scholarly journals Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells

1998 ◽  
Vol 72 (5) ◽  
pp. 3827-3836 ◽  
Author(s):  
Thomas F. Baumert ◽  
Susumu Ito ◽  
David T. Wong ◽  
T. Jake Liang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Vaccine Development ◽  
Cultured Cells ◽  
Recombinant Baculovirus ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Viruslike Particles ◽  
Hcv Structural Proteins

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

2010 ◽  
Vol 44 (1) ◽  
pp. 97-108 ◽  
Author(s):  
S. N. Belzhelarskaya ◽  
N. N. Koroleva ◽  
V. V. Popenko ◽  
V. L. Drutza ◽  
O. V. Orlova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Structural Proteins ◽  
Virus Like Particles ◽  
Infected Insect

scholarly journals Genetic Immunization of Wild-Type and Hepatitis C Virus Transgenic Mice Reveals a Hierarchy of Cellular Immune Response and Tolerance Induction against Hepatitis C Virus Structural Proteins

2001 ◽  
Vol 75 (24) ◽  
pp. 12121-12127 ◽  
Author(s):  
Jujin Satoi ◽  
Kazumoto Murata ◽  
Martin Lechmann ◽  
Elanchezhiyan Manickan ◽  
Zhensheng Zhang ◽  
...  
Keyword(s):  
Immune Response ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Transgenic Mice ◽  
Structural Proteins ◽  
Dna Immunization ◽  
Wild Type ◽  
Genetic Immunization ◽  
Cellular Immune ◽  
Hcv Structural Proteins

ABSTRACT To study the effect of genetic immunization on transgenic expression of hepatitis C virus (HCV) proteins, we evaluated the immunological response of HCV transgenic mice to HCV expression plasmids. FVB/n transgenic mice expressing HCV structural proteins (core, E1, and E2) and wild-type (WT) FVB/n mice were immunized intramuscularly with plasmids expressing core (pHCVcore) or core/E1/E2 (pHCVSt). After immunization, HCV-specific humoral and cellular immune response was studied. Both WT and transgenic mice immunized with either HCV construct produced antibodies and exhibited T-cell proliferative responses against core or envelope. In WT mice immunized with pHCVSt, cytotoxic T-lymphocyte (CTL) activities were detected against E2 but not against core or E1, whereas strong CTL activities against core could be detected in WT mice immunized with pHCVcore. In pHCVSt-immunized, transgenic mice, CTL activities against the core or envelope were completely absent, but core-specific CTL activities could be detected in pHCVcore-immunized transgenic mice. A similar pattern of immune responses was also observed in other mouse strains, including a transgenic line expressing human HLA-A2.1 molecules (AAD mice). Despite the presence of a peripheral cellular immunity against HCV, no liver pathology or lymphocytic infiltrate was observed in these transgenic mice. Our study suggests a hierarchy of CTL response against the HCV structural proteins (E2 > core > E1) in vivo when the proteins are expressed as a polyprotein. The HCV transgenic mice can be induced by DNA immunization to generate anti-HCV antibodies and anticore CTLs. However, they are tolerant at the CTL level against the E2 protein despite DNA immunization.

Secretion and purification of hepatitis C virus NS1 glycoprotein produced by recombinant baculovirus-infected insect cells

Gene ◽  
1993 ◽  
Vol 129 (2) ◽  
pp. 207-214 ◽  
Author(s):  
Tsukasa Nishihara ◽  
Chikateru Nozaki ◽  
Hiroshi Nakatake ◽  
Kazuya Hoshiko ◽  
Manko Esumi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Recombinant Baculovirus ◽  
Infected Insect

scholarly journals NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system

2014 ◽  
Vol 95 (11) ◽  
pp. 2427-2441 ◽  
Author(s):  
Matthew J. Bentham ◽  
Najat Marraiki ◽  
Christopher J. McCormick ◽  
David J. Rowlands ◽  
Stephen Griffin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Early Stage ◽  
Infectious Hepatitis ◽  
Structural Proteins ◽  
Structural Protein ◽  
Subgenomic Replicon ◽  
Particle Assembly ◽  
Baculovirus Expression

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

scholarly journals Effect of the 5′ non-translated region on self-assembly of hepatitis C virus genotype 1a structural proteins produced in insect cells

2004 ◽  
Vol 85 (12) ◽  
pp. 3659-3670 ◽  
Author(s):  
Christel Girard ◽  
Marc Ravallec ◽  
Marcel Mariller ◽  
Jean-Pierre Bossy ◽  
Annie Cahour ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Self Assembly ◽  
Insect Cells ◽  
Structural Proteins ◽  
Virus Genotype ◽  
Genotype 1A

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

2002 ◽  
Vol 76 (23) ◽  
pp. 12325-12334 ◽  
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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