Faculty Opinions recommendation of Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

ABSTRACT Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase ∼4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.

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

Journal of Virology ◽

10.1128/jvi.02684-09 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7535-7542 ◽

Cited By ~ 32

Author(s):

Lonneke van der Linden ◽

Hilde M. van der Schaar ◽

Kjerstin H. W. Lanke ◽

Johan Neyts ◽

Frank J. M. van Kuppeveld

Keyword(s):

Secretory Pathway ◽

Brefeldin A ◽

Rna Replication ◽

Human Enterovirus ◽

Subgenomic Replicon ◽

Golgi Vesicles ◽

Golgi Membranes ◽

The Family ◽

Pathway Function ◽

Molecular Mechanism Of Action

ABSTRACT 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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Faculty Opinions recommendation of Viral reorganization of the secretory pathway generates distinct organelles for RNA replication.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.3580966.3943054 ◽

2010 ◽

Author(s):

Johan Neyts ◽

Armando De Palma

Keyword(s):

Secretory Pathway ◽

Rna Replication

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Generation of Unique Poliovirus RNA Replication Organelles

mBio ◽

10.1128/mbio.00833-13 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 44

Author(s):

Alexsia L. Richards ◽

Jamária A. P. Soares-Martins ◽

Geoffrey T. Riddell ◽

William T. Jackson

Keyword(s):

Drug Targets ◽

Secretory Pathway ◽

Rna Replication ◽

Full Length ◽

Host Cells ◽

Cell Protein ◽

Genome Replication ◽

Dependent Manner ◽

Nucleotide Exchange ◽

Multiple Hypotheses

ABSTRACTPoliovirus (PV), a model for interactions of picornaviruses with host cells, replicates its genomic RNA in association with cellular membranes. The origin of PV replication membranes has not been determined. Hypotheses about the origin of replication membranes, based largely on localization of viral proteins, include modification of coat protein complex I (COPI) and/or COPII secretory pathway vesicles and subversion of autophagic membranes. Here, we use an antibody against double-stranded RNA (dsRNA) to identify replication complexes by detection of dsRNA replication intermediates. dsRNA signal is dependent on virus genome replication and colocalizes with the viral integral membrane protein 3A, which is part of the RNA replication complex. We show that early in infection, dsRNA does not colocalize with a marker for autophagic vesicles, making it unlikely that autophagosomes contribute to the generation of PV RNA replication membranes. We also find that dsRNA does not colocalize with a marker of the COPII coat, Sec31, and, in fact, we demonstrate proteasome-dependent loss of full-length Sec31 during PV infection. These data indicate that COPII vesicles are an unlikely source of PV replication membranes. We show that the Golgi resident G-protein Arf1 and its associated guanine nucleotide exchange factor (GEF), GBF1, transiently colocalize with dsRNA early in infection. In uninfected cells, Arf1 nucleates COPI coat formation, although during infection the COPI coat itself does not colocalize with dsRNA. Phosphatidylinositol-4-phosphate, which is associated with enterovirus-induced vesicles, tightly colocalizes with Arf1/GBF1 throughout infection. Our data point to a noncanonical role for some of the COPI-generating machinery in producing unique replication surfaces for PV RNA replication.IMPORTANCEPicornaviruses are a diverse and major cause of human disease, and their genomes replicate in association with intracellular membranes. There are multiple hypotheses to explain the nature and origin of these membranes, and a complete understanding of the host requirements for membrane rearrangement would provide novel drug targets essential for viral genome replication. Here, we study the model picornavirus, poliovirus, and show that some, but not all, components of the cellular machinery required for retrograde traffic from the Golgi apparatus to the endoplasmic reticulum are transiently present at the sites of viral RNA replication. We also show that the full-length Sec31 protein, which has been suggested to be present on PV RNA replication membranes, is lost during infection in a proteasome-dependent manner. This study helps to reconcile multiple hypotheses about the origin of poliovirus replication membranes and points to known host cell protein complexes that would make likely drug targets to inhibit picornavirus infections.

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

Journal of Virology ◽

10.1128/jvi.01190-09 ◽

2009 ◽

Vol 84 (2) ◽

pp. 773-787 ◽

Cited By ~ 99

Author(s):

Lucie Goueslain ◽

Khaled Alsaleh ◽

Pauline Horellou ◽

Philippe Roingeard ◽

Véronique Descamps ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Secretory Pathway ◽

Rna Replication ◽

Small Gtpases ◽

Hcv Rna ◽

Nucleotide Exchange ◽

Cellular Mechanisms ◽

Functional Connection ◽

Wide Range

ABSTRACT 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.

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