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

Tetsuro Shimakami, Masao Honda, Takashi Kusakawa, Takayuki Murata, Kunitada Shimotohno, Shuichi Kaneko, Seishi Murakami

研究成果: Article

32 引用 (Scopus)

抄録

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.

元の言語English
ページ(範囲)3332-3340
ページ数9
ジャーナルJournal of Virology
80
発行部数7
DOI
出版物ステータスPublished - 01-04-2006

Fingerprint

replicon
Replicon
Hepatitis C virus
Virus Replication
virus replication
Hepacivirus
Proteins
mutants
proteins
alanine
Alanine
amino acids
Amino Acids
small interfering RNA
Small Interfering RNA
RNA-directed RNA polymerase
RNA Replicase
nucleolin
protein binding
transfection

All Science Journal Classification (ASJC) codes

  • Immunology

これを引用

Shimakami, Tetsuro ; Honda, Masao ; Kusakawa, Takashi ; Murata, Takayuki ; Shimotohno, Kunitada ; Kaneko, Shuichi ; Murakami, Seishi. / Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon. :: Journal of Virology. 2006 ; 巻 80, 番号 7. pp. 3332-3340.
@article{3b407cd4ab8d4403aa9a4c5c580b5592,
title = "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.",
author = "Tetsuro Shimakami and Masao Honda and Takashi Kusakawa and Takayuki Murata and Kunitada Shimotohno and Shuichi Kaneko and Seishi Murakami",
year = "2006",
month = "4",
day = "1",
doi = "10.1128/JVI.80.7.3332-3340.2006",
language = "English",
volume = "80",
pages = "3332--3340",
journal = "Journal of Virology",
issn = "0022-538X",
publisher = "American Society for Microbiology",
number = "7",

}

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon. / Shimakami, Tetsuro; Honda, Masao; Kusakawa, Takashi; Murata, Takayuki; Shimotohno, Kunitada; Kaneko, Shuichi; Murakami, Seishi.

:: Journal of Virology, 巻 80, 番号 7, 01.04.2006, p. 3332-3340.

研究成果: Article

TY - JOUR

T1 - Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon

AU - Shimakami, Tetsuro

AU - Honda, Masao

AU - Kusakawa, Takashi

AU - Murata, Takayuki

AU - Shimotohno, Kunitada

AU - Kaneko, Shuichi

AU - Murakami, Seishi

PY - 2006/4/1

Y1 - 2006/4/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33645227630&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33645227630&partnerID=8YFLogxK

U2 - 10.1128/JVI.80.7.3332-3340.2006

DO - 10.1128/JVI.80.7.3332-3340.2006

M3 - Article

VL - 80

SP - 3332

EP - 3340

JO - Journal of Virology

JF - Journal of Virology

SN - 0022-538X

IS - 7

ER -