Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

ABSTRACT Replication and infectivity of hepatitis C virus (HCV) with a defective genome is ambiguous. We molecularly cloned 38 HCV isolates with defective genomes from 18 patient sera. The structural regions were widely deleted, with the 5′ untranslated, core, and NS3-NS5B regions preserved. All of the deletions were in frame, indicating that they are translatable to the authentic terminus. Phylogenetic analyses showed self-replication of the defective genomes independent of full genomes. We generated a defective genome of chimeric HCV to mimic the defective isolate in the serum. By using this, we demonstrated for the first time that the defective genome, as it is circulating in the blood, can be encapsidated as an infectious particle by trans complementation of the structural proteins.

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Naturally Occurring Hepatitis C Virus Subgenomic Deletion Mutants Replicate Efficiently in Huh-7 Cells and Are trans-Packaged In Vitro To Generate Infectious Defective Particles

Journal of Virology ◽

10.1128/jvi.00308-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9079-9093 ◽

Cited By ~ 32

Author(s):

Laura Pacini ◽

Rita Graziani ◽

Linda Bartholomew ◽

Raffaele De Francesco ◽

Giacomo Paonessa

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Line ◽

Genome Structure ◽

Structural Proteins ◽

Wild Type Virus ◽

Deletion Mutants ◽

Wild Type ◽

Naturally Occurring

ABSTRACT Naturally occurring hepatitis C virus (HCV) subgenomic RNAs have been found in several HCV patients. These subgenomic deletion mutants, mostly lacking the genes encoding envelope glycoproteins, were found in both liver and serum, where their relatively high abundance suggests that they are capable of autonomous replication and can be packaged and secreted in viral particles, presumably harboring the envelope proteins from wild type virus coinfecting the same cell. We recapitulated some of these natural subgenomic deletions in the context of the isolate JFH-1 and confirmed these hypotheses in vitro. In Huh-7.5 cells, these deletion-containing genomes show robust replication and can be efficiently trans-packaged and infect naïve Huh-7.5 cells when cotransfected with the full-length wild-type J6/JFH genome. The genome structure of these natural subgenomic deletion mutants was dissected, and the maintenance of both core and NS2 regions was proven to be significant for efficient replication and trans-packaging. To further explore the requirements needed to achieve trans-complementation, we provided different combinations of structural proteins in trans. Optimal trans-complementation was obtained when fragments of the polyprotein encompassing core to p7 or E1 to NS2 were expressed. Finally, we generated a stable helper cell line, constitutively expressing the structural proteins from core to p7, which efficiently supports trans-complementation of a subgenomic deletion encompassing amino acids 284 to 732. This cell line can produce and be infected by defective particles, thus representing a powerful tool to investigate the life cycle and relevance of natural HCV subgenomic deletion mutants in vivo.

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Hepatitis C Virus-Like Particle Morphogenesis

Journal of Virology ◽

10.1128/jvi.76.8.4073-4079.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4073-4079 ◽

Cited By ~ 77

Author(s):

Emmanuelle Blanchard ◽

Denys Brand ◽

Sylvie Trassard ◽

Alain Goudeau ◽

Philippe Roingeard

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Semliki Forest Virus ◽

Virus Like Particle ◽

Genes Encoding ◽

Host Cell Interactions ◽

Viral Morphogenesis ◽

First Time

ABSTRACT Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

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Synthesis of New Indanyl Nucleoside Analogues and their Biological Evaluation on Hepatitis C Virus (HCV) Replicon

Molecules ◽

10.3390/molecules24050990 ◽

2019 ◽

Vol 24 (5) ◽

pp. 990

Author(s):

Matías Gómez ◽

Emiliano Gentile ◽

M. Martini ◽

María Cuestas ◽

Verónica Mathet ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Biological Evaluation ◽

Computational Studies ◽

Replicon Cell ◽

Racemic Form ◽

Synthetic Procedure ◽

Replicon Cell Line ◽

First Time

Here, we report a convenient synthetic procedure for the preparation of four novel indanyl carbanucleoside derivatives in the racemic form. The action of these compounds against hepatitis C virus was evaluated in vitro using the replicon cell line, Huh7.5 SG. Contrary to our expectations, all these compounds did not inhibit, but rather promoted HCV genotype 1b (HCVg1b) replication. Similar effects have been reported for morphine in the replicon cell lines, Huh7 and Huh8. Several biological experiments and computational studies were performed to elucidate the effect of these compounds on HCVg1b replication. Based on all the experiments performed, we propose that the increase in HCVg1b replication could be mediated, at least in part, by a similar mechanism to that of morphine on the enhancement of this replication. The presence of opioid receptors in Huh7.5 SG cells was indirectly determined for the first time in this work.

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Generation of Hepatitis C Virus-Like Particles by Use of a Recombinant Vesicular Stomatitis Virus Vector

Journal of Virology ◽

10.1128/jvi.76.23.12325-12334.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12325-12334 ◽

Cited By ~ 46

Author(s):

Heather J. Ezelle ◽

Dubravka Markovic ◽

Glen N. Barber

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Vesicular Stomatitis Virus ◽

Etiologic Agent ◽

Hcv Infection ◽

Structural Proteins ◽

Coding Region ◽

Vesicular Stomatitis ◽

Hcv Structural Proteins

ABSTRACT Hepatitis C virus (HCV), a major etiologic agent of hepatocellular carcinoma, presently infects approximately 400 million people worldwide, making the development of protective measures against HCV infection a key objective. Here we have generated a recombinant vesicular stomatitis virus (VSV), which expresses the HCV structural proteins, by inserting the contiguous Core, E1, and E2 coding region of HCV into the VSV genome. Recombinant VSV expressing HCV Core, E1, and E2 (VSV-HCV-C/E1/E2) grew to high titers in vitro and efficiently expressed the incorporated HCV gene product, which became fully processed into the individual HCV structural proteins. Biochemical and biophysical analysis indicated that the HCV Core, E1, and E2 proteins assembled to form HCV-like particles (HCV-LPs) possessing properties similar to the ultrastructural properties of HCV virions. Mice immunized with VSV-HCV-C/E1/E2 generated cell-mediated immune responses to all of the HCV structural proteins, and humoral responses, particularly to E2, were also readily evident. Our data collectively indicate that engineered VSVs expressing HCV Core, E1, and E2 and/or HCV-LPs represent useful tools in vaccine and immunotherapeutic strategies designed to address HCV infection.

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