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Aquatic birds represent a vast reservoir from which new pandemic influenza A viruses can emerge.sup.1. Influenza viruses contain a negative-sense segmented RNA genome that is transcribed and replicated by the viral heterotrimeric RNA polymerase (FluPol) in the context of viral ribonucleoprotein complexes.sup.2,3. RNA polymerases of avian influenza A viruses (FluPolA) replicate viral RNA inefficiently in human cells because of species-specific differences in acidic nuclear phosphoprotein 32 (ANP32), a family of essential host proteins for FluPol activity.sup.4. Host-adaptive mutations, particularly a glutamic-acid-to-lysine mutation at amino acid residue 627 (E627K) in the 627 domain of the PB2 subunit, enable avian FluPolA 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-electron microscopy structures of influenza C virus polymerase (FluPolC) in complex with human and chicken ANP32A. In both structures, two FluPolC molecules form an asymmetric dimer bridged by the N-terminal leucine-rich repeat domain of ANP32A. The C-terminal low-complexity acidic region of ANP32A inserts between the two juxtaposed PB2 627 domains of the asymmetric FluPolA dimer, suggesting a mechanism for how the adaptive PB2(E627K) mutation enables the replication of viral RNA 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 viral ribonucleoprotein complex. Structural and biochemical studies of influenza virus RNA polymerase in complex with host acidic nuclear phosphoprotein 32 (ANP32) show how ANP32-mediated polymerase dimerization enables the replication of influenza viral RNA in a host-dependent manner., Author(s): Loïc Carrique [sup.1] , Haitian Fan [sup.2] , Alexander P. Walker [sup.2] , Jeremy R. Keown [sup.1] , Jane Sharps [sup.2] , Ecco Staller [sup.3] [sup.5] , Wendy S. [...]

Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans.sup.1,2. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol.sub.A) composed of PB1, PB2 and PA subunits.sup.3-5. Although the high-resolution crystal structure of FluPol.sub.A of bat influenza A virus has previously been reported.sup.6, there are no complete structures available for human and avian FluPol.sub.A. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication--which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase.sup.7-10--remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol.sub.A from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol.sub.A forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol.sub.A bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol.sub.A dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol.sub.A dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol.sub.A, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol.sub.A that could be targeted in the development of antiviral drugs. Structures of RNA polymerase of human and avian influenza A viruses reveal that the interface of the RNA polymerase dimer is required to initiate viral RNA synthesis in viral genome replication., Author(s): Haitian Fan [sup.1] , Alexander P. Walker [sup.1] , Loïc Carrique [sup.2] , Jeremy R. Keown [sup.2] , Itziar Serna Martin [sup.1] [sup.2] [sup.6] , Dimple Karia [sup.2] , [...]

The heterotrimeric influenza A virus RNA-dependent RNA polymerase complex, composed of PB1, PB2, and PA subunits, is responsible for transcribing and replicating the viral RNA genome. The N-terminal endonuclease domain of the PA subunit performs endonucleolytic cleavage of capped host RNAs to generate capped RNA primers for viral transcription. A surface-exposed flexible loop (PA51-72-loop) in the PA endonuclease domain has been shown to be dispensable for endonuclease activity. Interestingly, the PA51-72-loop was found to form different intramolecular interactions depending on the conformational arrangement of the polymerase. In this study, we show that a PA subunit lacking the PA51-72-loop assembles into a heterotrimeric polymerase with PB1 and PB2. We demonstrate that in a cellular context, the PA51-72-loop is required for RNA replication but not transcription by the viral polymerase. In agreement, recombinant viral polymerase lacking the PA51-72-loop is able to carry out cap-dependent transcription but is inhibited in de novo replication initiation in vitro. Furthermore, viral RNA (vRNA) synthesis is also restricted during ApG-primed extension, indicating that the PA51-72-loop is required not only for replication initiation but also for elongation on a cRNA template. We propose that the PA51-72-loop plays a role in the stabilization of the replicase conformation of the polymerase. Together, these results further our understanding of influenza virus RNA genome replication in general and highlight a role of the PA endonuclease domain in polymerase function in particular. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The RNA-dependent RNA polymerase complex of influenza A virus consists of three subunits, PB1, PB2 and PA. To investigate the function of the PA subunit, we mutated evolutionarily conserved amino acids to alanines in the C-terminal third of PA of influenza A/WSN/33 that shows the highest degree of conservation between PA proteins of influenza A, B and C viruses. A H510A mutation inhibited the endonuclease cleavage of capped RNAs, K539A downregulated replication, while R638A resulted in severe attenuation of viral growth in cell culture by promoting the synthesis of defective interfering (DI) RNAs. Our results show that PA is required for both transcription and replication and that, in addition to its role in endonuclease cleavage, it might also act as an elongation factor during viral RNA synthesis. To investigate the function of PB2 in cap binding, we mutated all evolutionarily conserved aromatic amino acids of PB2 of influenza A/WSN/33. Our results suggest that F363 and F404 bind the cap structure of mRNA in an aromatic sandwich as in the evolutionarily unrelated VP39, eIF4E and CBP20. [Copyright &y& Elsevier]