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Assembly and host interactions of influenza virus polymerase in the living cell

Final Report Summary - INVIVOPOL (Assembly and host interactions of influenza virus polymerase in the living cell)

Project context

The influenza virus constitutes a global health concern because of a permanent threat of the emergence of novel highly pathogenic strains capable of causing devastating pandemics. This is illustrated by numerous human fatal cases of avian influenza since 2003 and by the 'swine flu' pandemics in 2009. Moreover, complications related to seasonal influenza in 'non-pandemic' years cause 250 000-500 000 deaths per year – see for more details. A better understanding of the fundamental cell biology of influenza virus is required to enable society to better address the problem. Multiplication of the influenza virus requires a range of host cell factors, and adaptation to them is necessary for efficient inter-species transmission, which occurs, in particular, in human cases of avian H5N1. Strains of the influenza virus resistant to existing antiviral drugs frequently emerge, justifying the need for the development of novel therapeutic agents. They should preferentially be targeted at host factors, because the virus will hardly be able to develop resistance to such drugs.

Project objectives

The influenza virus genome consists of eight segments of negative-sense RNA (vRNA), each of which is encapsidated with multiple copies of the nucleoprotein (NP) and one trimeric polymerase complex (subunits PB1, PB2 and PA) to form ribonucleoprotein particles (RNPs). Both the replication and transcription of viral genome are performed by the polymerase in the nucleus of infected cells, and the mechanisms of traffic and assembly of the RNPs and their components are poorly understood. Advanced dynamic microscopy techniques provide now unprecedented opportunities to study biologically important phenomena directly in the live cells. These techniques were chosen to address the questions on influenza virus cell biology described above.

Project results

Studies of the cells expressing subunits of the viral polymerase revealed the mechanism of its nuclear import and assembly of the active trimer. Further progress in understanding the dynamics and trafficking of RNPs and their components required studies on the live cells infected with the virus. However, live microscopy of influenza-virus-infected cells is limited by the lack of a method enabling virus visualisation in the living cells, largely due to the limited coding capacity of the influenza virus genome, which did not permit us to introduce a conventional fluorescent fusion protein. To circumvent this problem, we adapted the split-GFP system to the influenza virus, which requires only a short tag to be added to a protein of interest, while the large fragment of the fluorescent protein is supplied independently. We produced and characterised in detail a quasi-wild-type recombinant A/WSN/33 influenza virus encoding a protein which became fluorescent in the infected cells.

The designed virus was used to characterise the intra-nuclear dynamics of PB2 and to visualise the trafficking of RNPs in live infected cells. It was observed that upon nuclear export, progeny RNPs accumulate in recycling endosome vesicles thanks to direct interaction with Rab11, a multi-functional cellular protein involved in vesicle trafficking. Single-particle-tracking analysis revealed intermittent directed motions of RNP-positive particles. Depolymerisation of either microtubules or actin filaments moderately reduced occurrence of these motions, while disruption of both cytoskeleton components in combination suppressed them entirely. The studies provided live-cell-based confirmation for the model of vRNP trafficking which assumes their accumulation in recycling endosomes through the direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.

Project outcomes

The most important outcome of the project is the development of the first unimpaired fluorescently labelled influenza virus. It can be used by the scientific community to study the influenza life cycle in the context of live infected cells, and thus will facilitate better understanding of influenza cell biology. Scientists from several laboratories have already expressed their interest in collaborating and using the split-GFP tagged influenza virus.

The new data concerning trafficking of viral RNPs and polymerase subunits, and involvement of host cell factors may help to identify novel targets for development of the drugs against influenza. Our work illustrates the great potential of the GFP11-tagged influenza virus and the split-GFP labelling in general for live-cell imaging of viral infections. Moreover, split-GFP labelled viruses may be used for the detection and quantification of viral replication in non-imaging applications, such as flow cytometry and live-cell-based high-throughput screening for inhibitors of viral replication.

The project helped to develop the scientific network of both the host laboratory and the fellow. For instance, long-term collaboration was established with the Department for Virology of the Institute Pasteur (Paris) and will continue beyond the project duration; collaboration with VirPath laboratory (University Claude Bernard, Lyon) has recently started; and finally, the fellow has established contacts with light microscopy experts at EMBL (Heidelberg), Institute Albert Bonniot (Grenoble, France) and Institute Pasteur (Paris).

A second paper, written by Sergiy V. Avilov, Dorothée Moisy, Nadia Naffakh and Stephen Cusack, entitled 'Influenza A virus progeny vRNP trafficking in live infected cells studied with the virus-encoded fluorescently tagged PB2 protein' was submitted in December 2011 for publication in the journal Vaccine.