scholarly journals Convergent use of phosphatidic acid for hepatitis C virus and SARS-CoV-2 replication organelle formation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Keisuke Tabata ◽  
Vibhu Prasad ◽  
David Paul ◽  
Ji-Young Lee ◽  
Minh-Tu Pham ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Phosphatidic Acid ◽  
Chronic Liver Disease ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Double Membrane ◽  
Targeting Therapy ◽  
Alternative Pathways

AbstractDouble membrane vesicles (DMVs) serve as replication organelles of plus-strand RNA viruses such as hepatitis C virus (HCV) and SARS-CoV-2. Viral DMVs are morphologically analogous to DMVs formed during autophagy, but lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV and SARS-CoV-2 replication and proper DMV formation. An intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways and their pharmacological inhibition also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as host cell lipid involved in proper replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. Host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

scholarly journals Convergent use of phosphatidic acid for Hepatitis C virus and SARS-CoV-2 replication organelle formation

2021 ◽  
Author(s):  
Keisuke Tabata ◽  
Vibhu Prasad ◽  
David Paul ◽  
Ji-Young Lee ◽  
Minh-Tu Pham ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Phosphatidic Acid ◽  
Rna Viruses ◽  
Light And Electron Microscopy ◽  
Membrane Vesicles ◽  
Double Knockout ◽  
Double Membrane ◽  
Targeting Therapy ◽  
Alternative Pathways

Double membrane vesicles (DMVs) are used as replication organelles by phylogenetically and biologically distant pathogenic RNA viruses such as hepatitis C virus (HCV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Viral DMVs are morphologically analogous to DMVs formed during autophagy, and although the proteins required for DMV formation are extensively studied, the lipids driving their biogenesis are largely unknown. Here we show that production of the lipid phosphatidic acid (PA) by acylglycerolphosphate acyltransferase (AGPAT) 1 and 2 in the ER is important for DMV biogenesis in viral replication and autophagy. Using DMVs in HCV-replicating cells as model, we found that AGPATs are recruited to and critically contribute to HCV replication and DMV formation. AGPAT1/2 double knockout also impaired SARS-CoV-2 replication and the formation of autophagosome-like structures. By using correlative light and electron microscopy, we observed the relocalization of AGPAT proteins to HCV and SARS-CoV-2 induced DMVs. In addition, an intracellular PA sensor accumulated at viral DMV formation sites, consistent with elevated levels of PA in fractions of purified DMVs analyzed by lipidomics. Apart from AGPATs, PA is generated by alternative pathways via phosphotidylcholine (PC) and diacylglycerol (DAG). Pharmacological inhibition of these synthesis pathways also impaired HCV and SARS-CoV-2 replication as well as formation of autophagosome-like DMVs. These data identify PA as an important lipid used for replication organelle formation by HCV and SARS-CoV-2, two phylogenetically disparate viruses causing very different diseases, i.e. chronic liver disease and COVID-19, respectively. In addition, our data argue that host-targeting therapy aiming at PA synthesis pathways might be suitable to attenuate replication of these viruses.

scholarly journals NS5A Domain 1 and Polyprotein Cleavage Kinetics Are Critical for Induction of Double-Membrane Vesicles Associated with Hepatitis C Virus Replication

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Inés Romero-Brey ◽  
Carola Berger ◽  
Stephanie Kallis ◽  
Androniki Kolovou ◽  
David Paul ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Viruses ◽  
Nonstructural Protein ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Concerted Action ◽  
Double Membrane ◽  
Positive Strand ◽  
Positive Strand Rna

ABSTRACTInduction of membrane rearrangements in the cytoplasm of infected cells is a hallmark of positive-strand RNA viruses. These altered membranes serve as scaffolds for the assembly of viral replication factories (RFs). We have recently shown that hepatitis C virus (HCV) infection induces endoplasmic reticulum-derived double-membrane vesicles (DMVs) representing the major constituent of the RF within the infected cell. RF formation requires the concerted action of nonstructural action of nonstructural protein (NS)3, -4A, protein (NS)3 -4A, -4B, -5A, and -5B. Although the sole expression of NS5A is sufficient to induce DMV formation, its efficiency is very low. In this study, we dissected the determinants within NS5A responsible for DMV formation and found that RNA-binding domain 1 (D1) and the amino-terminal membrane anchor are indispensable for this process. In contrast, deletion of NS5A D2 or D3 did not affect DMV formation but disrupted RNA replication and virus assembly, respectively. To identifycis- andtrans-acting factors of DMV formation, we established atranscleavage assay. We found that induction of DMVs requires full-length NS3, whereas a helicase-lacking mutant was unable to trigger DMV formation in spite of efficient polyprotein cleavage. Importantly, a mutation accelerating cleavage kinetics at the NS4B-5A site diminished DMV formation, while the insertion of an internal ribosome entry site mimicking constitutive cleavage at this boundary completely abolished this process. These results identify key determinants governing the biogenesis of the HCV RF with possible implications for our understanding of how RFs are formed in other positive-strand RNA viruses.IMPORTANCELike all positive-strand RNA viruses, hepatitis C virus (HCV) extensively reorganizes intracellular membranes to allow efficient RNA replication. Double-membrane vesicles (DMVs) that putatively represent sites of HCV RNA amplification are induced by the concerted action of viral and cellular factors. However, the contribution of individual proteins to this process remains poorly understood. Here we identify determinants in the HCV replicase that are required for DMV biogenesis. Major contributors to this process are domain 1 of nonstructural protein 5A and the helicase domain of nonstructural protein 3. In addition, efficient DMV induction depends onciscleavage of the viral polyprotein, as well as tightly regulated cleavage kinetics. These results identify key determinants governing the biogenesis of the HCV replication factory with possible implications for our understanding of how this central compartment is formed in other positive-strand RNA viruses.

scholarly journals Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Hossam Gewaid ◽  
Haruyo Aoyagi ◽  
Minetaro Arita ◽  
Koichi Watashi ◽  
Ryosuke Suzuki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Production ◽  
Rna Viruses ◽  
Ectopic Expression ◽  
Membrane Vesicles ◽  
Hcv Rna ◽  
Independent System ◽  
Double Membrane ◽  
Transfer Protein

ABSTRACT Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses. IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

scholarly journals Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Lingbao Kong ◽  
Haruyo Aoyagi ◽  
Zibing Yang ◽  
Tao Ouyang ◽  
Mami Matsuda ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Replication ◽  
Rna Viruses ◽  
Membrane Vesicles ◽  
Rna Replication ◽  
Double Membrane ◽  
Positive Strand ◽  
Replication Sites ◽  
Positive Strand Rna

ABSTRACT A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a “membranous web” structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication. IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

Hepatitis C virus replication in chronic liver disease

1991 ◽  
Vol 13 ◽  
pp. S40-S41
Author(s):  
S. Magrin ◽  
A. Craxì ◽  
C. Fabiano ◽  
G. Fiorentino ◽  
P. Almasio ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Chronic Liver Disease ◽  
Virus Replication ◽  
Chronic Liver

Low prevalence of hepatitis C virus antibodies in chronic liver disease in northwest China

1993 ◽  
Vol 41 (3) ◽  
pp. 247-250
Author(s):  
Xing Li ◽  
Norio Hayashi ◽  
Nobukazu Yuki ◽  
Kazuhiro Katayama ◽  
Akinori Kasahara ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Chronic Liver Disease ◽  
Northwest China ◽  
Chronic Liver ◽  
Low Prevalence

scholarly journals Chronic Hepatitis C Virus Infection After Treatment for Pediatric Malignancy

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1315-1320 ◽  
Author(s):  
Simone Cesaro ◽  
Maria Grazia Petris ◽  
Flavio Rossetti ◽  
Riccardo Cusinato ◽  
Corrado Pipan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Liver Disease ◽  
Hepatitis C ◽  
Virus Infection ◽  
Chronic Liver Disease ◽  
Chronic Liver ◽  
Hcv Infection ◽  
Pediatric Malignancy ◽  
Infection Markers

Abstract Sera of 658 patients who had completed treatment for pediatric malignancy were analyzed by a second-generation enzyme-linked immunosorbent assay and recombinant immunoblot assay test to assess the prevalence of hepatitis C virus (HCV)-seropositivity. All HCV-seropositive patients underwent detailed clinical, laboratory, virologic, and histologic study to analyze the course of HCV infection. One hundred seventeen of the 658 patients (17.8%) were positive for HCV infection markers. Among the 117 anti-HCV+ patients, 41 (35%) were also positive for markers of hepatitis B virus infection with or without delta virus infection markers, 91 (77.8%) had previously received blood product transfusions, and 25 (21.4%) showed a normal alanine aminotransferase (ALT) level during the last 5-year follow-up (11 of them never had abnormal ALT levels). The remaining 92 patients showed ALT levels higher than the upper limit of normal range. Eighty-one of 117 (70%) anti-HCV+ patients were HCV-RNA+, with genotype 1b being present in most patients (54%). In univariate analysis, no risk factor for chronic liver disease was statistically significant. In this study, the prevalence of HCV infection was high in patients who were treated for a childhood malignancy. In about 20% of anti-HCV+ patients, routes other than blood transfusions are to be considered in the epidemiology of HCV infection. After a 14-year median follow-up, chronic liver disease of anti-HCV+ positive patients did not show progression to liver failure.

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