Hepatitis C virus: viral proteins on the move

There is now increasing evidence that LDs (lipid droplets) play a central role in the production of infectious HCV (hepatitis C virus) and participate in virus assembly. Two viral proteins, namely core, which forms the capsid, and NS5A (non-structural 5A protein), a component of complexes engaged in viral RNA synthesis, are detected at LD surfaces in infected cells. Interactions between the two proteins may be critical for anchoring RNA replication sites to droplets for initiating virus assembly. The requirements for targeting of core in particular has received considerable attention since the nature of its interaction with LDs could play a key role in determining the efficiency of virion production. As well as attaching to droplets, core is able to alter their intracellular distribution and direct them towards the microtubule organizing centre. Inhibitors that disrupt microtubules block this redistribution by core and there is a concomitant decrease in virus production. Therefore altered dynamics of LDs may contribute to HCV assembly and release. The purpose of targeting LDs by HCV may be linked to their contribution to the formation of VLDLs (very-low-density lipoproteins) in hepatocytes since virus circulating in infected patients is associated with lipoprotein. Thus HCV may utilize the role played by LDs in the formation of lipoprotein particles as part of its life cycle and access this pathway by direct interaction of viral components with these intracellular storage organelles.

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Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

Journal of Virology ◽

10.1128/jvi.02053-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2120-2129 ◽

Cited By ~ 333

Author(s):

Pablo Gastaminza ◽

Guofeng Cheng ◽

Stefan Wieland ◽

Jin Zhong ◽

Wei Liao ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Assembly ◽

Buoyant Density ◽

Infectious Hepatitis ◽

Low Density ◽

Very Low Density Lipoproteins ◽

Independent Manner ◽

Viral Egress ◽

Vldl Assembly

ABSTRACT Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.

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A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

Journal of Virology ◽

10.1128/jvi.02406-08 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10788-10796 ◽

Cited By ~ 33

Author(s):

Mair Hughes ◽

Sarah Gretton ◽

Holly Shelton ◽

David D. Brown ◽

Christopher J. McCormick ◽

...

Keyword(s):

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Assembly ◽

Infectious Clone ◽

Consensus Sequence ◽

Virus Production ◽

Rna Replication ◽

Subgenomic Replicon ◽

Genotype 1B

ABSTRACT We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

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Adaptive mutations promote hepatitis C virus assembly by accelerating Core translocation to the endoplasmic reticulum

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.016010 ◽

2020 ◽

pp. jbc.RA120.016010

Author(s):

Fuxiang Zheng ◽

Ni Li ◽

Yi Xu ◽

Yuanping Zhou ◽

Yi-Ping Li

Keyword(s):

Endoplasmic Reticulum ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Virus Assembly ◽

Buoyant Density ◽

Virus Production ◽

Adaptive Mutations ◽

Huh7 Cells ◽

Density Analysis

The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved in are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ~1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain, and that mutations I853V, C2865F, I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

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Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

Journal of Virology ◽

10.1128/jvi.02043-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1666-1673 ◽

Cited By ~ 27

Author(s):

Martina Kopp ◽

Catherine L. Murray ◽

Christopher T. Jones ◽

Charles M. Rice

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Core Protein ◽

Virus Assembly ◽

Signal Sequence ◽

Virus Production ◽

Terminal Region ◽

Dual Processing ◽

Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

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Hepatitis C virus production by human hepatocytes dependent on assembly and secretion of very low-density lipoproteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0700760104 ◽

2007 ◽

Vol 104 (14) ◽

pp. 5848-5853 ◽

Cited By ~ 385

Author(s):

H. Huang ◽

F. Sun ◽

D. M. Owen ◽

W. Li ◽

Y. Chen ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Production ◽

Low Density Lipoproteins ◽

Human Hepatocytes ◽

Low Density ◽

Very Low Density Lipoproteins

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NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly

Journal of Virology ◽

10.1128/jvi.00724-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7624-7639 ◽

Cited By ~ 152

Author(s):

Yinghong Ma ◽

Jeremy Yates ◽

Yuqiong Liang ◽

Stanley M. Lemon ◽

MinKyung Yi

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplets ◽

Infectious Virus ◽

Core Protein ◽

Virus Assembly ◽

Virus Production ◽

Intermediate Step ◽

Particle Assembly ◽

Chimeric Rna

ABSTRACT A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

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Serodiagnostic Assay of Hepatitis C Virus Infection Using Viral Proteins Expressed inEscherichia coli

Japanese Journal of Cancer Research ◽

10.1111/j.1349-7006.1992.tb00098.x ◽

1992 ◽

Vol 83 (3) ◽

pp. 264-268 ◽

Cited By ~ 9

Author(s):

Shigehisa Mori ◽

Showgo Ohkoshi ◽

Makoto Hijikata ◽

Nobuyuki Kato ◽

Kunitada Shimotohno

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Viral Proteins ◽

Inescherichia Coli ◽

Hepatitis C Virus Infection

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Hepatitis C Virus Regulates its Replication by Maturing miR-122 Through Akt-Dependent Phosphorylation of KSRP

10.1101/654392 ◽

2019 ◽

Author(s):

Camille Baudesson ◽

Céline Amadori ◽

Hassan Danso ◽

Flora Donati ◽

Quentin Nevers ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Direct Interaction ◽

Micro Rna ◽

Host Protein ◽

Hcv Rna ◽

Serine Residue ◽

Reciprocal Regulation ◽

Mrna Targets ◽

Infected Cells

AbstractThe liver-specific micro-RNA-122 (miR-122) is required for the replication of hepatitis C virus (HCV). The direct interaction between miR-122 and the 5’ untranslated region of the HCV genome promotes viral replication and protects HCV RNA from degradation. Because HCV RNA is its own substrate for replication, infected cells are submitted to the sequestration of increasing levels of miR-122 and to global de-repression of host miR-122 mRNA targets. Whether and how HCV regulates miR-122 maturation to create an environment favorable to its replication remains unexplored. We discovered that Akt-dependent phosphorylation of KSRP host protein at Serine residue 193 is essential for miR-122 maturation in hepatocytes. Moreover, we showed the existence of a reciprocal regulation loop where HCV replication can modulate the proviral effect mediated by KSRP-dependent maturation of miR-122. These data support a mechanism by which HCV regulates the expression of miR-122 by hijacking KSRP, thereby fueling its own replication.

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25-Hydroxyvitamin D Inhibits Hepatitis C Virus Production in Hepatocellular Carcinoma Cell Line by a Vitamin D Receptor-Independent Mechanism

International Journal of Molecular Sciences ◽

10.3390/ijms20092367 ◽

2019 ◽

Vol 20 (9) ◽

pp. 2367 ◽

Cited By ~ 2

Author(s):

Amiram Ravid ◽

Noa Rapaport ◽

Assaf Issachar ◽

Arie Erman ◽

Larisa Bachmetov ◽

...

Keyword(s):

Vitamin D ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Vitamin D Receptor ◽

Vitamin D3 ◽

Mode Of Action ◽

Virus Production ◽

Independent Manner ◽

Hydroxyvitamin D ◽

Independent Mechanism

Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D3-treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D3 anti-hepatitis C virus effect. We show that vitamin D3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D3. This is inferred from the findings that 25(OH)D3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D3. Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D3 and 25(OH)D3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D3 and 25(OH)D3, being 3β-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D3 that is mediated by 25(OH)D3 in a vitamin D receptor-independent mechanism.

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