Expression of Hepatitis C Virus cDNA in Human Hepatoma Cell Line Mediated by a Hybrid Baculovirus–HCV Vector

Fipaldini, C; Bellei, Barbara; Monica, Nicola La

doi:10.1006/viro.1998.9565

Cited by 43 publications

(30 citation statements)

References 49 publications

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“…The most successful use of baculovirus for delivery and expression of genes in mammalian cells used a CMV promoter and transduction at 37OC (10)(11)(12)(13)(14)(15)(16)(17). We found that transduction of primary pancreatic dissociated islet cells and other cell lines using baculovirus at 37OC was four-to fivefold higher than that at room temperature (L.M., L.H.P., unpublished data).…”

Section: Discussionmentioning

confidence: 92%

“…Recently, it was shown that baculovirus can be transported into some primary cells and into many cell lines derived from mouse, rat, porcine, and human tissues (10)(11)(12)(13)(14)(15)(16)(17). Using mammalian promoters, baculovirus enabled efficient transient expression of reporter genes in mammalian cells (10)(11)(12)(13)(14)(15)(16)(17). Condreay et al (17) also reported stable baculovirus transduction of several mammalian cell types with expression of a reporter gene for multiple passages.…”

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confidence: 99%

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Baculovirus-mediated gene transfer into pancreatic islet cells.

Tamarina

Wang

et al. 2000

Diabetes

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Baculovirus transduction is a gene transfer method that uses a moth cell virus for mammalian cells in culture, which results in a high-level prolonged expression. Here we demonstrate that recombinant baculoviruses can serve as efficient gene transfer vehicles for delivering foreign genes driven by mammalian promoters into human and mouse pancreatic islet cells. Existing methods, such as various transfection and electroporation techniques, either suffer from low efficiency or cause extensive membrane damage. Viral vectors have emerged as an important tool for gene delivery and expression in mammalian cells but suffer from several drawbacks, such as lengthy construction time and expression of viral genes. The baculovirus Autographa californica multiple nuclear polyhedrosis virus is widely used as a vector for expression of foreign genes in insect cells and, more recently, in some mammalian cells. Using several green fluorescent protein- and LacZ-expressing constructs in a cytomegalovirus promoter cassette, we obtained efficient gene expression in primary human and mouse islet cells. There was no impairment of glucose-stimulated intracellular free calcium responses after baculovirus infection. The safety and the relative ease of construction and propagation of the virus makes the baculovirus system a useful tool for facilitating the transfer of foreign genes.

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Section: Discussionmentioning

confidence: 92%

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confidence: 99%

Baculovirus-mediated gene transfer into pancreatic islet cells.

Tamarina

Wang

et al. 2000

Diabetes

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“…The data are therefore consistent with an important role for MTs in HCV RNA synthesis. Furthermore, because MTs mediate intracellular membrane trafficking between the endoplasmic reticulum (ER) and the Golgi (5,12,26,33), and also because HCV replication complexes are known to be anchored in ER or ER-like membranes (3,8,9,16,17,20,21,24,25,27,30,31,35), our data suggest that HCV RNA synthesis may be dependent on MT-mediated delivery of membranes required to establish the functional HCV replication complex.…”

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confidence: 85%

Cytoskeletal Requirements for Hepatitis C Virus (HCV) RNA Synthesis in the HCV Replicon Cell Culture System

Bost

Venable

Liu

et al. 2003

J Virol

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Hepatitis C virus (HCV) induces microtubule aggregates in infected hepatocytes. To determine if cytoskeletal elements are important for HCV RNA synthesis, we examined the effect of cytoskeleton inhibitors on HCV replicon transcription in Huh7 cells. The data demonstrate that HCV replication complex-mediated RNA synthesis requires microtubule and actin polymerization.Hepatitis C virus (HCV) infection is the leading cause of liver transplantation in the United States, with sequelae including liver fibrosis, cirrhosis, and hepatocellular carcinoma (reviewed in reference 19). Identified in 1989 as a plus-strand RNA virus (4), HCV causes an infection that was originally distinguished by its characteristic induction of microtubule (MT) aggregates in infected hepatocytes, implicating MTs in HCV-associated disease (2,11,23,28,29,32,36). Studies with a related flavivirus (Kunjin virus) have demonstrated that MT aggregates similar to those induced by HCV are also important for Kunjin RNA synthesis (15,22). Moreover, MT paracrystals have recently been detected in cultured cells transfected with an HCV subgenomic replicon (21). Taken together, these studies suggest that cytoskeletal elements may be required for HCV replication. Yet, despite the historical emphasis on MTs and HCV from a clinical perspective, the role of MTs in the HCV life cycle at a molecular level remains poorly understood.To determine if HCV RNA synthesis requires functional actin or MT networks, we examined the effects of cytoskeleton inhibitors on the efficiency of HCV RNA synthesis in the HCV replicon cell system ( Fig. 1) (18). In this system, Huh7 cells stably transfected with an HCV replicon RNA are used to mimic the RNA synthesis that occurs in an ongoing, persistent infection with HCV. Because the replicon construct encodes a neomycin resistance gene for G418 (Geneticin) selection, HCV RNA synthesis in the replicon cells can be detected by quantitative PCR using a primer-probe set specific for the neomycin sequence. We have validated this approach by demonstrating a dose-dependent decrease in replicon RNA levels upon alpha interferon treatment (data not shown), using alpha interferon concentrations similar to those cited previously by other laboratories (1, 10, 18). In addition, we have confirmed that transcription of the replicon in our HCV replicon cells is resistant to actinomycin D, as originally demonstrated by Lohmann et al. (18), verifying that transcription of replicon RNA is specific to the RNA-dependent RNA polymerase activity of the HCV replication complex. A single HCV replicon cell line-a serially passaged line originally generated by stable transfection of Huh7 cells with a replicon with a sequence identical to that used by Lohmann et al. (18) (Fig. 1)-was used for all experiments.Because vinblastine sulfate (VS) is a well-characterized inhibitor of MT polymerization and has been shown to alter Kunjin virus replication (15, 22), we first determined the effect of VS on HCV replicon RNA synthesis. One day prior to addition of the inhib...

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“…Since the size of the nucleocapsid is flexible, recombinant baculovirus particles are able to expand to accommodate very large DNA fragments. 10 As examples of this feature of baculoviruses, are recent reports of the expression of type 1 poliovirus 18 and hepatitis C virus 24 as integrated parts of baculovirus genome as well as construction of hybrid baculovirus-adenoassociated virus. 21 The hybrid virus was capable of a sitespecific integration into human chromosome 19, which is typical for adeno-associated virus.…”

Section: Discussionmentioning

confidence: 99%

“…According to the best of our knowledge, this is the first report of in vivo gene transfer with baculoviruses. The ease of manipulation and rapid construction of recombinant baculoviruses, 10,22 the lack of cytotoxicity in mammalian cells even at a high multiplicity of infection, 17,18,23 an inherent incapability to replicate in mammalian cells, 11 and a large capacity for the insertion of foreign sequences 10,18,24 make baculoviruses attractive tools for in vivo gene therapy. …”

Section: Introductionmentioning

confidence: 99%