Crystal structure of the RNA-dependent RNA polymerase from influenza C virus.

Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.

Regulation of influenza A virus nucleoprotein oligomerization by phosphorylation.

In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.

Uncoupling of influenza A virus transcription and replication through mutation of the unpaired adenosine in the viral RNA promoter.

Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3' and 5' termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5' strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase.

Stabilization of influenza virus replication intermediates is dependent on the RNA-binding but not the homo-oligomerization activity of the viral nucleoprotein.

The influenza virus nucleoprotein (NP) is believed to play a central role in directing a switch from RNA genome transcription to replication by the viral RNA polymerase. However, this role has recently been disputed with the proposal of alternative regulatory mechanisms. It has been suggested that the expression of viral polymerase and NP allows genome replication by stabilization of cRNA replication intermediates and complementary ribonucleoprotein (cRNP) assembly. Here, we demonstrate that the RNA-binding activity of NP is necessary for stabilization of cRNA, whereas, surprisingly, homo-oligomerization of NP is not essential. However, both RNA binding and homo-oligomerization activities are essential for genome replication.

The influenza A virus NS1 protein interacts with the nucleoprotein of viral ribonucleoprotein complexes.

The influenza A virus genome consists of eight RNA segments that associate with the viral polymerase proteins (PB1, PB2, and PA) and nucleoprotein (NP) to form ribonucleoprotein complexes (RNPs). The viral NS1 protein was previously shown to associate with these complexes, although it was not clear which RNP component mediated the interaction. Using individual TAP (tandem affinity purification)-tagged PB1, PB2, PA, and NP, we demonstrated that the NS1 protein interacts specifically with NP and not the polymerase subunits. The region of NS1 that binds NP was mapped to the RNA-binding domain.

RIG-I detects viral genomic RNA during negative-strand RNA virus infection.

RIG-I is a key mediator of antiviral immunity, able to couple detection of infection by RNA viruses to the induction of interferons. Natural RIG-I stimulatory RNAs have variously been proposed to correspond to virus genomes, virus replication intermediates, viral transcripts, or self-RNA cleaved by RNase L. However, the relative contribution of each of these RNA species to RIG-I activation and interferon induction in virus-infected cells is not known. Here, we use three approaches to identify physiological RIG-I agonists in cells infected with influenza A virus or Sendai virus. We show that RIG-I agonists are exclusively generated by the process of virus replication and correspond to full-length virus genomes. Therefore, nongenomic viral transcripts, short replication intermediates, and cleaved self-RNA do not contribute substantially to interferon induction in cells infected with these negative strand RNA viruses. Rather, single-stranded RNA viral genomes bearing 5'-triphosphates constitute the natural RIG-I agonists that trigger cell-intrinsic innate immune responses during infection.

Model suggesting that replication of influenza virus is regulated by stabilization of replicative intermediates.

The RNA-dependent RNA polymerase of influenza A virus is responsible for both transcription and replication of negative-sense viral RNA. It is thought that a "switching" mechanism regulates the transition between these activities. We demonstrate that, in the presence of preexisting viral RNA polymerase and nucleoprotein (NP), influenza A virus synthesizes both mRNA (transcription) and cRNA (replication) early in infection. We suggest that there may be no switch regulating the initiation of RNA synthesis and present a model suggesting that nascent cRNA is degraded by host cell nucleases unless it is stabilized by newly synthesized viral RNA polymerase and NP.