The aim of this project is to structurally and functionally characterise the influenza virus nuclear export machinery that is responsible for the nuclear export of viral ribonucleoprotein (RNP) complexes for assembly into virions at the cell membrane. Structural and functional data about the influenza virus nuclear export protein (NEP) and its interaction with viral and cellular factors would greatly expand our limited knowledge of the mechanisms that influenza virus uses to transport its RNA genome out from the host cell nucleus.
The influenza virus genome consists of eight single stranded negative-sense RNAs that form viral ribonucleoprotein (vRNP) complexes with the viral RNA polymerase (PB1, PB2 and PA) and oligomeric nucleoprotein (NP). vRNPs display a double-helical arrangement resembling a large loop twisted into a helical filament. The RNA polymerase is responsible for replicating vRNPs in the nucleus of infected cells. The nuclear export of vRNPs is facilitated by the viral factor NEP that mediates the interaction between vRNPs and the cellular export factor Crm1/RanGTP in a viral matrix protein 1 (M1) dependent manner (see Figure). However, the structure of NEP and the molecular details of its interaction with vRNPs and Crm1/RanGTP, as well as the role or M1, remain poorly characterised at the molecular level.
Recently, we developed methods to express and purify mg quantities of NEP and obtained a preliminary structure using NMR (unpublished data). This revealed that the nuclear export signal (NES) of NEP is located on an N-terminal a-helix. Moreover, using mass spectrometry, we found a phosphorylation site in NEP (S24) that regulates its interaction with Crm1. Replacement of S24 with a phosphomimetic glutamic acid results in NEP forming stable complexes with Crm1/RanGTP. Initially, we aim to determine the structure of NEP in complex with Crm1/RanGTP using cryo-EM. However, ultimately we would like to extend these studies to complexes of vRNPs to reveal the atomic details of the interactions of the multiple viral and cellular factors involved.
Molecular and cell biology, virology, structural biology, x-ray crystallography, cryo-electron microscopy, small angle X-ray scattering, biophysical characterisation
Project reference number: 747
|Professor Jonathan M Grimes||Structural Biology||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Professor Ervin Fodor||Dunn School of Pathology||University of Oxford||GBRemail@example.com|
Influenza virus is a major human and animal pathogen causing seasonal epidemics and occasional pandemics in the human population that are associated with significant morbidity and mortality. Influenza A virus, a member of the orthomyxovirus family, contains an RNA genome with a coding capacity for a limited number of proteins. In addition to ensuring the structural integrity of virions, these viral proteins facilitate the replication of virus in the host cell. Consequently, viral proteins often evolve to perform multiple functions, the influenza A virus nuclear export protein (NEP) (also referred to as non-structural protein 2, or NS2) being an emerging example. NEP was originally implicated in mediating the nuclear export of viral ribonucleoprotein (RNP) complexes, which are synthesized in the infected cell nucleus and are assembled into progeny virions at the cell membrane. However, since then, new and unexpected roles for NEP during the influenza virus life cycle have started to emerge. These recent studies have shown NEP to be involved in regulating the accumulation of viral genomic vRNA and antigenomic cRNA as well as viral mRNA synthesized by the viral RNA-dependent RNA polymerase. Subsequently, this regulation of viral RNA transcription and replication by NEP was shown to be an important factor in the adaptation of highly pathogenic avian H5N1 influenza viruses to the mammalian host. Unexpectedly, NEP has also been implicated in recruiting a cellular ATPase to the cell membrane to aid the efficient release of budding virions. Accordingly, NEP is proposed to play multiple biologically important roles during the influenza virus life cycle. Hide abstract
Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses--to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins (PB2, PB1 and PA), glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation (PB2, M1, NP, NS1 and, through NP and NEP, of the viral RNA genome); and protein multimerisation (NS1 dimers, M2 tetramers and NP oligomers). Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets. Hide abstract