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 language | English |
|---|---|
| Pages (from-to) | 83-90 |
| Number of pages | 8 |
| Journal | Acta Virologica |
| Volume | 49 |
| Issue number | 2 |
| State | Published - 1 Dec 2005 |
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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 journal › Article › Research › peer-review
TY - JOUR
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
Y1 - 2005/12/1
N2 - 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.
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
KW - Negative RNA strand
KW - Positive RNA strand
KW - RNA
KW - RNA synthesis
KW - RNA-dependent RNA polymerase
KW - cis-acting element
UR - http://www.scopus.com/inward/record.url?scp=26044446438&partnerID=8YFLogxK
M3 - Article
VL - 49
SP - 83
EP - 90
JO - Acta Virologica
JF - Acta Virologica
SN - 0001-723X
IS - 2
ER -