Internal initiation of influenza virus replication of viral RNA and complementary RNA in vitro.
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 purified RNA polymerase (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.
Zhang
Shijian
S
Unit of Viral Genome Regulation, Institut Pasteur of Shanghai, Key Laboratory of Molecular Virology and Immunology, Chinese Academy of Sciences, 411 Hefei Road, Shanghai 200025, China.
Wang
Jinlan
J
Wang
Qiang
Q
Toyoda
Tetsuya
T
D009843:Oligoribonucleotides / Q000378:metabolism; D009975:Orthomyxoviridae / Q000502:physiology*; D018075:RNA, Complementary / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
D009843:Oligoribonucleotides; D018075:RNA, Complementary; D012367:RNA, Viral; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase