Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

Institut Pasteur of Shanghai, Chinese Academy of Science, Shanghai, 200025, China.
Journal of Virology (Impact Factor: 4.44). 03/2011; 85(5):2138-47. DOI: 10.1128/JVI.02313-10
Source: PubMed


Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components
of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope
proteins are deleted (HCVΔE). The lack of envelope proteins could be efficiently complemented by the expression of homologous
envelope proteins in trans. HCVΔE production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCVΔE
could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round
infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently
rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production.
VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV
replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious
HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy
for HCV vaccine development.

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Available from: Paul Zhou, Jan 07, 2014
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    • "TCID 50 / ml pWPI - CE1 - BSD and pWPI - SpE2p7NS2 - BSD Stable cell line Adair et al . , 2009 50 CFU / ml pFBM [ JFH1 ) C - p7 Baculovirus Li et al . , 2011 300 I . U . / ml pcDNA3 - JFH1 - E1 / E2 Stable cell line Corless et al . , 2010 1100 FFU / ml pcDNA - E1 - p7"
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    ABSTRACT: Complementation is a naturally occurring genetic mechanism that has been studied for a number of plus-strand RNA viruses. Although trans-complementation is well documented for Flaviviridae family viruses, the first such system for hepatitis C virus (HCV) was only described in 2005. Since then, the development of a number of HCV trans-complementation models has improved our knowledge of HCV protein functions and interactions, genome replication and viral particle assembly. These models have also been used to produce defective viruses and so improvements are necessary for vaccine assays. This review provides an update on HCV trans-complementation systems, the viral mechanisms studied therewith and the production and characterization of trans-encapsidated particles.
    Full-text · Article · Apr 2013 · Journal of Viral Hepatitis
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    • "Similarly to other groups, our infectious titres of transcomplemented viral particles were relatively low. In the literature, several strategies have been used to increase single-round particle infectious titres: (i) subcloning of a packaging cell line stably expressing the structural proteins (Steinmann et al., 2008), (ii) improvement of the transduction efficiency (Adair et al., 2009) and (iii) introduction of adaptive mutations (Adair et al., 2009; Li et al., 2011). In our present study, we took advantage of the ability to select cells infected with trans-complemented particles through expression of the puromycin resistance gene. "
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    ABSTRACT: A trans-packaging system for hepatitis C virus (HCV) replicons lacking envelope glycoproteins was developed. The replicons were efficiently encapsidated into infectious particles after expression in trans of homologous HCV envelope proteins under the control of an adenoviral vector. Interestingly, expression in trans of core or core, p7 and NS2 with envelope proteins did not enhance trans-encapsidation. Expression of heterologous envelope proteins, in the presence or absence of heterologous core, p7 and NS2, did not rescue single-round infectious particle production. To increase the titre of homologous, single-round infectious particles in our system, successive cycles of trans-encapsidation and infection were performed. Four cycles resulted in a hundred-fold increase in the yield of particles. Sequence analysis revealed a total of 16 potential adaptive mutations in two independent experiments. Except for a core mutation in one experiment, all the mutations were located in non-structural regions mainly in NS5A (four in domain III and two near the junction with the NS5B gene). Reverse genetics studies suggested that D2437A and S2443T adaptive mutations, which are located into the NS5A-B cleavage site did not affect viral replication but enhanced the single-round infectious particles assembly only in trans-encapsidation model. In conclusion, our trans-encapsidation system enables the production of HCV single-round infectious particles. This system is adaptable and can positively select variants. The adapted variants promote trans-encapsidation and should constitute a valuable tool in the development of replicon-based HCV vaccines.
    Full-text · Article · Jan 2013 · Journal of General Virology
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    ABSTRACT: Amphiregulin (AREG) is a ligand of the epidermal growth factor (EGF) receptor and may play a role in the development of cirrhosis and hepatocellular carcinoma in patients infected with hepatitis C virus (HCV). AREG showed an enhanced expression in HCV-infected human hepatoma cells according to gene array analysis. Therefore, we addressed the question about the role of AREG in HCV infection. AREG expression level was elevated in hepatoma cells containing a subgenomic HCV replicon or infected by HCV. Using a reporter assay, AREG promoter activity was found to be upregulated upon HCV infection. The enhanced AREG expression in hepatoma cells was partly caused by dsRNAs, HCV NS3 protein and autocrine stimulation. AREG was able to activate cellular signalling pathways including ERK, Akt and p38, promote cell proliferation, and protect cells from HCV-induced cell death. Further, knockdown of AREG expression increased the efficiency of HCV entry, as proven by HCV pseudoparticles reporter assay. However, the formation and release of infectious HCV particles were reduced by AREG silencing with a concomitant accumulation of intracellular HCV RNA pool, indicating that the assembly and release of HCV progeny may require AREG expression. Blocking the MAPK-ERK pathway by U0126 in Huh7.5.1 cells had a similar effect on HCV replication. In conclusion, HCV infection leads to an increase in AREG expression in hepatocytes. AREG expression is essential for efficient HCV assembly and virion release. Due to the activation of the cellular survival pathways, AREG may counteract HCV-induced apoptosis of infected hepatocytes and facilitate the development of liver cirrhosis and hepatocellular carcinoma.
    Full-text · Article · Jun 2011 · Journal of General Virology
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