Hepatitis C virus triggers Golgi fragmentation and autophagy through the immunity-related GTPase M

Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.

Download Full-text

Dissection of Membrane Dynamics of the ARF-Guanine Nucleotide Exchange Factor GBF1

Traffic ◽

10.1111/j.1600-0854.2005.00282.x ◽

2005 ◽

Vol 6 (5) ◽

pp. 374-385 ◽

Cited By ~ 59

Author(s):

Tomasz Szul ◽

Rafael Garcia-Mata ◽

Elizabeth Brandon ◽

Svetlana Shestopal ◽

Cecilia Alvarez ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factor ◽

Membrane Dynamics ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor

Download Full-text

Activation of ARF6 by ARNO stimulates epithelial cell migration through downstream activation of both Rac1 and phospholipase D

The Journal of Cell Biology ◽

10.1083/jcb.200104019 ◽

2001 ◽

Vol 154 (3) ◽

pp. 599-610 ◽

Cited By ~ 279

Author(s):

Lorraine C. Santy ◽

James E. Casanova

Keyword(s):

Epithelial Cells ◽

Cell Motility ◽

Phospholipase D ◽

Phospholipid Composition ◽

Nucleotide Exchange ◽

Guanine Nucleotide Exchange ◽

Pulldown Assay ◽

Nucleotide Exchange Factor ◽

Epithelial Cell Migration ◽

Arf Gtpases

Migration of epithelial cells is essential for tissue morphogenesis, wound healing, and metastasis of epithelial tumors. Here we show that ARNO, a guanine nucleotide exchange factor for ADP-ribosylation factor (ARF) GTPases, induces Madin-Darby canine kidney epithelial cells to develop broad lamellipodia, to separate from neighboring cells, and to exhibit a dramatic increase in migratory behavior. This transition requires ARNO catalytic activity, which we show leads to enhanced activation of endogenous ARF6, but not ARF1, using a novel pulldown assay. We further demonstrate that expression of ARNO leads to increased activation of endogenous Rac1, and that Rac activation is required for ARNO-induced cell motility. Finally, ARNO-induced activation of ARF6 also results in increased activation of phospholipase D (PLD), and inhibition of PLD activity also inhibits motility. However, inhibition of PLD does not prevent activation of Rac. Together, these data suggest that ARF6 activation stimulates two distinct signaling pathways, one leading to Rac activation, the other to changes in membrane phospholipid composition, and that both pathways are required for cell motility.

Download Full-text

SGEF, a RhoG Guanine Nucleotide Exchange Factor that Stimulates Macropinocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e04-02-0146 ◽

2004 ◽

Vol 15 (7) ◽

pp. 3309-3319 ◽

Cited By ~ 70

Author(s):

Shawn M. Ellerbroek ◽

Krister Wennerberg ◽

William T. Arthur ◽

Jill M. Dunty ◽

Dan R. Bowman ◽

...

Keyword(s):

Guanine Nucleotide Exchange Factor ◽

Membrane Dynamics ◽

Full Length ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Filamentous Actin ◽

Exchange Factor ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Constitutively Active

SGEF (SH3-containing Guanine Nucleotide Exchange Factor) is a RhoGEF of unknown function. We found the SGEF protein to be expressed in many established cell lines and highly expressed in human liver tissue. SGEF stimulated the formation of large interconnected membrane ruffles across dorsal surfaces when expressed in fibroblasts. SGEF required its proline-rich amino-terminus to generate dorsal, but not lateral, membrane ruffles and a functional SH3 domain to colocalize with filamentous actin at sites of membrane protrusion. Full-length SGEF activated RhoG, but not Rac, when expressed in fibroblasts. Further, recombinant SGEF DH/PH protein exchanged nucleotide on RhoG, but not on Rac1 or Rac3, in vitro. Scanning electron microscopy of fibroblasts demonstrated that SGEF induced dorsal ruffles that were morphologically similar to those generated by constitutively active RhoG, but not constitutively active Rac1. Transient expression of SGEF stimulated fibroblast uptake of 10-kDa dextran, a marker of macropinocytosis. This required the full-length protein and a catalytically active DH domain. Finally, activated RhoG was found to be more effective than activated Rac, and comparable to SGEF, in its ability to trigger dextran uptake. Together, this work establishes SGEF as a RhoG exchange factor and provides evidence that both SGEF and RhoG regulate membrane dynamics in promotion of macropinocytosis.

Download Full-text

Unfolded protein response regulates yeast small GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1518260113 ◽

2016 ◽

Vol 113 (12) ◽

pp. E1683-E1690 ◽

Cited By ~ 6

Author(s):

Jia-Wei Hsu ◽

Pei-Hua Tang ◽

I-Hao Wang ◽

Chia-Lun Liu ◽

Wen-Hui Chen ◽

...

Keyword(s):

Unfolded Protein Response ◽

Small Gtpase ◽

Nucleotide Exchange ◽

Unfolded Protein ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Active Transcription ◽

Arf Gtpases ◽

Protein Response ◽

Adp Ribosylation Factor

ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic at the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates Arf-like protein Arl1p, which was accompanied by accumulation of golgin Imh1p at late Golgi, but whether and how this function of Syt1p is regulated remains unclear. Here, we report that the inositol-requiring kinase 1 (Ire1p)-mediated unfolded protein response (UPR) modulated Arl1p activation at late Golgi. Arl1p activation was dependent on both kinase and endo-RNase activities of Ire1p. Moreover, constitutively active transcription factor Hac1p restored the Golgi localization of Arl1p and Imh1p inIRE1-deleted cells. Elucidating the mechanism of Ire1p–Hac1p axis actions, we found that it regulated phosphorylation of Syt1p, which enhances Arl1p activation, recruitment of Imh1p to the Golgi, and Syt1p interaction with Arl1p. Consistent with these findings, the induction of UPR by tunicamycin treatment increases phosphorylation of Syt1p, resulting in Arl1p activation. Thus, these findings clarify how the UPR influences the roles of Syt1p, Arl1p, and Imh1p in Golgi transport.

Download Full-text

Differential Roles of Lipin1 and Lipin2 in the Hepatitis C Virus Replication Cycle

Proceedings ◽

10.3390/proceedings2020050030 ◽

2020 ◽

Vol 50 (1) ◽

pp. 30

Author(s):

Victoria Castro ◽

Gema Calvo ◽

Ginés Ávila-Pérez ◽

Marlène Dreux ◽

Pablo Gastaminza

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Rna Viruses ◽

Hcv Infection ◽

Golgi Fragmentation ◽

Positive Strand Rna ◽

Virus Replication Cycle ◽

The Impact ◽

Late Stages

Although their origin, nature and structure are not identical, a common feature of positive-strand RNA viruses is their ability to subvert host lipids and intracellular membranes to generate replication and assembly complexes. Recently, lipin1, a cellular enzyme that converts phosphatidate into diacylglycerol, has been involved in the formation of the membranous web that hosts hepatitis C virus (HCV) replicase. In the liver, lipin1 cooperates with lipin2 to maintain glycerolipid homeostasis. We extended our previous study of the lipin family in HCV infection by determining the impact of the lipin2 silencing on viral replication. In contrast to the specific impact of lipin1 silencing on HCV replication, our data suggest a broader function of lipin2 not only in HCV infection, but also for the replication of other RNA viruses. Moreover, uninfected lipin2- but not lipin1-deficient cells display alterations in mitochondrial and Golgi morphology, suggesting that lipin2 contributes to the maintenance of the overall organelle architecture. Coinciding with Golgi fragmentation, our data reveal that lipin2 silencing mainly interferes with HCV virion secretion at late stages of the infection without significantly affecting viral replication or assembly. Overall, this study reveals distinctive functions of lipin1 and lipin2 in cells of hepatic origin, a context in which they are often considered functionally redundant.

Download Full-text