Abstract
ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp “snatches” capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to the ultimate or penultimate nucleotide of the segments for the initiation of viral mRNA synthesis. It has been proposed that this initiation process is not processive and that the RdRp uses a prime-realign mechanism during transcription. Here we provide in vitro evidence for the existence of this transcriptional prime-realign mechanism but show that it functions efficiently only for primers that are short or cannot stably base pair with the template. In addition, we demonstrate that transcriptional elongation is dependent on the priming loop of the PB1 subunit of the RdRp. We propose that the prime-realign mechanism may be used to rescue abortive transcription initiation events or cope with sequence variation among primers. Overall, these observations advance our mechanistic understanding of how influenza A virus initiates transcription correctly and efficiently.IMPORTANCE Influenza A virus causes severe disease in humans and is considered a major global health threat. The virus replicates and transcribes its genome by using an enzyme called the RNA polymerase. To ensure that the genome is amplified faithfully and abundant viral mRNAs are made for viral protein synthesis, the viral RNA polymerase must transcribe the viral genome efficiently. In this report, we characterize a structure inside the polymerase that contributes to the efficiency of viral mRNA synthesis.
Highlights
The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell
Ostensibly by default, the RdRp positions 2C of the viral RNA (vRNA) in the Ϫ1 position of the active site [13], which is ideal for transcription initiation with primers ending in 3= G from 3G of the template
Transcription products similar to the ones described above were previously observed in assays in which influenza A virus (IAV) RdRp purified from mammalian cells was used to extend an 11-nt-long capped primer [6, 7] or a -globin mRNA-derived primer [20,21,22,23,24] (Fig. 1D), suggesting that these three RNA species are typical IAV transcription products
Summary
The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. The N-terminal one-third of PB2, the PB1 subunit, and the C-terminal two-thirds of PA form the conserved core of the RdRp [3,4,5], while the remaining portions of PB2 and PA form flexible domains that have cap-binding and endonuclease activities, respectively (Fig. 1A) Another key functional structure in the RdRp is a conserved PB1 -hairpin called the priming loop, which resides downstream of the active site of the IAV RdRp and is important for viral replication initiation [4, 6, 7]. Te Velthuis and Oymans primer-dependent process for viral transcription initiation To produce this primer, the IAV RdRp must first bind to the C-terminal domain of an actively transcribing, serine 5-phosphorylated RNA polymerase II (Pol II) complex in the nucleus of an infected cell [8, 9]. Subsequent binding and cleavage of nascent Pol II transcripts produce 8- to jvi.asm.org 2
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