We have rescued influenza A virus by transfection of 12 plasmids into Vero cells. The eight individual negative-sense genomic viral RNAs were transcribed from plasmids containing human RNA polymerase I promoter and hepatitis delta virus ribozyme sequences. The three influenza virus polymerase proteins and the nucleoprotein were expressed from protein expression plasmids. This plasmid-based reverse genetics technique facilitates the generation of recombinant influenza viruses containing specific mutations in their genes.

Transcription by the influenza virus RNA-dependent RNA polymerase is dependent on cellular RNA processing activities that are known to be associated with cellular RNA polymerase II (Pol II) transcription, namely, capping and splicing. Therefore, it had been hypothesized that transcription by the viral RNA polymerase and Pol II might be functionally linked. Here, we demonstrate for the first time that the influenza virus RNA polymerase complex interacts with the large subunit of Pol II via its C-terminal domain. The viral polymerase binds hyperphosphorylated forms of Pol II, indicating that it targets actively transcribing Pol II. In addition, immunofluorescence analysis is consistent with a new model showing that influenza virus polymerase accumulates at Pol II transcription sites. The present findings provide a framework for further studies to elucidate the mechanistic principles of transcription by a viral RNA polymerase and have implications for the regulation of Pol II activities in infected cells.

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The influenza A virus RNA-dependent RNA polymerase is a heterotrimeric complex of polymerase basic protein 1 (PB1), PB2, and polymerase acidic protein (PA) subunits. It performs transcription and replication of the viral RNA genome in the nucleus of infected cells. We have identified a nuclear import factor, Ran binding protein 5 (RanBP5), also known as karyopherin ␤3, importin ␤3, or importin 5, as an interactor of the PB1 subunit. RanBP5 interacted with either PB1 alone or with a PB1-PA dimer but not with a PB1-PB2 dimer or the trimeric complex. The interaction between RanBP5 and PB1-PA was disrupted by RanGTP in vitro, allowing PB2 to bind to the PB1-PA dimer to form a functional trimeric RNA polymerase complex. We propose a model in which RanBP5 acts as an import factor for the newly synthesized polymerase by targeting the PB1-PA dimer to the nucleus. In agreement with this model, small interfering RNA (siRNA)-mediated knock-down of RanBP5 inhibited the nuclear accumulation of the PB1-PA dimer. Moreover, siRNA knock-down of RanBP5 resulted in the delayed accumulation of viral RNAs in infected cells, confirming that RanBP5 plays a biological role during the influenza virus life cycle.The genome of influenza A virus (family Orthomyxoviridae) consists of eight RNA segments of negative polarity. These RNA segments are bound by the viral RNA-dependent RNA polymerase (RdRp) and nucleoprotein (NP), forming viral ribonucleoprotein complexes (vRNPs). Upon infection of a cell and release of vRNPs into the cytoplasm, vRNPs are transported into the nucleus to catalyze primary viral RNA transcription (vRNA to mRNA synthesis). Subsequently, the newly synthesized viral RNA polymerase is transported from the cytoplasm into the nucleus and catalyzes secondary transcription and replication (vRNA to cRNA to vRNA synthesis). Newly formed vRNPs, in association with other viral proteins (M1 and NEP, the nuclear export protein), are exported into the cytoplasm and transported to the cell membrane, the site for viral assembly and budding (reviewed in references 16, 27, and 34).Since influenza virus transcribes and replicates its RNA genome in the nucleus of infected cells (reviewed in references 2 and 14), a number of viral proteins must enter the nucleus during the viral life cycle, namely the NP, the viral RdRp, the matrix protein M1, the NEP (formerly known as NS2), and the nonstructural protein NS1. Influenza virus therefore relies on the host cellular transport machinery for successful nuclear import and export of viral components during its life cycle.Transport into and out of the nucleus is an active, energydependent process for most proteins (reviewed in references 3, 20, and 28). Small molecules are able to diffuse freely across the nuclear pore complex (NPC), whereas larger proteins and large macromolecular complexes, such as messenger RNPs and ribosomes, require specialized transport receptors which direct them through the NPC. Nucleocytoplasmic transport is a highly selective process that involves the specific recogniti...

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A reverse genetics system for negative-strand RNA viruses was first successfully developed for influenza viruses. This technology involved the transfection of in vitro-reconstituted ribonucleoprotein (RNP) complexes into influenza virus-infected cells. We have now developed a method that allows intracellular reconstitution of RNP complexes from plasmid-based expression vectors. Expression of a viral RNA-like transcript is achieved from a plasmid containing a truncated human polymerase I (polI) promoter and a ribozyme sequence that generates the desired 3 end by autocatalytic cleavage. The polI-driven plasmid is cotransfected into human 293 cells with polII-responsive plasmids that express the viral PB1, PB2, PA, and NP proteins. This exclusively plasmid-driven system results in the efficient transcription and replication of the viral RNA-like reporter and allows the study of cis-and transacting signals involved in the transcription and replication of influenza virus RNAs. Using this system, we have also been able to rescue a synthetic neuraminidase gene into a recombinant influenza virus. This method represents a convenient alternative to the previously established RNP transfection system.

In February the World Health Organisation (WHO) recommends influenza viruses to be included in influenza vaccines for the forthcoming winter in the Northern Hemisphere. These recommendations are based on data collected by National Influenza Centres (NICs) through the WHO Global Influenza Surveillance and Response System (GISRS) and a more detailed analysis of representative and potential antigenically variant influenza viruses from the WHO Collaborating Centres for Influenza (WHO CCs) and Essential Regulatory Laboratories (ERLs). This article provides a detailed summary of the antigenic and genetic properties of viruses and additional background data used by WHO experts during development of the recommendations of the 2013-2014 Northern Hemisphere influenza vaccine composition.

Influenza viruses replicate and transcribe their segmented negative-sense single-stranded RNA genome in the nucleus of the infected host cell. All RNA synthesising activities associated with influenza virus are performed by the virally encoded RNA-dependent RNA polymerase (RdRp) that consists of three subunits, PA, PB1 and PB2. However, viral transcription is critically dependent on on-going cellular transcription, in particular, on activities associated with the cellular DNA-dependent RNA polymerase II (Pol II). Thus, the viral RdRp uses short 5' capped RNA fragments, derived from cellular Pol II transcripts, as primers for viral mRNA synthesis. These capped RNA primers are generated by cleavage of host Pol II transcripts by an endonuclease activity associated with the viral RdRp. Moreover, some viral transcripts require splicing and since influenza virus does not encode splicing machinery, it is dependent on host splicing, an activity also related to Pol II transcription. Despite these functional links between viral and host Pol II transcription, there has been no evidence that a physical association existed between the two transcriptional machineries. However, recently it was reported that there is a physical interaction between the trimeric viral RdRp and cellular Pol II. The viral RdRp was found to interact with the C-terminal domain (CTD) of initiating Pol II, at a stage in the transcription cycle when capping takes place. It was therefore proposed that this interaction may be required for the viral RNA (vRNA) polymerase to gain access to capped RNA substrates for endonucleolytic cleavage. The virus not only relies on cellular factors to support its own RNA synthesis, but also subverts cellular pathways in order to generate an environment optimised for viral multiplication. In this respect, the interaction of the viral NS1 protein with factors involved in cellular pre-mRNA processing is of particular relevance. The virus also alters the distribution of Pol II on cellular genes, leading to a reduction in elongating Pol II thereby contributing to the phenomenon known as host shut-off.