scholarly journals Hepatitis C Virus F Protein Is a Short-Lived Protein Associated with the Endoplasmic Reticulum

2003 ◽  
Vol 77 (2) ◽  
pp. 1578-1583 ◽  
Author(s):  
Zhenming Xu ◽  
Jinah Choi ◽  
Wen Lu ◽  
Jing-hsiung Ou
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Half Life ◽  
Proteasome Inhibitors ◽  
Subcellular Fractionation ◽  
Biological Properties ◽  
F Protein ◽  
Proteasome Pathway

ABSTRACT Hepatitis C virus (HCV) F protein is a newly discovered HCV gene product that is expressed by translational ribosomal frameshift. Little is known about the biological properties of this protein. By performing pulse-chase labeling experiments, we demonstrate here that the F protein is a labile protein with a half-life of <10 min in Huh7 hepatoma cells and in vitro. The half-life of the F protein could be substantially increased by proteasome inhibitors, suggesting that the rapid degradation of the F protein is mediated by the proteasome pathway. Further immunofluorescence staining and subcellular fractionation experiments indicate that the F protein is primarily associated with the endoplasmic reticulum. This subcellular localization is similar to those of HCV core and NS5A proteins, raising the possibility that the F protein may participate in HCV morphogenesis or replication.

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

Suggested role of the Golgi apparatus and endoplasmic reticulum for crucial sites of hepatitis C virus replication in human lymphoblastoid cells infected in vitro

2003 ◽  
Vol 70 (1) ◽  
pp. 31-41 ◽  
Author(s):  
Annalucia Serafino ◽  
Maria Beatrice Valli ◽  
Federica Andreola ◽  
Annalisa Crema ◽  
Giampietro Ravagnan ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Golgi Apparatus ◽  
Virus Replication ◽  
Lymphoblastoid Cells

scholarly journals Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome

2007 ◽  
Vol 88 (1) ◽  
pp. 134-142 ◽  
Author(s):  
G. Haqshenas ◽  
J. M. Mackenzie ◽  
X. Dong ◽  
E. J. Gowans
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Bilayers ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Rna Transcripts ◽  
Green Fluorescent ◽  
Viral Polyprotein

p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcripts. Confocal microscopy revealed that the tagged p7 protein was localized in the endoplasmic reticulum (ER) but not associated with mitochondria. Immunoelectron microscopy confirmed the p7 localization data and, moreover, showed that intracellular virus-like particles formed in the cells transfected with the wild-type, but not the recombinant, transcripts. Following a few passages of the transfected cells, the recombinant genome with the HA tag reverted to wild-type and the entire tag was deleted. Therefore, in this study, it has been demonstrated that the p7 protein in the context of the full-length polyprotein encoded by a replication competent genome is only localized to the ER and has a possible role in HCV particle formation.

scholarly journals Detection of a Novel Unglycosylated Form of Hepatitis C Virus E2 Envelope Protein That Is Located in the Cytosol and Interacts with PKR

2002 ◽  
Vol 76 (3) ◽  
pp. 1265-1272 ◽  
Author(s):  
Nicole Pavio ◽  
Deborah R. Taylor ◽  
Michael M. C. Lai
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Envelope Protein ◽  
Kinase Activity ◽  
Direct Interaction ◽  
Cellular Functions ◽  
Double Stranded Rna ◽  
Proteasome Pathway ◽  
The Stability

ABSTRACT The hepatitis C virus (HCV) envelope protein E2 has been shown to accumulate in the lumen of the endoplasmic reticulum (ER) as a properly folded glycoprotein as well as large aggregates of misfolded proteins. In the present study, we have identified an additional unglycosylated species, with an apparent molecular mass of 38 kDa (E2-p38). In contrast to the glycosylated E2, E2-p38 is significantly less stable and is degraded through the proteasome pathway. Correspondingly, E2-p38 is found to be ubiquitinated. E2-p38 is localized mostly in the cytosol, in contrast to the glycosylated form, which is exclusively membrane associated. Alpha interferon (IFN-α) treatment or overexpression of the double-stranded RNA-activated protein kinase (PKR) significantly increased the stability of E2-p38, consistent with a previous report (D. R. Taylor, S. T. Shi, P. R. Romano, G. N. Barber, and M. M. Lai, Science 285:107–110, 1999) that E2 interacts with PKR and inhibits its kinase activity. Direct interaction between PKR and E2-p38, but not the glycosylated form of E2, was also observed. These results show that E2-p38 is the form of E2 that interacts with PKR in the cytosol and may contribute to the resistance of HCV to IFN-α. Thus, an ER protein can exist in the cytosol as an unglycosylated species and impair cellular functions.

scholarly journals Hepatitis C Virus Lipoviroparticles Assemble in the Endoplasmic Reticulum (ER) and Bud off from the ER to the Golgi Compartment in COPII Vesicles

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Gulam H. Syed ◽  
Mohsin Khan ◽  
Song Yang ◽  
Aleem Siddiqui
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Immunogold Electron Microscopy ◽  
Hcv Rna ◽  
Core Proteins ◽  
Golgi Compartment ◽  
Copii Vesicles

ABSTRACT Hepatitis C virus (HCV) exists as a lipoprotein-virus hybrid lipoviroparticle (LVP). In vitro studies have demonstrated the importance of apolipoproteins in HCV secretion and infectivity, leading to the notion that HCV coopts the secretion of very-low-density lipoprotein (VLDL) for its egress. However, the mechanisms involved in virus particle assembly and egress are still elusive. The biogenesis of VLDL particles occurs in the endoplasmic reticulum (ER), followed by subsequent lipidation in the ER and Golgi compartment. The secretion of mature VLDL particles occurs through the Golgi secretory pathway. HCV virions are believed to latch onto or fuse with the nascent VLDL particle in either the ER or the Golgi compartment, resulting in the generation of LVPs. In our attempt to unravel the collaboration between HCV and VLDL secretion, we studied HCV particles budding from the ER en route to the Golgi compartment in COPII vesicles. Biophysical characterization of COPII vesicles fractionated on an iodixanol gradient revealed that HCV RNA is enriched in the highly buoyant COPII vesicle fractions and cofractionates with apolipoprotein B (ApoB), ApoE, and the HCV core and envelope proteins. Electron microscopy of immunogold-labeled microsections revealed that the HCV envelope and core proteins colocalize with apolipoproteins and HCV RNA in Sec31-coated COPII vesicles. Ultrastructural analysis also revealed the presence of HCV structural proteins, RNA, and apolipoproteins in the Golgi stacks. These findings support the hypothesis that HCV LVPs assemble in the ER and are transported to the Golgi compartment in COPII vesicles to embark on the Golgi secretory route. IMPORTANCE HCV assembly and release accompany the formation of LVPs that circulate in the sera of HCV patients and are also produced in an in vitro culture system. The pathway of HCV morphogenesis and secretion has not been fully understood. This study investigates the exact site where the association of HCV virions with host lipoproteins occurs. Using immunoprecipitation of COPII vesicles and immunogold electron microscopy (EM), we characterize the existence of LVPs that cofractionate with lipoproteins, viral proteins, RNA, and vesicular components. Our results show that this assembly occurs in the ER, and LVPs thus formed are carried through the Golgi network by vesicular transport. This work provides a unique insight into the HCV LVP assembly process within infected cells and offers opportunities for designing antiviral therapeutic cellular targets.

scholarly journals Differential reactivity of putative genotype 2 hepatitis C virus F protein between chronic and recovered infections

2008 ◽  
Vol 89 (8) ◽  
pp. 1890-1900 ◽  
Author(s):  
Wing Chia-Ming Chuang ◽  
Jean-Pierre Allain
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Cross Reactivity ◽  
Hcv Infection ◽  
Genotype 3 ◽  
F Protein ◽  
Genotype 2

To date, all studies regarding hepatitis C virus (HCV) F protein have been based on expression in vitro/in vivo of recombinant protein or monoclonal antibodies derived from genotype 1a or 1b sequences, but not from other genotypes. The objective of this study was to prepare a putative genotype 2 recombinant F protein and evaluate its reactivity in plasma from individuals with chronic HCV infection or who had recovered from infection. One genotype 2 strain was selected for F protein (F-2) and core expression in bacterial culture. An ELISA was developed and applied to samples from patients with chronic infection or recovered infection of various genotypes. The anti-F-2 response in 117 samples showed a significantly higher reactivity in chronic than in recovered HCV-infected blood donors (P<0.001), but no difference was found among genotypes. However, the correlation between anti-F and anti-core was more significant in genotypes 1 and 2 than in genotype 3. Anti-F-2 titres were also significantly higher in chronic than in recovered individuals (P<0.0001). Antibody titres to recombinant genotype 2 core protein or to genotype 1 multiple proteins used in commercial anti-HCV assays paralleled the anti-F-2 end-point antibody titre. This study thus demonstrated the antigenicity of genotype 2 HCV F protein, although the exact location of the natural frameshift position remains unknown. The difference in anti-F-2 response between chronic and recovered infection, the cross-reactivity irrespective of genotype and the correlation of antibody response with structural and non-structural antigens suggest that the immune response to F protein is an integral part of the natural HCV infection.

scholarly journals Hepatitis C virus F protein sequence reveals a lack of functional constraints and a variable pattern of amino acid substitution

2005 ◽  
Vol 86 (1) ◽  
pp. 115-120 ◽  
Author(s):  
Juan Cristina ◽  
Fernando Lopez ◽  
Gonzalo Moratorio ◽  
Lilia López ◽  
Silvia Vasquez ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Biological Properties ◽  
Functional Constraints ◽  
Ribosomal Frameshift ◽  
F Protein ◽  
Stop Codons ◽  
Conservation Patterns ◽  
Phylogenetic Lineages

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is an RNA molecule that is approximately 9·6 kb in length and encodes a polyprotein that is cleaved proteolytically to generate at least 10 mature viral proteins. Recently, a new HCV protein named F has been described, which is synthesized as a result of a ribosomal frameshift. Little is known about the biological properties of this protein, but the possibility that the F protein may participate in HCV morphology or replication has been raised. In this work, the presence of functional constraints in the F protein was investigated. It was found that the rate of amino acid substitutions along the F protein was significantly higher than the rate of synonymous substitutions, and comparisons involving genes that represented independent phylogenetic lineages yielded very different divergence/conservation patterns. The distribution of stop codons in the F protein across all HCV genotypes was also investigated; genotypes 2 and 3 were found to have more stop codons than genotype 1. The results of this work suggest strongly that the pattern of divergence in the F protein is not affected by functional constraints.

Funktion von CEACAM-1 im Rahmen der Therapie der Hepatitis C-Virus Infektion mit Interferon-alpha in vivo und in vitro

2006 ◽  
Vol 44 (08) ◽  
Author(s):  
P Hilgard ◽  
R Bröring ◽  
M Trippler ◽  
S Viazov ◽  
G Gerken ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Interferon Alpha
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