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

A functional link between the actin cytoskeleton and lipid rafts during budding of filamentous influenza virions

AbstractMorphogenesis of influenza virus is a poorly understood process that produces two types of enveloped virion: ∼100-nm spheres and similar diameter filaments that reach 20 μm 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 β-actin clusters to form novel annular membrane structures. HA in filamentous virions and jasplakinolide-induced annuli was detergent insoluble at 4°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

Potential of a sequence-based antigenic distance measure to indicate equine influenza vaccine strain efficacy

The calculation of pepitope values, a sequence-based measure of antigenic distance between strains, was developed for human influenza. The potential to apply the pepitope value to equine influenza vaccine strain selection was assessed. There was a negative correlation between pepitope value and vaccine efficacy for pairs of vaccine and challenge strains used in cross-protection studies in ponies that just reached statistical significance (p = 0.046) only if one pair of viruses was excluded from the analysis. Thus the pepitope value has potential to provide additional data to consider in the decision-making process for updating equine influenza vaccine strains. However, further work is required to define the epitopes of the equine H3N8 haemagglutinin protein recognised by equine antibodies, which could lead to refinement of the pepitope value calculation. Furthermore, other factors such as vaccine potency and virulence of circulating strains may also influence vaccine efficacy

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

Comparison of two modern vaccines and previous influenza infection against challenge with an equine influenza virus from the Australian 2007 outbreak

During 2007, large outbreaks of equine influenza (EI) caused by Florida sublineage Clade 1 viruses affected horse populations in Japan and Australia. The likely protection that would be provided by two modern vaccines commercially available in the European Union (an ISCOM-based and a canarypox-based vaccine) at the time of the outbreaks was determined. Vaccinated ponies were challenged with a representative outbreak isolate (A/eq/Sydney/2888-8/07) and levels of protection were compared. A group of ponies infected 18 months previously with a phylogenetically-related isolate from 2003 (A/eq/South Africa/4/03) was also challenged with the 2007 outbreak virus. After experimental infection with A/eq/Sydney/2888-8/07, unvaccinated control ponies all showed clinical signs of infection together with virus shedding. Protection achieved by both vaccination or long-term immunity induced by previous exposure to equine influenza virus (EIV) was characterised by minor signs of disease and reduced virus shedding when compared with unvaccinated control ponies. The three different methods of virus titration in embryonated hens’ eggs, EIV NP-ELISA and quantitative RT-PCR were used to monitor EIV shedding and results were compared. Though the majority of previously infected ponies had low antibody levels at the time of challenge, they demonstrated good clinical protection and limited virus shedding. In summary, we demonstrate that vaccination with current EIV vaccines would partially protect against infection with A/eq/Sydney/2888-8/07-like strains and would help to limit the spread of disease in our vaccinated horse population

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

Identification of the domains of the influenza A virus M1 matrix protein required for NP binding, oligomerization and incorporation into virions

The matrix (M1) protein of influenza A virus is a multifunctional protein that plays essential structural and functional roles in the virus life cycle. It drives virus budding and is the major protein component of the virion, where it forms an intermediate layer between the viral envelope and integral membrane proteins and the genomic ribonucleoproteins (RNPs). It also helps to control the intracellular trafficking of RNPs. These roles are mediated primarily via protein–protein interactions with viral and possibly cellular proteins. Here, the regions of M1 involved in binding the viral RNPs and in mediating homo-oligomerization are identified. In vitro, by using recombinant proteins, it was found that the middle domain of M1 was responsible for binding NP and that this interaction did not require RNA. Similarly, only M1 polypeptides containing the middle domain were able to bind to RNP–M1 complexes isolated from purified virus. When M1 self-association was examined, all three domains of the protein participated in homo-oligomerization although, again, the middle domain was dominant and self-associated efficiently in the absence of the N- and C-terminal domains. However, when the individual fragments of M1 were tagged with green fluorescent protein and expressed in virus-infected cells, microscopy of filamentous particles showed that only full-length M1 was incorporated into budding virions. It is concluded that the middle domain of M1 is primarily responsible for binding NP and self-association, but that additional interactions are required for efficient incorporation of M1 into virus particles

Codon conservation in the influenza A virus genome defines RNA packaging signals

Genome segmentation facilitates reassortment and rapid evolution of influenza A virus. However, segmentation complicates particle assembly as virions must contain all eight vRNA species to be infectious. Specific packaging signals exist that extend into the coding regions of most if not all segments, but these RNA motifs are poorly defined. We measured codon variability in a large dataset of sequences to identify areas of low nucleotide sequence variation independent of amino acid conservation in each segment. Most clusters of codons showing very little synonymous variation were located at segment termini, consistent with previous experimental data mapping packaging signals. Certain internal regions of conservation, most notably in the PA gene, may however signify previously unidentified functions in the virus genome. To experimentally test the bioinformatics analysis, we introduced synonymous mutations into conserved codons within known packaging signals and measured incorporation of the mutant segment into virus particles. Surprisingly, in most cases, single nucleotide changes dramatically reduced segment packaging. Thus our analysis identifies cis-acting sequences in the influenza virus genome at the nucleotide level. Furthermore, we propose that strain-specific differences exist in certain packaging signals, most notably the haemagglutinin gene; this finding has major implications for the evolution of pandemic viruses

Addressing the growing fossil record of subadult hominins by reaching across disciplines

The field of paleoanthropology lacks a coherent methodology to study ontogeny in extinct hominins. During the past two decades in this field, several factors have served as an impetus to better define this subfield of study within human evolution. First is the increased recovery of immature hominin remains that span multiple genera—Australopithecus, Paranthropus, and Homo