina G Viktorova, Elizabeth Sztul, Eunjoo Lee, Justyna M Meissner, Samuel Gabaglio, Seyedehmahsa Moghimi, George A BelovAbstract:The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.
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The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication.
Journal of virology, 2019Co-Authors: Jose L Martinez, Elizabeth Sztul, Eunjoo Lee, Francesca Arnoldi, Elisabeth M. Schraner, Catherine Eichwald, Daniela Silva-ayala, Oscar R. Burrone, Susana López, Carlos F AriasAbstract:Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.
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promiscuity of the catalytic sec7 domain within the guanine nucleotide exchange factor GBF1 in arf activation golgi homeostasis and effector recruitment
Molecular Biology of the Cell, 2019Co-Authors: Jay M Bhatt, George A Belov, Ekaterina G Viktorova, Eunjoo Lee, Justyna M Meissner, William D Hancock, Aneta Kaczmarczyk, Sasanka Ramanadham, Elizabeth SztulAbstract:Using a chimera in which the Sec7d of GBF1 was replaced with the Sec7d from ARNO, we show that 1) targeting of GEFs is independent of Sec7d, but Sec7d influences GEF substrate specificity to affect...
Paul Melançon - One of the best experts on this subject based on the ideXlab platform.
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bioid performed on golgi enriched fractions identify c10orf76 as a GBF1 binding protein essential for golgi maintenance and secretion
Molecular & Cellular Proteomics, 2019Co-Authors: Calvin J Chan, Kaylan Burns, Khadra Ahmed, Etienne Coyaud, Estelle M N Laurent, Brian Raught, Paul MelançonAbstract:The Golgi-specific Brefeldin-A resistance factor 1 (GBF1) is the only large GEF that regulates Arf activation at the cis-Golgi and is actively recruited to membranes on an increase in Arf-GDP. Recent studies have revealed that GBF1 recruitment requires one or more heat-labile and protease-sensitive protein factor(s) (Quilty et al., 2018, J. Cell Science, 132). Proximity-dependent biotinylation (BioID) and mass spectrometry from enriched Golgi fractions identified GBF1 proximal proteins that may regulate its recruitment. Knockdown studies revealed C10orf76 to be involved in Golgi maintenance. We find that C10orf76 interacts with GBF1 and rapidly cycles on and off GBF1-positive Golgi structures. More importantly, its depletion causes Golgi fragmentation, alters GBF1 recruitment, and impairs secretion. Homologs were identified in most species, suggesting its presence in the last eukaryotic common ancestor.
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the arf gdp regulated recruitment of GBF1 to golgi membranes requires domains hds1 and hds2 and a golgi localized protein receptor
Journal of Cell Science, 2019Co-Authors: Douglas Quilty, Calvin J Chan, Katherine Yurkiw, Alexandra Bain, Ghazal Babolmorad, Paul MelançonAbstract:We previously proposed a novel mechanism by which the enzyme Golgi-specific Brefeldin A resistance factor 1 (GBF1) is recruited to the membranes of the cis-Golgi, based on in vivo experiments. Here, we extended our in vivo analysis on the production of regulatory Arf-GDP and observed that ArfGAP2 and ArfGAP3 do not play a role in GBF1 recruitment. We confirm that Arf-GDP localization is critical, as a TGN-localized Arf-GDP mutant protein fails to promote GBF1 recruitment. We also reported the establishment of an in vitro GBF1 recruitment assay that supports the regulation of GBF1 recruitment by Arf-GDP. This in vitro assay yielded further evidence for the requirement of a Golgi-localized protein because heat denaturation or protease treatment of Golgi membranes abrogated GBF1 recruitment. Finally, combined in vivo and in vitro measurements indicated that the recruitment to Golgi membranes via a putative receptor requires only the HDS1 and HDS2 domains in the C-terminal half of GBF1.
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arf activation at the golgi is modulated by feed forward stimulation of the exchange factor GBF1
Journal of Cell Science, 2013Co-Authors: Douglas Quilty, Fraser Gray, Nathan Summerfeldt, Dan Cassel, Paul MelançonAbstract:ADP-ribosylation factors (Arfs) play central roles in the regulation of vesicular trafficking through the Golgi. Arfs are activated at the Golgi membrane by guanine-nucleotide-exchange factors (GEFs) that are recruited from cytosol. Here, we describe a novel mechanism for the regulation of recruitment and activity of the ArfGEF Golgi-specific BFA resistance factor 1 (GBF1). Conditions that alter the cellular Arf-GDP:Arf-GTP ratio result in GBF1 recruitment. This recruitment of GBF1 occurs selectively on cis-Golgi membranes in direct response to increased Arf-GDP. GBF1 recruitment requires Arf-GDP myristoylation-dependent interactions suggesting regulation of a membrane-bound factor. Once recruited, GBF1 causes increased Arf-GTP production at the Golgi, consistent with a feed-forward self-limiting mechanism of Arf activation. This mechanism is proposed to maintain steady-state levels of Arf-GTP at the cis-Golgi during cycles of Arf-dependent trafficking events.
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characterization of class i and ii adp ribosylation factors arfs in live cells gdp bound class ii arfs associate with the er golgi intermediate compartment independently of GBF1
Molecular Biology of the Cell, 2008Co-Authors: Justin Chun, John F Presley, Zoya Shapovalova, Selma Yilmaz Dejgaard, Paul MelançonAbstract:Despite extensive work on ADP-ribosylation factor (Arf) 1 at the Golgi complex, the functions of Arf2–5 in the secretory pathway, or for that of any Arf at the ER-Golgi intermediate compartment (ERGIC) remain uncharacterized. Here, we examined the recruitment of fluorescently tagged Arf1, -3, -4, and -5 onto peripheral ERGIC. Live cell imaging detected Arfs on peripheral puncta that also contained Golgi-specific brefeldin A (BFA) resistance factor (GBF) 1 and the ERGIC marker p58. Unexpectedly, BFA did not promote corecruitment of Arfs with GBF1 either at the Golgi complex or the ERGIC, but it uncovered striking differences between Arf1,3 and Arf4,5. Although Arf1,3 quickly dissociated from all endomembranes after BFA addition, Arf4,5 persisted on ERGIC structures, even after redistribution of GBF1 to separate compartments. The GDP-arrested Arf4(T31N) mutant localized to the ERGIC, even with BFA and Exo1 present. In addtion, loss of Arf · GTP after treatment with Exo1 caused rapid release of all Arfs from the Golgi complex and led to GBF1 accumulation on both Golgi and ERGIC membranes. Our results demonstrate that GDP-bound Arf4,5 associate with ERGIC membranes through binding sites distinct from those responsible for GBF1 recruitment. Furthermore, they provide the first evidence that GBF1 accumulation on membranes may be caused by loss of Arf · GTP, rather than the formation of an Arf · GDP · BFA · GBF1 complex.
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Distinct Functions for Arf Guanine Nucleotide Exchange Factors at the Golgi Complex: GBF1 and BIGs Are Required for Assembly and Maintenance of the Golgi Stack and trans-Golgi Network, Respectively
Molecular biology of the cell, 2007Co-Authors: Florin Manolea, Alejandro Claude, Justin Chun, Javier Rosas, Paul MelançonAbstract:We examined the relative function of the two classes of guanine nucleotide exchange factors (GEFs) for ADP-ribosylation factors that regulate recruitment of coat proteins on the Golgi complex. Complementary overexpression and RNA-based knockdown approaches established that GBF1 regulates COPI recruitment on cis-Golgi compartments, whereas BIGs appear specialized for adaptor proteins on the trans-Golgi. Knockdown of GBF1 and/or COPI did not prevent export of VSVGtsO45 from the endoplasmic reticulum (ER), but caused its accumulation into peripheral vesiculotubular clusters. In contrast, knockdown of BIG1 and BIG2 caused loss of clathrin adaptor proteins and redistribution of several TGN markers, but had no impact on COPI and several Golgi markers. Surprisingly, brefeldin A–inhibited guanine nucleotide exchange factors (BIGs) knockdown prevented neither traffic of VSVGtsO45 to the plasma membrane nor assembly of a polarized Golgi stack. Our observations indicate that COPII is the only coat required for sorting and export from the ER exit sites, whereas GBF1 but not BIGs, is required for COPI recruitment, Golgi subcompartmentalization, and cargo progression to the cell surface.
George A Belov - One of the best experts on this subject based on the ideXlab platform.
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a redundant mechanism of recruitment underlies the remarkable plasticity of the requirement of poliovirus replication for the cellular arfgef GBF1
Journal of Virology, 2019Co-Authors: Ekaterina G Viktorova, Elizabeth Sztul, Eunjoo Lee, Justyna M Meissner, Samuel Gabaglio, Seyedehmahsa Moghimi, George A BelovAbstract:The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.
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promiscuity of the catalytic sec7 domain within the guanine nucleotide exchange factor GBF1 in arf activation golgi homeostasis and effector recruitment
Molecular Biology of the Cell, 2019Co-Authors: Jay M Bhatt, George A Belov, Ekaterina G Viktorova, Eunjoo Lee, Justyna M Meissner, William D Hancock, Aneta Kaczmarczyk, Sasanka Ramanadham, Elizabeth SztulAbstract:Using a chimera in which the Sec7d of GBF1 was replaced with the Sec7d from ARNO, we show that 1) targeting of GEFs is independent of Sec7d, but Sec7d influences GEF substrate specificity to affect...
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highly conserved motifs within the large sec7 arf guanine nucleotide exchange factor GBF1 target it to the golgi and are critical for GBF1 activity
American Journal of Physiology-cell Physiology, 2018Co-Authors: Cristian A Pocognoni, George A Belov, Ekaterina G Viktorova, Justyna M Meissner, Garrett Sager, John Wright, Elizabeth SztulAbstract:Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by GBF1, a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of the Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4 and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based "replacement" assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion and sustaining cellular viability. We show that cells treated with the pharmacological GEF inhibitor Brefeldin A...
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highly conserved motifs within the large sec7 arf guanine nucleotide exchange factor GBF1 target it to the golgi and are critical for GBF1 activity
American Journal of Physiology-cell Physiology, 2018Co-Authors: Cristian A Pocognoni, George A Belov, Ekaterina G Viktorova, Eunjoo Lee, J T Wright, Justyna M Meissner, Garrett Sager, Elizabeth SztulAbstract:Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catal...
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oligomerization of the sec7 domain arf guanine nucleotide exchange factor GBF1 is dispensable for golgi localization and function but regulates degradation
American Journal of Physiology-cell Physiology, 2016Co-Authors: Jay M Bhatt, George A Belov, Ekaterina G Viktorova, Theodore Busby, Paulina Wyrozumska, Laura E Newman, Helen Lin, Eunjoo Lee, J T Wright, Richard A KahnAbstract:Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.
Catherine L. Jackson - One of the best experts on this subject based on the ideXlab platform.
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identification of GBF1 as a cellular factor required for hepatitis e virus rna replication
Cellular Microbiology, 2018Co-Authors: Rayan Farhat, Catherine L. Jackson, Yves Rouillé, Jean Dubuisson, Nadjet Lebsir, Maliki Ankavay, Jerome Gouttenoire, C Wychowski, Darius Moradpour, Laurence CocquerelAbstract:The hepatitis E virus (HEV) genome is a single-stranded, positive-sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1 and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose-dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wildtype GBF1 or a BFA-resistant GBF1 mutant rescuing HEV replication in BFA-treated cells, confirmed that GBF1 is the only BFA-sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalize with the ORF1 protein and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1-regulated mechanisms.
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interaction between the triglyceride lipase atgl and the arf1 activator GBF1
PLOS ONE, 2011Co-Authors: Emy Njoh Ellong, Krishnakant G Soni, Mariepierre Golinellicohen, Rachid Sougrat, Catherine L. JacksonAbstract:The Arf1 exchange factor GBF1 (Golgi Brefeldin A resistance factor 1) and its effector COPI are required for delivery of ATGL (adipose triglyceride lipase) to lipid droplets (LDs). Using yeast two hybrid, co-immunoprecipitation in mammalian cells and direct protein binding approaches, we report here that GBF1 and ATGL interact directly and in cells, through multiple contact sites on each protein. The C-terminal region of ATGL interacts with N-terminal domains of GBF1, including the catalytic Sec7 domain, but not with full-length GBF1 or its entire N-terminus. The N-terminal lipase domain of ATGL (called the patatin domain) interacts with two C-terminal domains of GBF1, HDS (Homology downstream of Sec7) 1 and HDS2. These two domains of GBF1 localize to lipid droplets when expressed alone in cells, but not to the Golgi, unlike the full-length GBF1 protein, which localizes to both. We suggest that interaction of GBF1 with ATGL may be involved in the membrane trafficking pathway mediated by GBF1, Arf1 and COPI that contributes to the localization of ATGL to lipid droplets.
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poliovirus replication requires the n terminus but not the catalytic sec7 domain of arfgef GBF1
Cellular Microbiology, 2010Co-Authors: George A Belov, Catherine L. Jackson, Gennadiy Kovtunovych, Ellie EhrenfeldAbstract:Viruses are intracellular parasites whose reproduction relies on factors provided by the host. The cellular protein GBF1 is critical for poliovirus replication. Here we show that the contribution of GBF1 to virus replication is different from its known activities in uninfected cells. Normally GBF1 activates the ADP-ribosylation factor (Arf) GTPases necessary for formation of COPI transport vesicles. GBF1 function is modulated by p115 and Rab1b. However, in polio-infected cells, p115 is degraded and neither p115 nor Rab1b knock-down affects virus replication. Poliovirus infection is very sensitive to brefeldin A (BFA), an inhibitor of Arf activation by GBF1. BFA targets the catalytic Sec7 domain of GBF1. Nevertheless the BFA block of polio replication is rescued by expression of only the N-terminal region of GBF1 lacking the Sec7 domain. Replication of BFA-resistant poliovirus in the presence of BFA is uncoupled from Arf activation but is dependent on GBF1. Thus the function(s) of this protein essential for viral replication can be separated from those required for cellular metabolism.
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GBF1 a guanine nucleotide exchange factor for arf is crucial for coxsackievirus b3 rna replication
Journal of Virology, 2009Co-Authors: Kjerstin Lanke, Ellie Ehrenfeld, Catherine L. Jackson, Hilde M Van Der Schaar, George A Belov, Qian Feng, Daniel Duijsings, Frank J M Van KuppeveldAbstract:The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.
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a critical role of a cellular membrane traffic protein in poliovirus rna replication
PLOS Pathogens, 2008Co-Authors: George A Belov, Catherine L. Jackson, Qian Feng, Krisztina Nikovics, Ellie EhrenfeldAbstract:Replication of many RNA viruses is accompanied by extensive remodeling of intracellular membranes. In poliovirus-infected cells, ER and Golgi stacks disappear, while new clusters of vesicle-like structures form sites for viral RNA synthesis. Virus replication is inhibited by brefeldin A (BFA), implicating some components(s) of the cellular secretory pathway in virus growth. Formation of characteristic vesicles induced by expression of viral proteins was not inhibited by BFA, but they were functionally deficient. GBF1, a guanine nucleotide exchange factor for the small cellular GTPases, Arf, is responsible for the sensitivity of virus infection to BFA, and is required for virus replication. Knockdown of GBF1 expression inhibited virus replication, which was rescued by catalytically active protein with an intact N-terminal sequence. We identified a mutation in GBF1 that allows growth of poliovirus in the presence of BFA. Interaction between GBF1 and viral protein 3A determined the outcome of infection in the presence of BFA.
Frank J M Van Kuppeveld - One of the best experts on this subject based on the ideXlab platform.
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GBF1 and acbd3 independent recruitment of pi4kiiiβ to replication sites by rhinovirus 3a proteins
Journal of Virology, 2015Co-Authors: Kjerstin Lanke, Hilde M Van Der Schaar, George A Belov, Frank J M Van Kuppeveld, Cristina M Dorobantu, Lauren A Fordsiltz, Simone P SittigAbstract:PI4KIIIβ recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIIIβ to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIβ to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIIIβ recruitment occurred independently of both GBF1 and ACBD3.
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recruitment of pi4kiiiβ to coxsackievirus b3 replication organelles is independent of acbd3 GBF1 and arf1
Journal of Virology, 2014Co-Authors: Cristina M Dorobantu, Hilde M Van Der Schaar, George A Belov, Lauren A Ford, Jeroen R P M Strating, Rachel Ulferts, Ying Fang, Frank J M Van KuppeveldAbstract:Members of the Enterovirus (poliovirus [PV], coxsackieviruses, and human rhinoviruses) and Kobuvirus (Aichi virus) genera in the Picornaviridae family rely on PI4KIIIβ (phosphatidylinositol-4-kinase IIIβ) for efficient replication. The small membrane-anchored enteroviral protein 3A recruits PI4KIIIβ to replication organelles, yet the underlying mechanism has remained elusive. Recently, it was shown that kobuviruses recruit PI4KIIIβ through interaction with ACBD3 (acyl coenzyme A [acyl-CoA]-binding protein domain 3), a novel interaction partner of PI4KIIIβ. Therefore, we investigated a possible role for ACBD3 in recruiting PI4KIIIβ to enterovirus replication organelles. Although ACBD3 interacted directly with coxsackievirus B3 (CVB3) 3A, its depletion from cells by RNA interference did not affect PI4KIIIβ recruitment to replication organelles and did not impair CVB3 RNA replication. Enterovirus 3A was previously also proposed to recruit PI4KIIIβ via GBF1/Arf1, based on the known interaction of 3A with GBF1, an important regulator of secretory pathway transport and a guanine nucleotide exchange factor (GEF) of Arf1. However, our results demonstrate that inhibition of GBF1 or Arf1 either by pharmacological inhibition or depletion with small interfering RNA (siRNA) treatment did not affect the ability of 3A to recruit PI4KIIIβ. Furthermore, we show that a 3A mutant that no longer binds GBF1 was capable of recruiting PI4KIIIβ, even in ACBD3-depleted cells. Together, our findings indicate that unlike originally envisaged, coxsackievirus recruits PI4KIIIβ to replication organelles independently of ACBD3 and GBF1/Arf1. IMPORTANCE A hallmark of enteroviral infection is the generation of new membranous structures to support viral RNA replication. The functionality of these “replication organelles” depends on the concerted actions of both viral nonstructural proteins and co-opted host factors. It is thus essential to understand how these structures are formed and which cellular components are key players in this process. GBF1/Arf1 and ACBD3 have been proposed to contribute to the recruitment of the essential lipid-modifying enzyme PI4KIIIβ to enterovirus replication organelles. Here we show that the enterovirus CVB3 recruits PI4KIIIβ by a mechanism independent of both GBF1/Arf1 and ACBD3. This study shows that the strategy employed by coxsackievirus to recruit PI4KIIIβ to replication organelles is far more complex than initially anticipated.
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differential effects of the putative GBF1 inhibitors golgicide a and ag1478 on enterovirus replication
Journal of Virology, 2010Co-Authors: Kjerstin Lanke, Hilde M Van Der Schaar, Lonneke Van Der Linden, Johan Neyts, Frank J M Van KuppeveldAbstract:The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication.
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GBF1 a guanine nucleotide exchange factor for arf is crucial for coxsackievirus b3 rna replication
Journal of Virology, 2009Co-Authors: Kjerstin Lanke, Ellie Ehrenfeld, Catherine L. Jackson, Hilde M Van Der Schaar, George A Belov, Qian Feng, Daniel Duijsings, Frank J M Van KuppeveldAbstract:The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.
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mouse hepatitis coronavirus rna replication depends on GBF1 mediated arf1 activation
PLOS Pathogens, 2008Co-Authors: Monique H Verheije, Frank J M Van Kuppeveld, Matthijs Raaben, Muriel Mari, Eddie Te G Lintelo, Fulvio Reggiori, Peter J M Rottier, Cornelis A M De HaanAbstract:Coronaviruses induce in infected cells the formation of double membrane vesicles, which are the sites of RNA replication. Not much is known about the formation of these vesicles, although recent observations indicate an important role for the endoplasmic reticulum in the formation of the mouse hepatitis coronavirus (MHV) replication complexes (RCs). We now show that MHV replication is sensitive to brefeldin A (BFA). Consistently, expression of a dominant-negative mutant of ARF1, known to mimic the action of the drug, inhibited MHV infection profoundly. Immunofluorescence analysis and quantitative electron microscopy demonstrated that BFA did not block the formation of RCs per se, but rather reduced their number. MHV RNA replication was not sensitive to BFA in MDCK cells, which are known to express the BFA-resistant guanine nucleotide exchange factor GBF1. Accordingly, individual knockdown of the Golgi-resident targets of BFA by transfection of small interfering RNAs (siRNAs) showed that GBF1, but not BIG1 or BIG2, was critically involved in MHV RNA replication. ARF1, the cellular effector of GBF1, also appeared to be involved in MHV replication, as siRNAs targeting this small GTPase inhibited MHV infection significantly. Collectively, our results demonstrate that GBF1-mediated ARF1 activation is required for efficient MHV RNA replication and reveal that the early secretory pathway and MHV replication complex formation are closely connected.
