Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication

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 wild type 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 colocalise 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.     

Differential Effects of the Putative GBF1 Inhibitors Golgicide A and AG1478 on Enterovirus Replication

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.The Enterovirus genus, belonging to the Picornaviridae family, includes many important human pathogens, such as poliovirus, human rhinovirus, echovirus, and coxsackievirus. These are small, nonenveloped viruses that contain a single-stranded RNA genome of positive polarity. The genome is approximately 7.5 kb in length and encodes a single large polyprotein, which is processed into capsid proteins, encoded in the P1 region, and the nonstructural P2 and P3 region proteins that mediate viral RNA replication.Brefeldin A (BFA), a fungal metabolite, is a well-known inhibitor of enteroviruses. BFA blocks transport of cargo from the endoplasmic reticulum (ER) to the Golgi vesicles by disrupting the Golgi vesicles and ER-Golgi intermediate compartment (ERGIC) integrity through inhibition of several guanine nucleotide exchange factors (GEFs), including Golgi-specific BFA resistance factor 1 (GBF1), BFA-inhibited GEF 1 (BIG1), and BIG2 (3, 18). These GEFs regulate the activity of GTPase ADP-ribosylation factor 1 (Arf1) by stimulating GTP exchange. Upon activation, Arf1-GTP binds to Golgi membranes where it induces formation of secretory vesicles via recruitment of coatomer protein complex I (COP-I), a coatomer protein involved in the transport between the Golgi vesicles and the ER. The inhibitory effect of BFA on enterovirus replication is attributed to the inhibition of GBF1 and does not seem to involve BIG1 or BIG2 (2, 11). Besides enteroviruses, other plus-strand RNA viruses, such as mouse hepatitis virus and hepatitis C virus, also seem to rely on GBF1 for efficient replication (2, 8, 11, 21). The viral protein 3A of the enteroviruses poliovirus and coxsackievirus B3 (CVB3) has been shown to interact directly with GBF1 (22, 22a, 23), but the exact function of this interaction remains to be established.Recently, two compounds, AG1478 and Golgicide A (GCA), have been proposed to specifically inhibit GBF1. AG1478 was identified by screening a library of compounds for their ability to induce Golgi complex disassembly (13). AG1478, known as an inhibitor of the epidermal growth factor receptor (EGFR), had effects on the Golgi membranes highly similar to those of BFA through a mechanism not involving the inhibition of EGFR. Arf1-GTP pulldown assays showed that AG1478 inhibited Arf1 activation. Furthermore, overexpression of GBF1 was shown to counter the effect of AG1478 on COP-I localization. Based on these results, AG1478 was proposed to be a GBF1 inhibitor.GCA was identified in a high-throughput screen for small molecules that protected Vero cells from the effects of Shiga toxin (15). Similar to AG1478 and BFA, GCA was reported to fragment the Golgi vesicles and to inhibit Arf1 activation. Furthermore, overexpression of either wild-type GBF1 or the BFA-resistant mutant GBF1-M832L relieved the effects of GCA. In addition, the authors constructed a structural model of the catalytic Sec7 domain of GBF1 in complex with GCA, showing that GCA binds GBF1 at the same site as BFA. Collectively, their results provided convincing lines of evidence that GCA specifically inhibits GBF1 in a manner similar to BFA and does not act on BIG1 and BIG2.BFA has been instrumental in elucidating the membrane requirements for enterovirus replication. Therefore, we investigated the effects of AG1478 and GCA on enterovirus replication after first characterizing the effects of these drugs on BGM cells, the cell line that we routinely use in our studies on coxsackievirus B3 replication. Treatment with other AG1478 or GCA fragmented the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet these drugs had different effects on GBF1 localization. Interestingly, the effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1. Next, GCA was found to abrogate enterovirus replication, whereas surprisingly AG1478 did not affect replication at all. Together these results indicate that AG1478 on one hand and GCA and BFA on the other hand have different mechanisms of action, leading to a disparate effect on enterovirus replication.     

A Critical Role of a Cellular Membrane Traffic Protein in Poliovirus RNA Replication

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.     

Poliovirus replication requires the N‐terminus but not the catalytic Sec7 domain of ArfGEF GBF1

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.     

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.     

GBF1, a Guanine Nucleotide Exchange Factor for Arf,Is Crucial for Coxsackievirus B3 RNA Replication

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.Enteroviruses are small, nonenveloped, positive-stranded RNA viruses that include many important pathogens, such as poliovirus (PV), coxsackievirus, echovirus, and human rhinovirus. Following virus entry and uncoating, the 7.5-kb enteroviral RNA genome is directly translated into a large polyprotein. This polyprotein is proteolytically processed by the virus-encoded proteases 2A^pro, 3C^pro, and 3CD^pro into the structural P1 region proteins and the nonstructural P2 and P3 region proteins that are involved in viral RNA replication.All RNA viruses with a positive-stranded genome induce the remodeling of cellular membranes to create a scaffold for genomic RNA replication. The organelle origin and morphology of these membranous replication sites, however, appear to vary for different viruses. Enteroviruses replicate their RNA genomes in nucleoprotein complexes that are associated with small vesicular membrane structures (6). The enteroviral proteins 2B, 2C, and 3A have been implicated in vesicle formation (4, 6, 27), but the mechanism and pathway of membrane reorganization are poorly understood. There are strong indications that these vesicular membranous structures, which are referred to here as “vesicles,” are derived from the early secretory pathway. Vesicles produced in PV-infected cells may form at the endoplasmic reticulum (ER) by the cellular COP-II budding machinery and may therefore share components with the membranous vesicles mediating ER-to-Golgi network transport (26). Further support for the involvement of the secretory pathway stems from the observation that brefeldin A (BFA), a well-known inhibitor of ER-to-Golgi network transport, completely inhibits enteroviral RNA replication (17, 20). In addition, the autophagocytic pathway appears to contribute to the formation of the membrane vesicles, many of which exhibit a double-membrane morphology characteristic of autophagosomes (18, 27). The utilization of individual components or reactions from different membrane metabolic pathways, rather than subversion of an entire pathway in toto, may represent a common strategy for building viral replication machinery.BFA inhibits activation of the small monomeric GTPase ADP ribosylation factor 1 (Arf1), a major regulator of intracellular protein transport (2). Arf1 cycles between an inactive, GDP-bound, cytosolic state and an active, GTP-bound, membrane-associated state, and this cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (13). BFA blocks the activities of the large GEFs GBF1, BIG1, and BIG2 by stabilizing an intermediate, abortive complex with inactive Arf1 (23), thus efficiently preventing activation of Arf1 and eventually formation of transport intermediates.Not only the fact that BFA blocks enteroviral replication suggests a role for Arf1 and/or its large GEFs in this process; recently, it was shown that Arf1 accumulates on membranes during PV infection (3). Arf1 translocation to membranes can be induced independently by enterovirus protein 3A or 3CD in vitro (5), but the underlying mechanisms seem to differ; the 3A protein specifically triggers the recruitment of GBF1 to membranes, most likely through a direct interaction with this GEF (32, 33), whereas 3CD recruits BIG1 and BIG2 to membranes (3). Here, we report the involvement of Arf1 and its large BFA-sensitive GEFs in coxsackievirus B3 (CVB3) replication.     

Poliovirus proteins induce membrane association of GTPase ADP-ribosylation factor

Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication.     

The brefeldin A-inhibited guanine nucleotide-exchange protein, BIG2, regulates the constitutive release of TNFR1 exosome-like vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.     

LG186: An Inhibitor of GBF1 Function that Causes Golgi Disassembly in Human and Canine Cells

Brefeldin A‐mediated inhibition of ADP ribosylation factor (Arf) GTPases and their guanine nucleotide exchange factors, Arf‐GEFs, has been a cornerstone of membrane trafficking research for many years. Brefeldin A (BFA) is relatively non‐selective inhibiting at least three targets in human cells, Golgi brefeldin A resistance factor 1 (GBF1), brefeldin A inhibited guanine nucleotide exchange factor 1 (BIG1) and brefeldin A inhibited guanine nucleotide exchange factor 2 (BIG2). Here, we show that the previously described compound Exo2 acts through inhibition of Arf‐GEF function, but causes other phenotypic changes that are not GBF1 related. We describe the engineering of Exo2 to produce LG186, a more selective, reversible inhibitor of Arf‐GEF function. Using multiple‐cell‐based assays and GBF1 mutants, our data are most consistent with LG186 acting by selective inhibition of GBF1. Unlike other Arf‐GEF and reported GBF1 inhibitors including BFA, Exo2 and Golgicide A, LG186 induces disassembly of the Golgi stack in both human and canine cells.     

Inhibition of poliovirus RNA synthesis by brefeldin A.

Brefeldin A (BFA), a fungal metabolite that blocks transport of newly synthesized proteins from the endoplasmic reticulum, was found to inhibit poliovirus replication 10(5)- to 10(6)-fold. BFA does not inhibit entry of poliovirus into the cell or translation of viral RNA. Poliovirus RNA synthesis, however, is completely inhibited by BFA. A specific class of membranous vesicles, with which the poliovirus replication complex is physically associated, is known to proliferate in poliovirus-infected cells. BFA may inhibit poliovirus replication by preventing the formation of these vesicles.     

Valosin-containing protein (VCP/p97) is required for poliovirus replication and is involved in cellular protein secretion pathway in poliovirus infection

Poliovirus (PV) modifies membrane-trafficking machinery in host cells for its viral RNA replication. To date, ARF1, ACBD3, BIG1/BIG2, GBF1, RTN3, and PI4KB have been identified as host factors of enterovirus (EV), including PV, involved in membrane traffic. In this study, we performed small interfering RNA (siRNA) screening targeting membrane-trafficking genes for host factors required for PV replication. We identified valosin-containing protein (VCP/p97) as a host factor of PV replication required after viral protein synthesis, and its ATPase activity was essential for PV replication. VCP colocalized with viral proteins 2BC/2C and 3AB/3B in PV-infected cells and showed an interaction with 2BC and 3AB but not with 2C and 3A. Knockdown of VCP did not suppress the replication of coxsackievirus B3 or Aichi virus. A VCP-knockdown-resistant PV mutant had an A4881G (a mutation of E253G in 2C) mutation, which is known as a determinant of a secretion inhibition-negative phenotype. However, knockdown of VCP did not affect the inhibition of cellular protein secretion caused by overexpression of each individual viral protein. These results suggested that VCP is a host factor required for viral RNA replication of PV among membrane-trafficking proteins and provides a novel link between cellular protein secretion and viral RNA replication.     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.     

CSFV proliferation is associated with GBF1 and Rab2

The Golgi apparatus and its resident proteins are utilized and regulated by viruses to facilitate their proliferation. In this study, we investigated Classical swine fever virus (CSFV) proliferation when the function of the Golgi was disturbed. Golgi function was disturbed using chemical inhibitors, namely, brefeldin A (BFA) and golgicide A (GCA), and RNA interfering targets, such as the Golgi-specific BFA-resistance guanine nucleotide exchange factor 1 (GBF1) and Rab2 GTPases. CSFV proliferation was significantly inhibited during RNA replication and viral particle generation after BFA and GCA treatment. CSFV multiplication dynamics were retarded in cells transfected with GBF1 and Rab2 shRNA. Furthermore, CSFV proliferation was promoted by GBF1 and Rab2 overexpression using a lentiviral system. Hence, Golgi function is important for CSFV multiplication, and GBF1 and Rab2 participate in CSFV proliferation. Further studies must investigate Golgi-resident proteins to elucidate the mechanism underlying CSFV replication.     

Localization of large ADP-ribosylation factor-guanine nucleotide exchange factors to different Golgi compartments: evidence for distinct functions in protein traffic.

Activation of several ADP-ribosylation factors (ARFs) by guanine nucleotide exchange factors (GEFs) regulates recruitment of coat proteins (COPs) on the Golgi complex and is generally assumed to be the target of brefeldin A (BFA). The large ARF-GEFs Golgi-specific BFA resistance factor 1 (GBF1) and BFA-inhibited GEFs (BIGs) localize to this organelle but catalyze exchange preferentially on class II and class I ARFs, respectively. We now demonstrate using quantitative confocal microscopy that these GEFs show a very limited overlap with each other (15 and 23%). In contrast, GBF1 colocalizes with the cis-marker p115 (86%), whereas BIGs overlap extensively with TGN38 (83%). Consistent with these distributions, GBF1, but not BIG1, partially relocalized to peripheral sites after incubation at 15 degrees C. The new GBF1 structures represent peripheral vesicular tubular clusters (VTCs) because 88% of structures analyzed stained for both GBF1 and p115. Furthermore, as expected of VTCs, they rapidly reclustered to the Golgi complex in a microtubule-dependent manner upon warm-up. These observations suggest that GBF1 and BIGs activate distinct subclasses of ARFs in specific locations to regulate different types of reactions. In agreement with this possibility, COPI overlapped to a greater extent with GBF1 (64%) than BIG1 (31%), whereas clathrin showed limited overlap with BIG1, and virtually none with GBF1.     

Identification of GBF1 as a Cellular Factor Required for Hepatitis C Virus RNA Replication

In infected cells, hepatitis C virus (HCV) induces the formation of membrane alterations referred to as membranous webs, which are sites of RNA replication. In addition, HCV RNA replication also occurs in smaller membrane structures that are associated with the endoplasmic reticulum. However, cellular mechanisms involved in the formation of HCV replication complexes remain largely unknown. Here, we used brefeldin A (BFA) to investigate cellular mechanisms involved in HCV infection. BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARF), which can lead to a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways. Our data show that HCV RNA replication is highly sensitive to BFA. Individual knockdown of the cellular targets of BFA using RNA interference and the use of a specific pharmacological inhibitor identified GBF1, a guanine nucleotide exchange factor for small GTPases of the ARF family, as a host factor critically involved in HCV replication. Furthermore, overexpression of a BFA-resistant GBF1 mutant rescued HCV replication in BFA-treated cells, indicating that GBF1 is the BFA-sensitive factor required for HCV replication. Finally, immunofluorescence and electron microscopy analyses indicated that BFA does not block the formation of membranous web-like structures induced by expression of HCV proteins in a nonreplicative context, suggesting that GBF1 is probably involved not in the formation of HCV replication complexes but, rather, in their activity. Altogether, our results highlight a functional connection between the early secretory pathway and HCV RNA replication.Hepatitis C virus (HCV) is an important human pathogen. It mainly infects human hepatocytes, and this often leads to chronic hepatitis, cirrhosis, or hepatocarcinoma. HCV studies have been hampered for many years by the difficulty in propagating this virus in vitro. Things have recently changed with the development of a cell culture model referred to as HCVcc (34, 60, 65), which allows the study of the HCV life cycle in cell culture and facilitates studies of the interactions between HCV and the host cell.HCV is an enveloped positive-strand RNA virus belonging to the family Flaviviridae (35). The viral genome contains a single open reading frame, which is flanked by two noncoding regions that are required for translation and replication. All viral proteins that are produced after proteolytic processing of the initially synthesized polyprotein are membrane associated (15, 43). This reflects the fact that virtually all steps of the viral life cycle occur in close association with cellular membranes.Interactions of HCV with cell membranes begin during entry. Several receptors, coreceptors, and other entry factors have been discovered over the years, which link HCV entry to specialized domains of the plasma membrane, such as tetraspanin-enriched microdomains and tight junctions (8, 16, 59). The internalization of the viral particle occurs by clathrin-mediated endocytosis (5, 40). The fusion of the viral envelope with the membrane of an acidic endosome likely mediates the transfer of the viral genome to the cytosol of the cell (5, 40, 57). However, little is known regarding the pre- and postfusion intracellular transport steps of entering viruses in the endocytic pathway.HCV RNA replication is also associated with cellular membranes. Replication begins with the translation of the genomic RNA of an incoming virus. This leads to the production of viral proteins, which in turn initiate the actual replication of the viral RNA. Mechanisms regulating the transition from the translation of the genomic RNA to its replication are not yet known. All viral proteins are not involved in RNA replication. Studies performed with subgenomic replicons demonstrated that proteins NS3-4A, NS4B, NS5A, and NS5B are necessary and sufficient for replication (6, 27, 37). RNA replication proceeds through the synthesis of a cRNA strand (negative strand), catalyzed by the RNA-dependent RNA polymerase activity of NS5B, which is then used as a template for the synthesis of new positive strands.Electron microscopy studies using a subgenomic replicon model suggested that replication takes place in membrane structures made of small vesicles, referred to as “membranous webs,” which are induced by the virus (26). Membranous webs are detectable not only in cells carrying subgenomic replicons but also in infected cells (50). They appear to be associated with the endoplasmic reticulum (ER) (26). In addition to the membranous webs, a second type of ER-associated replicase that is smaller and more mobile has recently been described (63). Cellular mechanisms leading to these membrane alterations are still poorly understood. In cells replicating and secreting infectious viruses effectively, the situation appears to be even more complex, since replicase components appear to be, at least in part, associated with cytoplasmic lipid droplets (41, 50, 56). This association depends on the capsid protein (41) and may reflect a coupling between replication and assembly. Indeed, HCV assembly and secretion show some similarities with very-low-density lipoprotein (VLDL) maturation and secretion (24, 64).Our knowledge of the cellular membrane mechanisms involved in the HCV life cycle is still limited. The expression of NS4B alone induces membrane alterations that are reminiscent of membranous webs (19). However, cellular factors that participate in this process are still unknown. On the other hand, several cellular proteins potentially involved in the HCV life cycle have been identified through their interactions with viral proteins. For some of these proteins, a functional role in infection was recently confirmed using RNA interference (48). It is very likely that other cellular factors critical to HCV infection have yet to be identified.To gain more insight into cellular mechanisms underlying HCV infection, we made use of brefeldin A (BFA), a macrocyclic lactone of fungal origin that exhibits a wide range of inhibitory actions on membrane-associated mechanisms of the secretory and endocytic pathways (30). BFA acts on cell membranes by interfering with the activation of several members of the family of ADP-ribosylation factors (ARFs). ARFs are small GTP-binding proteins of the Ras superfamily. They function as regulators of vesicular traffic, actin remodeling, and phospholipid metabolism by recruiting effectors to membranes. BFA does not actually interfere directly with ARF GTPases but rather interferes with their activation by regulators known as guanine nucleotide exchange factors (GEFs) (14, 25). We now report the identification of an ARF GEF as a cellular BFA-sensitive factor that is required for HCV replication.     

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor,specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF-GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.     

RNA replication of mouse hepatitis virus takes place at double-membrane vesicles

The replication complexes (RCs) of positive-stranded RNA viruses are intimately associated with cellular membranes. To investigate membrane alterations and to characterize the RC of mouse hepatitis virus (MHV), we performed biochemical and ultrastructural studies using MHV-infected cells. Biochemical fractionation showed that all 10 of the MHV gene 1 polyprotein products examined pelleted with the membrane fraction, consistent with membrane association of the RC. Furthermore, MHV gene 1 products p290, p210, and p150 and the p150 cleavage product membrane protein 1 (MP1, also called p44) were resistant to extraction with Triton X-114, indicating that they are integral membrane proteins. The ultrastructural analysis revealed double-membrane vesicles (DMVs) in the cytoplasm of MHV-infected cells. The DMVs were found either as separate entities or as small clusters of vesicles. To determine whether MHV proteins and viral RNA were associated with the DMVs, we performed immunocytochemistry electron microscopy (IEM). We found that the DMVs were labeled using an antiserum directed against proteins derived from open reading frame 1a of MHV. By electron microscopy in situ hybridization (ISH) using MHV-specific RNA probes, DMVs were highly labeled for both gene 1 and gene 7 sequences. By combined ISH and IEM, positive-stranded RNA and viral proteins localized to the same DMVs. Finally, viral RNA synthesis was detected by labeling with 5-bromouridine 5'-triphosphate. Newly synthesized viral RNA was found to be associated with the DMVs. We conclude from these data that the DMVs carry the MHV RNA replication complex and are the site of MHV RNA synthesis.