Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography

ABSTRACT Recently, complete replication of hepatitis C virus (HCV) in tissue culture was established using the JFH1 isolate. To analyze determinants of HCV genome packaging and virion assembly, we developed a system that supports particle production based on trans-packaging of subgenomic viral RNAs. Using JFH1 helper viruses, we show that subgenomic JFH1 replicons lacking the entire core to NS2 coding region are efficiently encapsidated into infectious virus-like particles. Similarly, chimeric helper viruses with heterologous structural proteins trans-package subgenomic JFH1 replicons. Like authentic cell culture-produced HCV (HCVcc) particles, these trans-complemented HCV particles (HCVTCP) penetrate target cells in a CD81 receptor-dependent fashion. Since HCVTCP production was limited by competition between the helper and subgenomic RNA and to avoid contamination of HCVTCP stocks with helper viruses, we created HCV packaging cells. These cells encapsidate various HCV replicons with high efficiency, reaching infectivity titers up to 106 tissue culture infectious doses 50 per milliliter. The produced particles display a buoyant density comparable to HCVcc particles and can be propagated in the packaging cell line but support only a single-round infection in naïve cells. Together, this work demonstrates that subgenomic HCV replicons are assembly competent, thus excluding cis-acting RNA elements in the core-to-NS2 genomic region essential for RNA packaging. The experimental system described here should be helpful to decipher the mechanisms of HCV assembly and to identify RNA elements and viral proteins involved in particle formation. Similar to other vector systems of plus-strand RNA viruses, HCVTCP may prove valuable for gene delivery or vaccination approaches.

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In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

Journal of Virology ◽

10.1128/jvi.77.6.3669-3679.2003 ◽

2003 ◽

Vol 77 (6) ◽

pp. 3669-3679 ◽

Cited By ~ 85

Author(s):

Caterina Trozzi ◽

Linda Bartholomew ◽

Alessandra Ceccacci ◽

Gabriella Biasiol ◽

Laura Pacini ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Serine Protease ◽

In Vitro Selection ◽

Three Dimensional ◽

Host Cells ◽

Dimensional Structure ◽

In Vitro Systems

ABSTRACT The hepatitis C virus (HCV) serine protease is necessary for viral replication and represents a valid target for developing new therapies for HCV infection. Potent and selective inhibitors of this enzyme have been identified and shown to inhibit HCV replication in tissue culture. The optimization of these inhibitors for clinical development would greatly benefit from in vitro systems for the identification and the study of resistant variants. We report the use HCV subgenomic replicons to isolate and characterize mutants resistant to a protease inhibitor. Taking advantage of the replicons' ability to transduce resistance to neomycin, we selected replicons with decreased sensitivity to the inhibitor by culturing the host cells in the presence of the inhibitor and neomycin. The selected replicons replicated to the same extent as those in parental cells. Sequence analysis followed by transfection of replicons containing isolated mutations revealed that resistance was mediated by amino acid substitutions in the protease. These results were confirmed by in vitro experiments with mutant enzymes and by modeling the inhibitor in the three-dimensional structure of the protease.

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Analysis of Antigenicity and Topology of E2 Glycoprotein Present on Recombinant Hepatitis C Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.76.15.7672-7682.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7672-7682 ◽

Cited By ~ 110

Author(s):

Reginald F. Clayton ◽

Ania Owsianka ◽

Jim Aitken ◽

Susan Graham ◽

David Bhella ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Mammalian Cells ◽

Function Analysis ◽

Recombinant Baculovirus ◽

Microscopic Analysis ◽

Surface Topology ◽

Viral Glycoprotein ◽

Electron Microscopic ◽

Virus Like Particles

ABSTRACT Purification of hepatitis C virus (HCV) from sera of infected patients has proven elusive, hampering efforts to perform structure-function analysis of the viral components. Recombinant forms of the viral glycoproteins have been used instead for functional studies, but uncertainty exists as to whether they closely mimic the virion proteins. Here, we used HCV virus-like particles (VLPs) generated in insect cells infected with a recombinant baculovirus expressing viral structural proteins. Electron microscopic analysis revealed a population of pleomorphic VLPs that were at least partially enveloped with bilayer membranes and had viral glycoprotein spikes protruding from the surface. Immunogold labeling using specific monoclonal antibodies (MAbs) demonstrated these protrusions to be the E1 and E2 glycoproteins. A panel of anti-E2 MAbs was used to probe the surface topology of E2 on the VLPs and to compare the antigenicity of the VLPs with that of truncated E2 (E2660) or the full-length (FL) E1E2 complex expressed in mammalian cells. While most MAbs bound to all forms of antigen, a number of others showed striking differences in their abilities to recognize the various E2 forms. All MAbs directed against hypervariable region 1 (HVR-1) recognized both native and denatured E2660 with comparable affinities, but most bound either weakly or not at all to the FL E1E2 complex or to VLPs. HVR-1 on VLPs was accessible to these MAbs only after denaturation. Importantly, a subset of MAbs specific for amino acids 464 to 475 and 524 to 535 recognized E2660 but not VLPs or FL E1E2 complex. The antigenic differences between E2660, FL E1E2, and VLPs strongly point to the existence of structural differences, which may have functional relevance. Trypsin treatment of VLPs removed the N-terminal part of E2, resulting in a 42-kDa fragment. In the presence of detergent, this was further reduced to a trypsin-resistant 25-kDa fragment, which could be useful for structural studies.

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Probing and pressing surfaces of hepatitis C virus-like particles

Journal of Colloid and Interface Science ◽

10.1016/j.jcis.2019.03.022 ◽

2019 ◽

Vol 545 ◽

pp. 259-268 ◽

Cited By ~ 8

Author(s):

Simon Collett ◽

Joseph Torresi ◽

Linda Earnest-Silveira ◽

Dale Christiansen ◽

Aaron Elbourne ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Like Particles

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Amino Acid Substitutions Associated with Treatment Failure for Hepatitis C Virus Infection

Journal of Clinical Microbiology ◽

10.1128/jcm.01985-20 ◽

2020 ◽

Vol 58 (12) ◽

Cited By ~ 1

Author(s):

María Eugenia Soria ◽

Carlos García-Crespo ◽

Brenda Martínez-González ◽

Lucía Vázquez-Sirvent ◽

Rebeca Lobo-Vega ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Treatment Failure ◽

Antiviral Agents ◽

Three Dimensional ◽

Sustained Viral Response ◽

Amino Acid Substitutions ◽

Hepatitis C Virus Infection ◽

Bona Fide

ABSTRACT Despite the high virological response rates achieved with current directly acting antiviral agents (DAAs) against hepatitis C virus (HCV), around 2% to 5% of treated patients do not achieve a sustained viral response. The identification of amino acid substitutions associated with treatment failure requires analytical designs, such as subtype-specific ultradeep sequencing (UDS) methods, for HCV characterization and patient management. Using this procedure, we have identified six highly represented amino acid substitutions (HRSs) in NS5A and NS5B of HCV, which are not bona fide resistance-associated substitutions (RAS), from 220 patients who failed therapy. They were present frequently in basal and posttreatment virus of patients who failed different DAA-based therapies. Contrary to several RAS, HRSs belong to the acceptable subset of substitutions according to the PAM250 replacement matrix. Their mutant frequency, measured by the number of deep sequencing reads within the HCV quasispecies that encode the relevant substitutions, ranged between 90% and 100% in most cases. They also have limited predicted disruptive effects on the three-dimensional structures of the proteins harboring them. Possible mechanisms of HRS origin and dominance, as well as their potential predictive value for treatment response, are discussed.

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65 Induction of HCV-specific immune responses by hepatitis C virus-like particles in non-human primates: implications for prevention and therapy of hepatitis C

Hepatology ◽

10.1016/s0270-9139(03)80108-8 ◽

2003 ◽

Vol 38 ◽

pp. 186-186

Author(s):

S JEONG ◽

M QIAO ◽

M NASCIMBENI ◽

B REHERMANN ◽

K MURTHY ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Immune Responses ◽

Virus Like Particles ◽

Prevention And Therapy

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Visualization of Double-Stranded RNA in Cells Supporting Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.01565-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2182-2195 ◽

Cited By ~ 126

Author(s):

Paul Targett-Adams ◽

Steeve Boulant ◽

John McLauchlan

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Virus ◽

Three Dimensional ◽

Rna Replication ◽

Hcv Rna ◽

Double Stranded Rna ◽

Core Proteins ◽

Virus Rna ◽

Infected Cells

ABSTRACT The mechanisms involved in hepatitis C virus (HCV) RNA replication are unknown, and this aspect of the virus life cycle is not understood. It is thought that virus-encoded nonstructural proteins and RNA genomes interact on rearranged endoplasmic reticulum (ER) membranes to form replication complexes, which are believed to be sites of RNA synthesis. We report that, through the use of an antibody specific for double-stranded RNA (dsRNA), dsRNA is readily detectable in Huh-7 cells that contain replicating HCV JFH-1 genomes but is absent in control cells. Therefore, as that of other RNA virus genomes, the replication of the HCV genome may involve the generation of a dsRNA replicative intermediate. In Huh-7 cells supporting HCV RNA replication, dsRNA was observed as discrete foci, associated with virus-encoded NS5A and core proteins and identical in morphology and distribution to structures containing HCV RNA visualized by fluorescence-based hybridization methods. Three-dimensional reconstruction of deconvolved z-stack images of virus-infected cells provided detailed insight into the relationship among dsRNA foci, NS5A, the ER, and lipid droplets (LDs). This analysis revealed that dsRNA foci were located on the surface of the ER and often surrounded, partially or wholly, by a network of ER-bound NS5A protein. Additionally, virus-induced dsRNA foci were juxtaposed to LDs, attached to the ER. Thus, we report the visualization of HCV-induced dsRNA foci, the likely sites of virus RNA replication, and propose that HCV genome synthesis occurs at LD-associated sites attached to the ER in virus-infected cells.

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