TY - JOUR
T1 - Internal initiation of influenza virus replication of viral RNA and complementary RNA in vitro
AU - Zhang, Shijian
AU - Wang, Jinlan
AU - Wang, Qiang
AU - Toyoda, Tetsuya
N1 - Copyright:
Copyright 2011 Elsevier B.V., All rights reserved.
PY - 2010/12/24
Y1 - 2010/12/24
N2 - Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and realignment model for influenza virus genome replication has been recently proposed (Deng, T., Vreede, F. T., and Brownlee, G. G. (2006) J. Virol. 80, 2337-2348). We obtained new results, which led us to propose a novel model for the initiation of viral RNA (vRNA) replication. In our study, we analyzed the initiation mechanisms of influenza virus vRNA and complementary RNA (cRNA) synthesis in vitro, using purifiedRNApolymerase (RdRp) and 84-nt model RNA templates. We found that, for vRNA → cRNA →, RdRp initiated replication from the second nucleotide of the 3′-end. Therefore, host RNA-specific ribonucleotidyltransferases are required to add one nucleotide (purine residues are preferred) to the 3′-end of vRNA to make the complete copy of vRNA. This hypothesis was experimentally proven using poly(A) polymerase. For cRNA → vRNA, the dinucleotide primer AG was synthesized from UC (fourth and fifth from the 3′-end) by RdRp pausing at the sixth U of UUU and realigning at the 3′-end of cRNA template; then RdRp was able to read through the entire template RNA. The RdRp initiation complex was not stable until it had read through the UUU of cRNA and the UUUU of vRNA at their respective 3′-ends. This was because primers overlapping with the first U of the clusters did not initiate transcription efficiently, and the initiation product of v84+G (the v84 template with an extra G at its 3′-end), AGC, realigned to the 3′-end.
AB - Influenza virus transcription is a prototype of primer-dependent initiation. Its replication mechanism is thought to be primer-independent. The internal initiation and realignment model for influenza virus genome replication has been recently proposed (Deng, T., Vreede, F. T., and Brownlee, G. G. (2006) J. Virol. 80, 2337-2348). We obtained new results, which led us to propose a novel model for the initiation of viral RNA (vRNA) replication. In our study, we analyzed the initiation mechanisms of influenza virus vRNA and complementary RNA (cRNA) synthesis in vitro, using purifiedRNApolymerase (RdRp) and 84-nt model RNA templates. We found that, for vRNA → cRNA →, RdRp initiated replication from the second nucleotide of the 3′-end. Therefore, host RNA-specific ribonucleotidyltransferases are required to add one nucleotide (purine residues are preferred) to the 3′-end of vRNA to make the complete copy of vRNA. This hypothesis was experimentally proven using poly(A) polymerase. For cRNA → vRNA, the dinucleotide primer AG was synthesized from UC (fourth and fifth from the 3′-end) by RdRp pausing at the sixth U of UUU and realigning at the 3′-end of cRNA template; then RdRp was able to read through the entire template RNA. The RdRp initiation complex was not stable until it had read through the UUU of cRNA and the UUUU of vRNA at their respective 3′-ends. This was because primers overlapping with the first U of the clusters did not initiate transcription efficiently, and the initiation product of v84+G (the v84 template with an extra G at its 3′-end), AGC, realigned to the 3′-end.
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U2 - 10.1074/jbc.M110.130062
DO - 10.1074/jbc.M110.130062
M3 - Article
C2 - 20858902
AN - SCOPUS:78650403082
SN - 0021-9258
VL - 285
SP - 41194
EP - 41201
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 52
ER -