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

Abstract

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.HCV | Golgi fragmentation | autophagy | IRGM | membranous web H epatitis C virus (HCV) is a positive-sense RNA virus in the family Flaviviridae that is a major cause of chronic liver disease. All positive-strand RNA viruses studied until now, including HCV, replicate their genomes in association with cellular membrane rearrangements. In this process, viruses remodel intracellular membranes [e.g., of mitochondria, endoplasmic reticulum (ER), and plasma membrane] to generate membrane structures such as single-or double-membrane vesicles that contribute to viral replication complexes (VRCs). HCV replication takes place at a unique subcellular compartment, the membranous web (MW), which has been proposed to be derived from the ER (1, 2). The HCV MW has a complex morphology consisting of clusters of single-, double-, and multimembrane vesicles and probably includes autophagosomes and lipid droplets (1, 3, 4). Recent findings reveal that the MW is produced by distinct HCV nonstructural (NS) proteins acting through sequential interaction with several host factors, such as the virus-targeted phosphatidylinositol-4 kinase III α (PI4KIIIα) (2, 3), but the full spectrum of host components and precise membrane composition that supports HCV replication are not fully defined.Autophagy is an evolutionarily conserved cellular mechanism that involves intracellular membrane trafficking and degradation to maintain cell homeostasis. Viruses, including HCV, have been reported to exploit autophagy for replication purposes (4-6), but the mechanism by which this exploitation occurs is largely unknown. De novo synthesis of autophagosomes is a complex process that involves the formation of a phagophore membrane and its elongation. Initiation of autophagy is regulated by the mammalian target of rapamycin complex 1 (mTORC1), which neg...

Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein

“…This change in GBF1 intracellular localization is consistent with an alteration in Golgi structure, which has already been reported for HCV-infected cells (53)(54)(55). This change in Golgi morphology is due to the effects of HCV infection on GBF1 function (55). Importantly, we found that NS3-4A expression induced a change of GBF1 intracellular localization from Golgi membranes to NS3-positive structures and that this association with NS3-positive structures was insensitive to BFA, indicating that it was independent of Arf activation.…”

“…An alteration in the intracellular localization of GBF1 was observed both in inducible NS3-4A-expressing cells and in replicon-containing Huh-7 cells expressing higher levels of NS3, confirming the impact of NS3 on GBF1 intracellular localization in a more physiological model. This change in GBF1 intracellular localization is consistent with an alteration in Golgi structure, which has already been reported for HCV-infected cells (53)(54)(55). This change in Golgi morphology is due to the effects of HCV infection on GBF1 function (55).…”

Manipulation of autophagy by (+) RNA viruses

“…A link between autophagosomes and virus-induced vesicles was proposed by George Palade by EM imaging of poliovirus containing vesicles that resembled autophagosomal membranes [6]. Over the past few decades, a growing body of research has defined the critical role of this pathway in facilitating infection by numerous +RNA RNA viruses, including poliovirus (PV) [7,8], Coxsackievirus B3 (CVB3) [9,10], CVB4 [11], Enterovirus 71 (EV71) [12], Human rhinovirus (HRV) [13], Foot-and-mouth disease virus (FMDV) [14], encephalomyocarditis virus (EMCV) [15], Dengue virus (DENV) [16,17], Zika virus (ZIKV) [18,19], Hepatitis C virus (HCV) [20], Mouse hepatitic virus (MHV), Newcastle disease virus (NDV) [21], Severe and acute respiratory syndrome coronavirus (SARS-CoV) [22], Chikungunya virus (ChikV) [23], and Japanese encephalitis virus (JEV) [24]. In many of the above cases, pharmacological or genetic manipulation of autophagy in vitro confirmed an inhibition in replication and/or spread of these viruses, whereas induction of autophagy resulted in increased production of progeny virions [21,25].…”

PI4KB on Inclusion Bodies Formed by ER Membrane Remodeling Facilitates Replication of Human Parainfluenza Virus Type 3

“…HCV ROs (Romero-Brey et al, 2012;Welsch et al, 2009). However, a recent study showed that the Golgi and autophagosome membrane are important for HCV replication and highlighted the complexity of the cellular membrane structures of positive-strand viruses (Hansen et al, 2017). Interestingly, the autophagy marker LC3 was also found on HPIV3 IBs (data not shown), suggesting that autophagy may also play a role in IB formation.…”