Abstract
We describe a mutational analysis of the 3′ nontranslated RNA (3′NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3′NTR, namely, the highly conserved 3′ terminal 98-nucleotide (nt) segment (3′X), an upstream poly(U)-poly (UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5′ end of the 3′NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3′X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3′ end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3′-most 150 nt of the HCV-N genome [the 3′X region and the 3′52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3′NTR plays a facilitating role in replication but is not absolutely required.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 3557-3568 |
| Number of pages | 12 |
| Journal | Journal of Virology |
| Volume | 77 |
| Issue number | 6 |
| DOIs | |
| State | Published - Mar 2003 |
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ASJC Scopus subject areas
- Immunology
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3′ nontranslated RNA signals required for replication of hepatitis C virus RNA. / Yi, Min Kyung; Lemon, Stanley M.
In: Journal of Virology, Vol. 77, No. 6, 03.2003, p. 3557-3568.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - 3′ nontranslated RNA signals required for replication of hepatitis C virus RNA
AU - Yi, Min Kyung
AU - Lemon, Stanley M.
PY - 2003/3
Y1 - 2003/3
N2 - We describe a mutational analysis of the 3′ nontranslated RNA (3′NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3′NTR, namely, the highly conserved 3′ terminal 98-nucleotide (nt) segment (3′X), an upstream poly(U)-poly (UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5′ end of the 3′NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3′X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3′ end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3′-most 150 nt of the HCV-N genome [the 3′X region and the 3′52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3′NTR plays a facilitating role in replication but is not absolutely required.
AB - We describe a mutational analysis of the 3′ nontranslated RNA (3′NTR) signals required for replication of subgenomic hepatitis C virus (HCV) RNAs. A series of deletion mutants was constructed within the background of an HCV-N replicon that induces the expression of secreted alkaline phosphatase in order to examine the requirements for each of the three domains comprising the 3′NTR, namely, the highly conserved 3′ terminal 98-nucleotide (nt) segment (3′X), an upstream poly(U)-poly (UC) [poly(U/UC)] tract, and the variable region (VR) located at the 5′ end of the 3′NTR. Each of these domains was found to contribute to efficient replication of the viral RNA in transiently transfected hepatoma cells. Replication was not detected when any of the three putative stem-loop structures within the 3′X region were deleted. Similarly, complete deletion of the poly(U/UC) tract abolished replication. Replacement of a minimum of 50 to 62 nt of poly(U/UC) sequence was required for detectable RNA replication when the native sequence was restored in a stepwise fashion from its 3′ end. Lengthier poly(U/UC) sequences, and possibly pure homopolymeric poly(U) tracts, were associated with more efficient RNA amplification. Finally, while multiple deletion mutations were tolerated within VR, each led to a partial loss of replication capacity. The impaired replication capacity of the deletion mutants could not be explained by reduced translational activity or by decreased stability of the RNA, suggesting that each of these mutations may impair recognition of the RNA by the viral replicase during an early step in negative-strand RNA synthesis. The results indicate that the 3′-most 150 nt of the HCV-N genome [the 3′X region and the 3′52 nt of the poly(U/UC) tract] contain RNA signals that are essential for replication, while the remainder of the 3′NTR plays a facilitating role in replication but is not absolutely required.
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U2 - 10.1128/JVI.77.6.3557-3568.2003
DO - 10.1128/JVI.77.6.3557-3568.2003
M3 - Article
VL - 77
SP - 3557
EP - 3568
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 6
ER -