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.

Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus.

Negative-strand RNA viruses represent a significant class of important pathogens that cause substantial morbidity and mortality in human and animal hosts worldwide. A defining feature of these viruses is that their single-stranded RNA genomes are of opposite polarity to messenger RNA and are replicated through a positive-sense intermediate. The replicative intermediate is thought to exist as a complementary ribonucleoprotein (cRNP) complex. However, isolation of such complexes from infected cells has never been accomplished. Here we report the development of an RNA-based affinity-purification strategy for the isolation of cRNPs of influenza A virus from infected cells. This technological advance enabled the structural and functional characterization of this elusive but essential component of the viral RNA replication machine. The cRNP exhibits a filamentous double-helical organization with defined termini, containing the viral RNA-dependent RNA polymerase (RdRp) at one end and a loop structure at the other end. In vitro characterization of cRNP activity yielded mechanistic insights into the workings of this RNA synthesis machine. In particular, we found that cRNPs show activity in vitro only in the presence of added RdRp. Intriguingly, a replication-inactive RdRp mutant was also able to activate cRNP-templated viral RNA synthesis. We propose a model of influenza virus genome replication that relies on the trans-activation of the cRNP-associated RdRp. The described purification strategy should be applicable to other negative-strand RNA viruses and will promote studies into their replication mechanisms.

Host restriction of influenza virus polymerase activity by PB2 627E is diminished on short viral templates in a nucleoprotein-independent manner.

Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.

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.

The PB2 subunit of the influenza virus RNA polymerase affects virulence by interacting with the mitochondrial antiviral signaling protein and inhibiting expression of beta interferon.

The PB2 subunit of the influenza virus RNA polymerase is a major virulence determinant of influenza viruses. However, the molecular mechanisms involved remain unknown. It was previously shown that the PB2 protein, in addition to its nuclear localization, also accumulates in the mitochondria. Here, we demonstrate that the PB2 protein interacts with the mitochondrial antiviral signaling protein, MAVS (also known as IPS-1, VISA, or Cardif), and inhibits MAVS-mediated beta interferon (IFN-beta) expression. In addition, we show that PB2 proteins of influenza viruses differ in their abilities to associate with the mitochondria. In particular, the PB2 proteins of seasonal human influenza viruses localize to the mitochondria while PB2 proteins of avian influenza viruses are nonmitochondrial. This difference in localization is caused by a single amino acid polymorphism in the PB2 mitochondrial targeting signal. In order to address the functional significance of the mitochondrial localization of the PB2 protein in vivo, we have generated two recombinant human influenza viruses encoding either mitochondrial or nonmitochondrial PB2 proteins. We found that the difference in the mitochondrial localization of the PB2 proteins does not affect the growth of these viruses in cell culture. However, the virus encoding the nonmitochondrial PB2 protein induces higher levels of IFN-beta and, in an animal model, is attenuated compared to the isogenic virus encoding a mitochondrial PB2. Overall this study implicates the PB2 protein in the regulation of host antiviral innate immune pathways and suggests an important role for the mitochondrial association of the PB2 protein in determining virulence.

Splicing of influenza A virus NS1 mRNA is independent of the viral NS1 protein.

RNA segment 8 (NS) of influenza A virus encodes two proteins. The NS1 protein is translated from the unspliced primary mRNA transcript, whereas the second protein encoded by this segment, NS2/NEP, is translated from a spliced mRNA. Splicing of influenza NS1 mRNA is thought to be regulated so that the levels of NS2 spliced transcripts are approximately 10 % of total NS mRNA. Regulation of splicing of the NS1 mRNA has been studied at length, and a number of often-contradictory control mechanisms have been proposed. In this study, we used (32)P-labelled gene-specific primers to investigate influenza A NS1 mRNA splicing regulation. It was found that the efficiency of splicing of NS1 mRNA was maintained at similar levels in both virus infection and ribonucleoprotein-reconstitution assays, and NS2 mRNA comprised approximately 15 % of total NS mRNA in both assays. The effect of NS1 protein expression on the accumulation of viral NS2 mRNA and spliced cellular beta-globin mRNA was analysed, and it was found that NS1 protein expression reduced spliced beta-globin mRNA levels, but had no effect on the accumulation of NS2 mRNA. We conclude that the NS1 protein specifically inhibits the accumulation of cellular RNA polymerase II-driven mRNAs, but does not affect the splicing of its own viral NS1 mRNA.

Role of initiating nucleoside triphosphate concentrations in the regulation of influenza virus replication and transcription.

The mechanisms regulating the synthesis of mRNA, cRNA, and viral genomic RNA (vRNA) by the influenza A virus RNA-dependent RNA polymerase are not fully understood. Previous studies in our laboratory have shown that virion-derived viral ribonucleoprotein complexes synthesize both mRNA and cRNA in vitro and early in the infection cycle in vivo. Our continued studies showed that de novo synthesis of cRNA in vitro is more sensitive to the concentrations of ATP, CTP, and GTP than capped-primer-dependent synthesis of mRNA. Using rescued recombinant influenza A/WSN/33 viruses, we now demonstrate that the 3'-terminal sequence of the vRNA promoter dictates the requirement for a high nucleoside triphosphate (NTP) concentration during de novo-initiated replication to cRNA, whereas this is not the case for the extension of capped primers during transcription to mRNA. In contrast to some other viral polymerases, for which only the initiating NTP is required at high concentrations, influenza virus polymerase requires high concentrations of the first three NTPs. In addition, we show that base pair mutations in the vRNA promoter can lead to nontemplated dead-end mutations during replication to cRNA in vivo. Based on our observations, we propose a new model for the de novo initiation of influenza virus replication.

The role and assembly mechanism of nucleoprotein in influenza A virus ribonucleoprotein complexes.

The nucleoprotein of negative-strand RNA viruses forms a major component of the ribonucleoprotein complex that is responsible for viral transcription and replication. However, the precise role of nucleoprotein in viral RNA transcription and replication is not clear. Here we show that nucleoprotein of influenza A virus is entirely dispensable for replication and transcription of short viral RNA-like templates in vivo, suggesting that nucleoprotein represents an elongation factor for the viral RNA polymerase. We also find that the recruitment of nucleoprotein to nascent ribonucleoprotein complexes during replication of full-length viral genes is mediated through nucleoprotein-nucleoprotein homo-oligomerization in a 'tail loop-first' orientation and is independent of RNA binding. This work demonstrates that nucleoprotein does not regulate the initiation and termination of transcription and replication by the viral polymerase in vivo, and provides new mechanistic insights into the assembly and regulation of viral ribonucleoprotein complexes.

The role of the influenza virus RNA polymerase in host shut-off.

Viruses induce an antiviral host response by activating the expression of antiviral host genes. However, viruses have evolved a wide range of strategies to counteract antiviral host responses. One of the strategies used by many viruses is the general inhibition of host gene expression, also referred to as a host shut-off mechanism. Here we discuss our recent findings that influenza virus infection results in the inhibition and degradation of host RNA polymerase II (Pol II) and that the viral RNA polymerase plays a critical role in this process. In particular, we found that Pol II is ubiquitylated in influenza virus infected cells and ubiquitylation can be induced by the expression of the RNA polymerase. Moreover, the expression of an antiviral host gene could be inhibited by the over-expression of the RNA polymerase. Both ubiquitylation and the inhibition of the host gene were dependent on the ability of the RNA polymerase to bind to Pol II. Further studies will be required to understand the interplay between the host and viral transcriptional machineries and to elucidate the exact molecular mechanisms that lead to the inhibition and degradation of Pol II as a result of viral RNA polymerase binding. These findings extend our understanding of how influenza virus counteracts antiviral host responses and underpin studies into the mechanisms by which the RNA polymerase determines virulence.

Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells.

Influenza viruses induce a host shut off mechanism leading to the general inhibition of host gene expression in infected cells. Here, we report that the large subunit of host RNA polymerase II (Pol II) is degraded in infected cells and propose that this degradation is mediated by the viral RNA polymerase that associates with Pol II. We detect increased ubiquitylation of Pol II in infected cells and upon the expression of the viral RNA polymerase suggesting that the proteasome pathway plays a role in Pol II degradation. Furthermore, we find that expression of the viral RNA polymerase results in the inhibition of Pol II transcription. We propose that Pol II inhibition and degradation in influenza virus infected cells could represent a viral strategy to evade host antiviral defense mechanisms. Our results also suggest a mechanism for the temporal regulation of viral mRNA synthesis.

NS2/NEP protein regulates transcription and replication of the influenza virus RNA genome.

The influenza virus RNA polymerase transcribes the negative-sense viral RNA segments (vRNA) into mRNA and replicates them via complementary RNA (cRNA) intermediates into more copies of vRNA. It is not clear how the relative amounts of the three RNA products, mRNA, cRNA and vRNA, are regulated during the viral life cycle. We found that in viral ribonucleoprotein (vRNP) reconstitution assays involving only the minimal components required for viral transcription and replication (the RNA polymerase, the nucleoprotein and a vRNA template), the relative levels of accumulation of RNA products differed from those observed in infected cells, suggesting a regulatory role for additional viral proteins. Expression of the viral NS2/NEP protein in RNP reconstitution assays affected viral RNA levels by reducing the accumulation of transcription products and increasing the accumulation of replication products to more closely resemble those found during viral infection. This effect was functionally conserved in influenza A and B viruses and was influenza-virus-type-specific, demonstrating that the NS2/NEP protein changes RNA levels by specific alteration of the viral transcription and replication machinery, rather than through an indirect effect on the host cell. Although NS2/NEP has been shown previously to play a role in the nucleocytoplasmic export of viral RNPs, deletion of the nuclear export sequence region that is required for its transport function did not affect the ability of the protein to regulate RNA levels. A role for the NS2/NEP protein in the regulation of influenza virus transcription and replication that is independent of its viral RNP export function is proposed.

Different de novo initiation strategies are used by influenza virus RNA polymerase on its cRNA and viral RNA promoters during viral RNA replication.

Various mechanisms are used by single-stranded RNA viruses to initiate and control their replication via the synthesis of replicative intermediates. In general, the same virus-encoded polymerase is responsible for both genome and antigenome strand synthesis from two different, although related promoters. Here we aimed to elucidate the mechanism of initiation of replication by influenza virus RNA polymerase and establish whether initiation of cRNA and viral RNA (vRNA) differed. To do this, two in vitro replication assays, which generated transcripts that had 5' triphosphate end groups characteristic of authentic replication products, were developed. Surprisingly, mutagenesis screening suggested that the polymerase initiated pppApG synthesis internally on the model cRNA promoter, whereas it initiated pppApG synthesis terminally on the model vRNA promoter. The internally synthesized pppApG could subsequently be used as a primer to realign, by base pairing, to the terminal residues of both the model cRNA and vRNA promoters. In vivo evidence, based on the correction of a mutated or deleted residue 1 of a cRNA chloramphenicol acetyltransferase reporter construct, supported this internal initiation and realignment model. Thus, influenza virus RNA polymerase uses different initiation strategies on its cRNA and vRNA promoters. To our knowledge, this is novel and has not previously been described for any viral RNA-dependent RNA polymerase. Such a mechanism may have evolved to maintain genome integrity and to control the level of replicative intermediates in infected cells.

Influenza virus inhibits RNA polymerase II elongation.

The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus, influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding to the CTD of initiating Pol II and subsequent cleavage of the capped 5' end of the nascent transcript, triggers premature Pol II termination.

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.