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MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR

MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism su... Full description

Journal Title: Proceedings of the National Academy of Sciences 22 March 2011, Vol.108(12), p.4991
Main Author: Yi-Ping Li
Other Authors: Judith M. Gottwein , Troels K. Scheel , Tanja B. Jensen , Jens Bukh
Format: Electronic Article Electronic Article
Language: English
Subjects:
ID: ISSN: 0027-8424 ; E-ISSN: 1091-6490 ; DOI: 10.1073/pnas.1016606108
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recordid: pnas_s108_12_4991
title: MicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR
format: Article
creator:
  • Yi-Ping Li
  • Judith M. Gottwein
  • Troels K. Scheel
  • Tanja B. Jensen
  • Jens Bukh
subjects:
  • Sciences (General)
ispartof: Proceedings of the National Academy of Sciences, 22 March 2011, Vol.108(12), p.4991
description: MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1–6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1–6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.
language: eng
source:
identifier: ISSN: 0027-8424 ; E-ISSN: 1091-6490 ; DOI: 10.1073/pnas.1016606108
fulltext: fulltext_linktorsrc
issn:
  • 0027-8424
  • 00278424
  • 1091-6490
  • 10916490
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titleMicroRNA-122 antagonism against hepatitis C virus genotypes 1–6 and reduced efficacy by host RNA insertion or mutations in the HCV 5′ UTR
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descriptionMicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1–6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1–6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.
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MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1–6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1–6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.

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MicroRNA-122 (miR-122) is believed to stimulate hepatitis C virus (HCV) replication through interaction with two adjacent sites downstream of stem loop I (SLI) within the HCV 5′ untranslated region (5′ UTR). Recently, it was demonstrated that locked nucleic acid SPC3649-induced miR-122 antagonism suppressed HCV genotype 1a and 1b infection in vivo. However, virus-producing culture systems with 5′ UTR of different HCV genotypes have not been available for testing 5′ UTR-based treatment approaches. Using JFH1-based Core-NS2 genotype recombinants, we developed 5′ UTR-NS2 recombinants of HCV genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, and 6a with efficient growth in Huh7.5 cells. Deletion mutagenesis studies demonstrated that the 5′ UTR SLI was essential for genotypes 1–6 infection. However, lack of SLI could be compensated for by insertion of other structured HCV or host RNA sequences, including U3 small nucleolar RNA. We demonstrated that SPC3649-induced miR-122 antagonism had a potent antiviral effect against HCV genotypes 1–6 5′ UTR-NS2 viruses. Strikingly, HCV recombinant virus with substitution of SLI and miR-122 binding site 1 (S1) by the U3 RNA sequence was not affected by miR-122 antagonism; this was attributed to the lack of an intact S1 by reverse genetics studies. Therefore, we engineered the corresponding U3 RNA sequences into S1 and demonstrated that HCV recombinants with wild-type SLI and single or combined mutations at four of eight nucleotides of S1 were viable in Huh7.5 cells. These mutations reduced the efficacy of SPC3649 treatment, indicating that escape variants to miR-122 antagonism-based HCV therapy could potentially occur.

pubNational Acad Sciences
doi10.1073/pnas.1016606108
urlhttp://www.pnas.org/content/108/12/4991.abstract
lad01Proceedings of the National Academy of Sciences
pages4991-4996
date2011-03-22