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Influenza virus genome replication requires the virus-encoded nucleoprotein (NP), partly because it is necessary to encapsidate the viral genomic RNA (vRNA) and antigenomic cRNA segments into ribonucleoproteins (RNPs). However, there is also evidence that NP actively regulates viral RNA synthesis and there is a long-standing hypothesis that increased concentrations of NP in the cell are responsible for a switch from genome transcription to replication. Here, this hypothesis is tested in a recombinant setting and in the context of virus infection. In a plasmid-based system for reconstituting active viral RNPs in cells, titration of increasing amounts of NP did not promote higher levels of genome replication relative to transcription, but in fact caused the opposite effect. An approximately fourfold reduction in the ratio of genomic and antigenomic RNAs to mRNA was seen across an 80-fold range of NP plasmid concentrations. When cells were transfected with the same amounts of NP plasmid to establish a concentration gradient of NP prior to virus superinfection, no change in the ratio of cRNA to mRNA was seen for segments 5 and 7, or for the ratio of segment 5 vRNA to mRNA. A slight reduction in the ratio of segment 7 vRNA to mRNA was seen. These findings do not support the simple hypothesis that increased intracellular concentrations of NP promote influenza virus genome replication.

Influenza virus genomic RNA segments are packaged into ribonucleoprotein (RNP) structures by the PB1, PB2, and PA subunits of an RNA polymerase and a single-strand RNA-binding nucleoprotein (NP). Assembly and function of these ribonucleoproteins depend on a complex set of protein-protein and protein-RNA interactions. Here, we identify new functional domains of PB2. We show that PB2 contains two regions that bind NP and also identify a novel PB1 binding site. The regions of PB2 responsible for binding NP and PB1 show considerable overlap, and binding of NP to the PB2 fragments could be outcompeted by PB1. The binding domains of PB2 acted as trans-dominant inhibitors of viral gene expression, and consistent with the in vitro binding data, their inhibitory activity depended on the concentration of wild-type PB2, NP, and PB1. This provides evidence for functionally significant and potentially regulatory interactions between PB2 and NP.

The current model for influenza virus mRNA transcription involves the sequential interaction of the viral polymerase with the 5'- and 3'-ends of vRNA, with each RNA-protein interaction triggering a polymerase function necessary for cap-primed transcription. Here we show that the order in which this ternary complex is assembled is in fact important. Polymerase bound simultaneously to a pre-annealed duplex of the 5'- and 3'-ends of vRNA had greatly increased levels of primer binding and endonuclease activities compared to a sequentially assembled complex. Increased primer binding was due to the activation of a high affinity binding site with a preference for primer length RNAs. This correlated with enhanced levels of cap-primed transcription. Polymerase that was bound initially to just 5' vRNA had low primer binding activity, but was endonucleolytically active. Neither activity was significantly increased by the subsequent addition of 3' vRNA, and this sequentially assembled complex had correspondingly low mRNA transcription activity. Nevertheless, both routes of assembly led to complexes that were highly competent for dinucleotide ApG-primed transcription. Therefore, polymerase complexes assembled on pre-annealed 5' and 3' terminal viral RNA sequences have distinct properties from those assembled by sequential loading of polymerase onto the 5'-end followed by the 3'-end. This suggests a mechanism by which the virus couples transcription initiation and termination during mRNA transcription.

Classical swine fever virus (CSFV) is a member of the pestivirus family, which shares many features in common with hepatitis C virus (HCV). It is shown here that CSFV has an exceptionally efficient cis-acting internal ribosome entry segment (IRES), which, like that of HCV, is strongly influenced by the sequences immediately downstream of the initiation codon, and is optimal with viral coding sequences in this position. Constructs that retained 17 or more codons of viral coding sequence exhibited full IRES activity, but with only 12 codons, activity was approximately 66% of maximum in vitro (though close to maximum in transfected BHK cells), whereas with just 3 codons or fewer, the activity was only approximately 15% of maximum. The minimal coding region elements required for high activity were exchanged between HCV and CSFV. Although maximum activity was observed in each case with the homologous combination of coding region and 5' UTR, the heterologous combinations were sufficiently active to rule out a highly specific functional interplay between the 5' UTR and coding sequences. On the other hand, inversion of the coding sequences resulted in low IRES activity, particularly with the HCV coding sequences. RNA structure probing showed that the efficiency of internal initiation of these chimeric constructs correlated most closely with the degree of single-strandedness of the region around and immediately downstream of the initiation codon. The low activity IRESs could not be rescued by addition of supplementary eIF4A (the initiation factor with ATP-dependent RNA helicase activity). The extreme sensitivity to secondary structure around the initiation codon is likely to be due to the fact that the eIF4F complex (which has eIF4A as one of its subunits) is not required for and does not participate in initiation on these IRESs.

Morphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: approximately 100-nm spheres and similar diameter filaments that reach 20 microm in length. Spherical particles assemble at plasma membrane lipid rafts in a process independent of microfilaments. The budding site of filamentous virions is hitherto uncharacterised but their formation involves the actin cytoskeleton. We confirm microfilament involvement in filamentous budding and show that after disruption of cortical actin by jasplakinolide, HA, NP, and M1 redistributed around beta-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4 degrees C. Furthermore, in both cases HA partitioned into low buoyant density detergent-insoluble glycolipid domains, indicating that filamentous virions and annuli contain reorganised lipid rafts. We propose that the actin cytoskeleton is required to maintain the correct organisation of lipid rafts for incorporation into budding viral filaments.

All viruses with negative-sense RNA genomes encode a single-strand RNA-binding nucleoprotein (NP). The primary function of NP is to encapsidate the virus genome for the purposes of RNA transcription, replication and packaging. The purpose of this review is to illustrate using the influenza virus NP as a well-studied example that the molecule is much more than a structural RNA-binding protein, but also functions as a key adapter molecule between virus and host cell processes. It does so through the ability to interact with a wide variety of viral and cellular macromolecules, including RNA, itself, two subunits of the viral RNA-dependent RNA polymerase and the viral matrix protein. NP also interacts with cellular polypeptides, including actin, components of the nuclear import and export apparatus and a nuclear RNA helicase. The evidence for the existence of each of these activities and their possible roles in transcription, replication and intracellular trafficking of the virus genome is considered.

The first 11 nt at the 5' end of influenza virus genomic RNA were shown to be both necessary and sufficient for specific binding by the influenza virus polymerase. A novel in vitro transcription assay, in which the polymerase was bound to paramagnetic beads via a biotinylated 5'-vRNA oligonucleotide, was used to study the activities of different forms of the polymerase. Complexes composed of co-expressed PB1/PB2/PA proteins and a sub-complex composed of PB1/PA bound to the 5'-vRNA oligonucleotide, whereas PB1 expressed alone did not. The enriched 5'-vRNA/PB1/PB2/PA complex was highly active for ApG and globin mRNA primed transcription on a model 3'-vRNA template. RNA synthesis in the absence of added primers produced products with 5'-terminal tri- or diphosphate groups, indicating that genuine unprimed initiation of transcription also occurred. No transcriptase activity was detected for the PB1/PA complex. These results demonstrate a role for PA in the enhancement of 5' end binding activity of PB1, a role for PB2 in the assembly of a polymerase complex able to perform both cap-dependent and -independent synthesis and that NP is not required for the initiation of replicative transcription.

All three influenza virus polymerase (P) proteins were expressed in Xenopus oocytes from microinjected in vitro transcribed mRNA analogs, with yields of up to 100 ng per oocyte. To examine the functional state of the Xenopus-expressed P proteins, the polypeptides were tested for their ability to form stable complexes with each other. As seen in virus-infected cells, all three P proteins associated into an immunoprecipitable complex, suggesting that the system has considerable promise for the reconstruction of an active influenza RNA polymerase. Examination of the ability of paired combinations of the P proteins to associate indicated that PB1 contained independent binding sites for PB2 and PA, and so probably formed the backbone of the complex. Sedimentation analysis of free and complexed P proteins indicated that PB1 and PB2 did not exist as free monomers, and that similarly, complexes of all three P proteins did not simply consist of one copy of each protein. The heterodisperse sedimentation rate seen for complexes of all three P proteins did not appear to result from their binding to RNA, suggesting the incorporation of additional polypeptides in the polymerase complex.