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Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

ABSTRACT: We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

SUBMITTER: Shimakami T

PROVIDER: S-EPMC1440399 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

Shimakami Tetsuro T Honda Masao M Kusakawa Takashi T Murata Takayuki T Shimotohno Kunitada K Kaneko Shuichi S Murakami Seishi S

Journal of virology 20060401 7

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to ...[more]

PMID: 16537600

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Project description:To construct a tricistronic hepatitis C virus (HCV) replicon with double internal ribosome entry sites (IRESes) of only 22 nucleotides for each, substituting the encephalomyocarditis virus (EMCV) IRESes, which are most often used as the translation initiation element to form HCV replicons.The alternative 22-nucleotide IRES, RNA-binding motif protein 3 IRES (Rbm3 IRES), was used to form a tricistronic HCV replicon, to facilitate constructing HCV-harboring stable cell lines and successive antiviral screening using a luciferase marker. Briefly, two sequential Rbm3 IRESes were inserted into bicistronic pUC19-HCV plasmid, consequently forming a tricistronic HCV replicon (pHCV-rep-NeoR-hRluc), initiating the translation of humanized Renilla luciferase and HCV non-structural gene, along with HCV authentic IRES initiating the translation of neomycin resistance gene. The sH7 cell lines, in which the novel replicon RNA stably replicated, were constructed by neomycin and luciferase activity screening. The intracellular HCV replicon RNA, expression of inserted foreign genes and HCV non-structural gene, as well as response to anti-HCV agents, were measured in sH7 cells and cells transiently transfected with tricistronic replicon RNA.The intracellular HCV replicon RNA and expression of inserted foreign genes and HCV non-structural gene in sH7 cells and cells transiently transfected with tricistronic replicon RNA were comparable to those in cells stably or transiently transfected with traditional bicistronic HCV replicons. The average relative light unit in pHCV-rep-NeoR-hRluc group was approximately 2-fold of those in the pUC19-HCV-hRLuc and Tri-JFH1 groups (1.049 × 10(8) ± 2.747 × 10(7) vs 5.368 × 10(7) ± 1.016 × 10(7), P < 0.05; 1.049 × 10(8) ± 2.747 × 10(7) vs 5.243 × 10(7) ± 1.194 × 10(7), P < 0.05), suggesting that the translation initiation efficiency of the first Rbm3 IRES in the two sequential IRESes was stronger than the HCV authentic IRES and EMCV IRES. The fold changes of 72 h/4 h relative light units in the pHCV-rep-NeoR-hRluc and pUC19-HCV-hRLuc groups were similar (159.619 ± 9.083 vs 163.536 ± 24.031, P = 0.7707), and were both higher than the fold change in the Tri-JFH1 group 159.619± 9.083 vs 140.811 ± 9.882, P < 0.05; 163.536 ± 24.031 vs 140.811 ± 9.882, P < 0.05), suggesting that the replication potency of the Rbm3 IRES tricistronic replicon matched the replication of bicistronic replicon and exceeded the potency of EMCV IRES replicon. Replication of tricistronic replicons was suppressed by ribavirin, simvastatin, atorvastatin, telaprevir and boceprevir. Interferon-alpha 2b could not block replication of the novel replicon RNA in sH7 cells. After interferon stimulation, MxA mRNA and protein levels were lower in sH7 than in parental cells.Tricistronic HCV replicon with double Rbm3 IRESes could be applied to evaluate the replication inhibition efficacy of anti-HCV agents.

Dataset Information

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

Publications

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

Similar Datasets

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