Influenza A viruses (IAV) are responsible for seasonal epidemics, and pandemics can arise from novel zoonotic influenza A viruses transmitting to humans 1,2 . IAV contain a segmented negative sense RNA genome that is transcribed and replicated by the viral RNA-dependent RNA polymerase, composed of the PB1, PB2, and PA subunits [3][4][5] . Although the high-resolution crystal structure of bat IAV polymerase (FluPol A ) has been reported 6 , there are no complete structures available for human and avian FluPol A . Furthermore, the molecular mechanisms of viral RNA (vRNA) replication, which proceeds through a complementary RNA (cRNA) replicative intermediate and requires polymerase oligomerisation 7-10 , remain largely unknown. Here we Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The influenza A virus RNA-dependent RNA polymerase consists of three subunits-PB1, PB2, and PA. The PB1 subunit is the catalytically active polymerase, catalyzing the sequential addition of nucleotides to the growing RNA chain. The PB2 subunit is a cap-binding protein that plays a role in initiation of viral mRNA synthesis by recruiting capped RNA primers. The function of PA is unknown, but previous studies of temperature-sensitive viruses with mutations in PA have implied a role in viral RNA replication. In this report we demonstrate that the PA subunit is required not only for replication but also for transcription of viral RNA. We mutated evolutionarily conserved amino acids to alanines in the C-terminal region of the PA protein, since the C-terminal region shows the highest degree of conservation between PA proteins of influenza A, B, and C viruses. We tested the effects of these mutations on the ability of RNA polymerase to transcribe and replicate viral RNA. We also tested the compatibility of these mutations with viral viability by using reverse-genetics techniques. A mutant with a histidine-to-alanine change at position 510 (H510A) in the PA protein of influenza A/WSN/33 virus showed a differential effect on transcription and replication. This mutant was able to perform replication (vRNA3cRNA3vRNA), but its transcriptional activity (vRNA3mRNA) was negligible. In vitro analyses of the H510A recombinant polymerase, by using transcription initiation, vRNA-binding, capped-RNAbinding, and endonuclease assays, suggest that the primary defect of this mutant polymerase is in its endonuclease activity.Influenza A virus is a negative-strand RNA virus containing eight segments of single-stranded RNA as its genome (39). The RNA genome is transcribed and replicated by the viral RNA-dependent RNA polymerase in the cell nucleus (21). The viral RNAs (vRNA) are transcribed into mRNAs and replicated through a cRNA intermediate to produce more vRNA molecules. Synthesis of these three RNA species requires different modes of initiation and termination (reviewed in references 23 and 34). Synthesis of mRNAs is primed by short capped RNA fragments that are generated from cellular pre-mRNAs by endonucleolytic cleavage. Consequently, viral mRNA molecules contain a 9-to 17-nucleotide (nt) capped host-derived RNA sequence at their 5Ј ends. On the other hand, the synthesis of cRNA and vRNA molecules is initiated in a primer-independent manner, resulting in triphosphorylated 5Ј ends. Synthesis of mRNAs is prematurely terminated 16 to 17 nucleotides from the 5Ј end of the vRNA template at a sequence of 5 to 7 uridines that acts as a polyadenylation signal (30,47,49). The poly(A) tail is synthesized by the viral RNA polymerase by repeated copying of the U sequence (47). During the synthesis of cRNA molecules, the polyadenylation signal is ignored, resulting in full-length copies of vRNA.All three reactions, i.e., vRNA3mRNA (transcription), vRNA3cRNA (first step of replication), and cRNA3vRNA (second step of replication) are catalyzed b...

Influenza virus RNA-dependent RNA polymerase is a heterotrimeric complex of PB1, PB2, and PA. We show that the individually expressed PB2 subunit can be assembled with the coexpressed PB1-PA dimer in vitro into a transcriptionally active complex. Furthermore, we demonstrate that a model viral RNA promoter can bind to the PB1-PA dimer prior to assembly with PB2. Our results are consistent with a recently proposed model for the sequential assembly of viral RNA polymerase complex in which the PB1-PA dimeric complex and the PB2 monomer are transported into the nucleus separately and then assembled in the nucleus.

Aquatic birds represent a vast reservoir from which novel pandemic influenza A viruses can emerge 1 . Influenza viruses contain a negative-sense segmented RNA genome which is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein (vRNP) complexes 2 , 3 . RNA polymerases of avian influenza A viruses (FluPol A ) replicate viral RNA poorly in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity 4 . Interestingly, host adaptive mutations, particularly a glutamic acid to lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit (PB2 627 ), allow avian FluPol A to overcome this restriction and efficiently replicate viral RNA in the presence of human ANP32 proteins. However, the molecular mechanisms of genome replication and the interplay with ANP32 proteins remain largely unknown. Here, we report cryo-EM structures of influenza C virus polymerase (FluPol C ) in complex with human and chicken ANP32A. In both structures, two FluPol C molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain (LRR) of ANP32A. The C-terminal low complexity acidic region (LCAR) of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, providing insight into the mechanism behind the PB2E 627K adaptive mutation in mammalian hosts. We propose that this complex represents a replication platform for the viral RNA genome, in which one of the FluPol molecules acts as a replicase while the other initiates the assembly of the nascent replication product into a vRNP.

The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-β. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.