Functional domains of the influenza A virus PB2 protein:identification of NP- and PB1-binding sites

AbstractInfluenza 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 genetics of virus particle shape in equine influenza A virus

Background Many human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross-section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic. Objectives/Methods Here, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells. Results and Conclusions The majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non-filamentous strains into the non-filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses

Interaction of the influenza virus nucleoprotein with the cellular CRM1-mediated nuclear export pathway

Influenza virus transcription occurs in the nuclei of infected cells, where the viral genomic RNAs are complexed with a nucleoprotein (NP) to form ribonucleoprotein (RNP) structures. Prior to assembly into progeny virions, these RNPs exit the nucleus and accumulate in the cytoplasm. The mechanisms responsible for RNP export are only partially understood but have been proposed to involve the viral M1 and NS2 polypeptides. We found that the drug leptomycin B (LMB), which specifically inactivates the cellular CRM1 polypeptide, caused nuclear retention of NP in virus-infected cells, indicating a role for the CRM1 nuclear export pathway in RNP egress. However, no alteration was seen in the cellular distribution of M1 or NS2, even in the case of a mutant virus which synthesizes greatly reduced amounts of NS2. Furthermore, NP was distributed throughout the nuclei of infected cells at early times postinfection but, when retained in the nucleus at late times by LMB treatment, was redistributed to the periphery of the nucleoplasm. No such change was seen in the nuclear distribution of M1 or NS2 after drug treatment. Similar to the behavior of NP, M1 and NS2 in infected cells, LMB treatment of cells expressing each polypeptide in isolation caused nuclear retention of NP but not M1 or NS2. Conversely, overexpression of CRM1 caused increased cytoplasmic accumulation of NP but had little effect on M1 or NS2 distribution. Consistent with this, NP bound CRM1 in vitro. Overall, these data raise the possibility that RNP export is mediated by a direct interaction between NP and the cellular CRM1 export pathway

Evidence that the C-terminal PB2-binding region of the influenza A virus PB1 protein is a discrete alpha-helical domain

AbstractThe influenza A virus RNA-dependent RNA polymerase is a heterotrimer composed of PB1, PB2 and PA subunits and essential for viral replication. However, little detailed structural information is available for this important enzyme. We show by circular dichroism spectroscopy that polypeptides from the C-terminus of PB1 that are capable of binding efficiently to PB2 fold into stable α-helical structures. Structure prediction analysis of this region of PB1 indicates that it likely consists of a three-helical bundle. Deletion of any of the helices abrogated transcriptional function. Thus, PB1 contains a C-terminal α-helical PB2-binding domain that is essential for nucleotide polymerization activity

Development of a surveillance scheme for equine influenza in the UK and characterisation of viruses isolated in Europe, Dubai and the USA from 2010-2012

Equine influenza viruses are a major cause of respiratory disease in horses worldwide and undergo antigenic drift. Several outbreaks of equine influenza occurred worldwide during 2010-2012, including in vaccinated animals, highlighting the importance of surveillance and virus characterisation. Virus isolates were characterised from more than 20 outbreaks over a 3-year period, including strains from the UK, Dubai, Germany and the USA. The haemagglutinin-1 (HA1) sequence of all isolates was determined and compared with OIE-recommended vaccine strains. Viruses from Florida clades 1 and 2 showed continued divergence from each other compared with 2009 isolates. The antigenic inter-relationships among viruses were determined using a haemagglutination-inhibition (HI) assay with ferret antisera and visualised using antigenic cartography. All European isolates belonged to Florida clade 2, all those from the USA belonged to Florida clade 1. Two subpopulations of clade 2 viruses were isolated, with either substitution A144V or I179V. Isolates from Dubai, obtained from horses shipped from Uruguay, belonged to Florida clade 1 and were similar to viruses isolated in the USA the previous year. The neuraminidase (NA) sequence of representative strains from 2007 and 2009 to 2012 was also determined and compared with that of earlier isolates dating back to 1963. Multiple changes were observed at the amino acid level and clear distinctions could be made between viruses belonging to Florida clade 1 and clade 2

Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway

The mechanism of membrane scission during influenza A virus budding has been the subject of controversy. We confirm that influenza M1 binds VPS28, a subunit of the ESCRT-1 complex. However, confocal microscopy of infected cells showed no marked colocalisation between M1 and VPS28 or VPS4 ESCRT proteins, or relocalisation of the cellular proteins. Trafficking of HA and M1 appeared normal when endosomal sorting was impaired by expression of inactive VPS4. Overexpression of either isoform of VPS28 or wildtype or dominant negative VPS4 proteins did not alter production of filamentous virions. SiRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication. Furthermore, cells expressing wildtype or dominant-negative VPS4 replicated filamentous and non-filamentous strains of influenza to similar titres, indicating that influenza release is VPS4-independent. Overall, we see no role for the ESCRT pathway in influenza virus budding and the significance of the M1-VPS28 interaction remains to be determined. (C) 2009 Elsevier Inc. All rights reserved

Temperature-Sensitive Lesions in Two Influenza A Viruses Defective for Replicative Transcription Disrupt RNA Binding by the Nucleoprotein

The negative-sense segmented RNA genome of influenza virus is transcribed into capped and polyadenylated mRNAs, as well as full-length replicative intermediates (cRNAs). The mechanism that regulates the two forms of transcription remains unclear, although several lines of evidence imply a role for the viral nucleoprotein (NP). In particular, temperature-shift and biochemical analyses of the temperature-sensitive viruses A/WSN/33 ts56 and A/FPV/Rostock/34/Giessen tsG81 containing point mutations within the NP coding region have indicated specific defects in replicative transcription at the nonpermissive temperature. To identify the functional defect, we introduced the relevant mutations into the NP of influenza virus strain A/PR/8/34. Both mutants were temperature sensitive for influenza virus gene expression in transient-transfection experiments but localized and accumulated normally in transfected cells. Similarly, the mutants retained the ability to self-associate and interact with the virus polymerase complex whether synthesized at the permissive or the nonpermissive temperatures. In contrast, the mutant NPs were defective for RNA binding when expressed at the nonpermissive temperature but not when expressed at 30°C. This suggests that the RNA-binding activity of NP is required for replicative transcription

‘Genome gating’; polarized intranuclear trafficking of influenza virus RNPs

Many viruses exploit cellular polarity to constrain the assembly and release of progeny virions to a desired surface. Influenza virus particles are released only from the apical surface of epithelial cells and this polarization is partly owing to specific targeting of the viral membrane proteins to the apical plasma membrane. The RNA genome of the virus is transcribed and replicated in the nucleus, necessitating nuclear export of the individual ribonucleoprotein (RNP) segments before they can be incorporated into budding virus particles. We show that the process of polarized virus assembly begins in the nucleus with the RNPs adopting a novel asymmetric distribution at the inner nuclear membrane prior to their export to the cytoplasm. The viral nucleoprotein, the major protein component of RNPs, displays the same polarized intranuclear distribution in the absence of other influenza virus components, suggesting the existence of a hitherto unrecognized polarity within the mammalian cell nucleus

Identification of Amino Acid Residues of Influenza Virus Nucleoprotein Essential for RNA Binding

The influenza virus nucleoprotein (NP) is a single-strand-RNA-binding protein associated with genome and antigenome RNA and is one of the four virus proteins necessary for transcription and replication of viral RNA. To better characterize the mechanism by which NP binds RNA, we undertook a physical and mutational analysis of the polypeptide, with the strategy of identifying first the regions in direct contact with RNA, then the classes of amino acids involved, and finally the crucial residues by mutagenesis. Chemical fragmentation and amino acid sequencing of NP that had been UV cross linked to radiolabelled RNA showed that protein-RNA contacts occur throughout the length of the polypeptide. Chemical modification experiments implicated arginine but not lysine residues as important for RNA binding, while RNA-dependent changes in the intrinsic fluorescence spectrum of NP suggested the involvement of tryptophan residues. Supporting these observations, single-codon mutagenesis identified five tryptophan, one phenylalanine, and two arginine residues as essential for high-affinity RNA binding at physiological temperature. In addition, mutants unable to bind RNA in vitro were unable to support virus gene expression in vivo. The mutationally sensitive residues are not localized to any particular region of NP but instead are distributed throughout the protein. Overall, these data are inconsistent with previous models suggesting that the NP-RNA interaction is mediated by a discrete N-terminal domain. Instead, we propose that high-affinity binding of RNA by NP requires the concerted interaction of multiple regions of the protein with RNA and that the individual protein-RNA contacts are mediated by a combination of electrostatic interactions between positively charged residues and the phosphate backbone and planar interactions between aromatic side chains and bases