Role of Phosphatidylinositol 4-Phosphate (PI4P) and Its Binding Protein GOLPH3 in Hepatitis C Virus Secretion

Bishé, Bryan; Syed, Gulam Hussain; Field, Seth J.; Siddiqui, Aleem

doi:10.1074/jbc.m112.346569

Cited by 82 publications

(84 citation statements)

References 61 publications

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“…Furthermore, rotaviruses are known to bypass the Golgi apparatus during assembly and release, although the mechanisms are poorly understood and the biology of rotaviruses strongly differs from that of HCV (79). Other possible explanations for the phenotypes that we observed are virus-induced alterations of the Golgi complex, including its dispersion, as previously proposed (23,27), and classical secretion without processing of the HCV glycoproteins by Golgi-residing enzymes due to their putative inaccessibility when they are associated with lipids (33). Given the findings that a proportion of HCV glycoproteins indeed carries complex glycans (46) (Fig.…”

Section: Discussionsupporting

confidence: 65%

“…Our model is not at odds with studies postulating an important role of the Golgi apparatus in HCV release (27,28,76). It is entirely conceivable that Golgi-derived factors or Golgi components are important for assembly and/or participate in the release of assembled particles.…”

Section: Discussioncontrasting

confidence: 50%

mentioning

confidence: 99%

“…Therefore, a similar mechanism has been postulated for HCV. In a recent study, PI4P-binding protein GOLPH3 was suggested to have a role in HCV budding since silencing of this protein led to reduced levels of HCV release (27). Similarly, a small interfering previously described (38), and plasmids were coelectroporated with RNA of HCV Jc1 genomes using a Bio-Rad Gene Pulser Xcell system.…”

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confidence: 99%

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Hepatitis C Virus Is Released via a Noncanonical Secretory Route

Bayer

Banning

Bruss

et al. 2016

J Virol

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We analyzed hepatitis C virus (HCV) morphogenesis using viral genomes encoding a mCherry-tagged E1 glycoprotein. HCV-E1-mCherry polyprotein expression, intracellular localization, and replication kinetics were comparable to those of untagged HCV, and E1-mCherry-tagged viral particles were assembled and released into cell culture supernatants. Expression and localization of structural E1 and nonstructural NS5A followed a temporospatial pattern with a succinct decrease in the number of replication complexes and the appearance of E1-mCherry punctae. Interaction of the structural proteins E1, Core, and E2 increased at E1-mCherry punctae in a time-dependent manner, indicating that E1-mCherry punctae represent assembled or assembling virions. E1-mCherry did not colocalize with Golgi markers. Furthermore, the bulk of viral glycoproteins within released particles revealed an EndoH-sensitive glycosylation pattern, indicating an absence of viral glycoprotein processing by the Golgi apparatus. In contrast, HCV-E1-mCherry trafficked with Rab9-positive compartments and inhibition of endosomes specifically suppressed HCV release. Our data suggest that assembled HCV particles are released via a noncanonical secretory route involving the endosomal compartment. IMPORTANCEThe goal of this study was to shed light on the poorly understood trafficking and release routes of hepatitis C virus (HCV). For this, we generated novel HCV genomes which resulted in the production of fluorescently labeled viral particles. We used live-cell microscopy and other imaging techniques to follow up on the temporal dynamics of virus particle formation and trafficking in HCV-expressing liver cells. While viral particles and viral structural protein were found in endosomal compartments, no overlap of Golgi structures could be observed. Furthermore, biochemical and inhibitor-based experiments support a HCV release route which is distinguishable from canonical Golgi-mediated secretion. Since viruses hijack cellular pathways to generate viral progeny, our results point toward the possible existence of a not-yet-described cellular secretion route. Hepatitis C virus (HCV) belongs to the Flavivirus genus and has a positive-strand RNA genome. This encodes a polyprotein which is posttranslationally cleaved into six nonstructural (NS) proteins, the ion channel p7 protein, and the structural proteins Core, E1, and E2 (1). The NS proteins reside at the outer leaflet of the endoplasmic reticulum (ER) membrane where NS4B and NS5A in particular induce membrane alterations resulting in the formation of the membranous web, which is the major site for HCV replication (2-5). Core is targeted to adjacent lipid droplets (LDs) (6, 7), which represent intracellular lipid deposits and are considered important for production of infectious particles (1,7,8). The E1 and E2 envelope proteins are incorporated into ER membranes with ectodomains facing the ER lumen (9, 10). Later, they are recruited to assembly sites via the NS2 complex (11,12). Upon recruitment of all requi...

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Section: Discussionsupporting

confidence: 65%

Section: Discussioncontrasting

confidence: 50%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Hepatitis C Virus Is Released via a Noncanonical Secretory Route

Bayer

Banning

Bruss

et al. 2016

J Virol

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“…2E). Previously, an increased level of Golgi fragmentation in HCV-infected cells was observed (34), but the cellular factors implicated in this process have not been addressed. We next performed a detailed analysis and quantification of the dispersed Golgi phenotype observed by confocal imaging.…”

Section: Resultsmentioning

confidence: 99%

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

Hansen

Johnsen

Stiberg

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

119

127

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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...

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