Two cis-acting elements in negative RNA strand of hepatitis C virus involved in synthesis of positive RNA strand in vitro

L. Ye, Khalid A. Timani, L. Ye, L. Kong, X. Yang, Q. Liao, J. Wu

Research output: Contribution to journalArticleResearchpeer-review

8 Citations (Scopus)

Abstract

Sequences at the 3′-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3′-terminal region of negative RNA strand ((-)3′T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3′-cytidylate (3′C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3′-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3′-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3′C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3′-proximal stem-loop and the first 3′C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.

Original languageEnglish
Pages (from-to)83-90
Number of pages8
JournalActa Virologica
Volume49
Issue number2
StatePublished - 1 Dec 2005

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Hepacivirus
RNA
In Vitro Techniques
RNA Replicase
Mutagenesis

Keywords

  • Hepatitis C virus
  • Negative RNA strand
  • Positive RNA strand
  • RNA
  • RNA synthesis
  • RNA-dependent RNA polymerase
  • cis-acting element

Cite this

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title = "Two cis-acting elements in negative RNA strand of hepatitis C virus involved in synthesis of positive RNA strand in vitro",
abstract = "Sequences at the 3′-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3′-terminal region of negative RNA strand ((-)3′T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3′-cytidylate (3′C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3′-proximal stem-loop resulted in an approximately 90{\%} decrease in RNA synthesis. Disruption of the 3′-proximal stem-loop structure by nucleotide substitutions led to a 70-80{\%} decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3′C by guanylate (G) led to a 90{\%} decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80{\%} decrease in the RNA synthesis only. These findings demonstrate that the 3′-proximal stem-loop and the first 3′C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.",
keywords = "Hepatitis C virus, Negative RNA strand, Positive RNA strand, RNA, RNA synthesis, RNA-dependent RNA polymerase, cis-acting element",
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Two cis-acting elements in negative RNA strand of hepatitis C virus involved in synthesis of positive RNA strand in vitro. / Ye, L.; Timani, Khalid A.; Ye, L.; Kong, L.; Yang, X.; Liao, Q.; Wu, J.

In: Acta Virologica, Vol. 49, No. 2, 01.12.2005, p. 83-90.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - Two cis-acting elements in negative RNA strand of hepatitis C virus involved in synthesis of positive RNA strand in vitro

AU - Ye, L.

AU - Timani, Khalid A.

AU - Ye, L.

AU - Kong, L.

AU - Yang, X.

AU - Liao, Q.

AU - Wu, J.

PY - 2005/12/1

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AB - Sequences at the 3′-ends of both positive and negative strands of Hepatitis C virus (HCV) RNA harbor cis-acting elements required for RNA replication. However, little is known about the properties of the negative RNA strand as a template for the synthesis of positive RNA strand. In this study, a purified recombinant HCV RNA-dependent RNA polymerase (RdRp) was used to investigate the synthesis of positive RNA strand using the 3′-terminal region of negative RNA strand ((-)3′T RNA) as template. A mutagenesis analysis was performed to evaluate the role of the 3'-proximal stem-loop and the first 3′-cytidylate (3′C) of the negative RNA strand in the synthesis of the positive RNA strand. A negative RNA strand of wild type (wt) HCV as template was able to direct the synthesis of a full-length positive RNA strand. Deletion of the 3′-proximal stem-loop resulted in an approximately 90% decrease in RNA synthesis. Disruption of the 3′-proximal stem-loop structure by nucleotide substitutions led to a 70-80% decrease in RNA synthesis. However, the restoration of the stem-loop by compensatory mutations in the stem region restored also the RNA synthesis. Likewise, the deletion or substitution of the first 3′C by guanylate (G) led to a 90% decrease in the RNA synthesis; while the substitution by adenylate (A) or uridylate (U) resulted in a 60-80% decrease in the RNA synthesis only. These findings demonstrate that the 3′-proximal stem-loop and the first 3′C of the negative RNA strand of HCV are two cis-acting elements involved in the synthesis of the positive RNA strand.

KW - Hepatitis C virus

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KW - Positive RNA strand

KW - RNA

KW - RNA synthesis

KW - RNA-dependent RNA polymerase

KW - cis-acting element

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JO - Acta Virologica

JF - Acta Virologica

SN - 0001-723X

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