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

The significance of seniority for women managers’ interpretations of organizational restructuring

This paper examines the impact of restructuring within the transport and logistics sector on women managers working at senior and less senior (middle/junior management) levels of the organization. The majority of women experienced increased performance pressures and heavier workloads as well as an increase in working hours. At the same time, there were pressures to work at home (i.e. week-ends and evenings) and reduced opportunities to work from home (i.e. during normal office hours). Management level emerged as an important factor in how these changes were interpreted. Senior managers perceived more positive outcomes in terms of increased motivation and loyalty. Despite a longer working week, they were less likely to report low morale as an outcome from long hours. In fact, irrespective of management level, women working shorter hours were more likely to report low morale as an outcome. Results are discussed in relation to literature on restructuring and careers, in terms of perceptual framing and in relation to different levels of investment in the organization

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

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

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Host Cell Nucleolin Is Required To Maintain the Architecture of Human Cytomegalovirus Replication Compartments

Drastic reorganization of the nucleus is a hallmark of herpesvirus replication. This reorganization includes the formation of viral replication compartments, the subnuclear structures in which the viral DNA genome is replicated. The architecture of replication compartments is poorly understood. However, recent work with human cytomegalovirus (HCMV) showed that the viral DNA polymerase subunit UL44 concentrates and viral DNA synthesis occurs at the periphery of these compartments. Any cellular factors involved in replication compartment architecture are largely unknown. Previously, we found that nucleolin, a major protein component of nucleoli, associates with HCMV UL44 in infected cells and is required for efficient viral DNA synthesis. Here, we show that nucleolin binds to purified UL44. Confocal immunofluorescence analysis demonstrated colocalization of nucleolin with UL44 at the periphery of replication compartments. Pharmacological inhibition of viral DNA synthesis prevented the formation of replication compartments but did not abrogate association of UL44 and nucleolin. Thus, association of UL44 and nucleolin is unlikely to be a nonspecific effect related to development of replication compartments. No detectable colocalization of 5-ethynyl-2′-deoxyuridine (EdU)-labeled viral DNA with nucleolin was observed, suggesting that nucleolin is not directly involved in viral DNA synthesis. Small interfering RNA (siRNA)-mediated knockdown of nucleolin caused improper localization of UL44 and a defect in EdU incorporation into viral DNA. We propose a model in which nucleolin anchors UL44 at the periphery of replication compartments to maintain their architecture and promote viral DNA synthesis

Identification of morphological differences between avian influenza A viruses grown in chicken and duck cells

Although wild ducks are considered to be the major reservoirs for most influenza A virus subtypes, they are typically resistant to the effects of the infection. In contrast, certain influenza viruses may be highly pathogenic in other avian hosts such as chickens and turkeys, causing severe illness and death. Following in vitro infection of chicken and duck embryo fibroblasts (CEF and DEF) with low pathogenic avian influenza (LPAI) viruses, duck cells die more rapidly and produce fewer infectious virions than chicken cells. In the current study, the morphology of viruses produced from CEF and DEF cells infected with low pathogenic avian H2N3 was examined. Transmission electron microscopy showed that viruses budding from duck cells were elongated, while chicken cells produced mostly spherical virions; similar differences were observed in viral supernatants. Sequencing of the influenza genome of chicken- and duck-derived H2N3 LPAI revealed no differences, implicating host cell determinants as responsible for differences in virus morphology. Both DEF and CEF cells produced filamentous virions of equine H3N8 (where virus morphology is determined by the matrix gene). DEF cells produced filamentous or short filament virions of equine H3N8 and avian H2N3, respectively, even after actin disruption with cytochalasin D. These findings suggest that cellular factors other than actin are responsible for the formation of filamentous virions in DEF cells. The formation of elongated virions in duck cells may account for the reduced number of infectious virions produced and could have implications for virus transmission or maintenance in the reservoir host

SWI/SNF complexes are required for full activation of the DNA-damage response

SWI/SNF complexes utilize BRG1 (also known as SMARCA4) or BRM (also known as SMARCA2) as alternative catalytic subunits with ATPase activity to remodel chromatin. These chromatin-remodeling complexes are required for mammalian development and are mutated in ~20% of all human primary tumors. Yet our knowledge of their tumor-suppressor mechanism is limited. To investigate the role of SWI/SNF complexes in the DNA-damage response (DDR), we used shRNAs to deplete BRG1 and BRM and then exposed these cells to a panel of 6 genotoxic agents. Compared to controls, the shRNA knockdown cells were hypersensitive to certain genotoxic agents that cause double-strand breaks (DSBs) associated with stalled/collapsed replication forks but not to ionizing radiation-induced DSBs that arise independently of DNA replication. These findings were supported by our analysis of DDR kinases, which demonstrated a more prominent role for SWI/SNF in the activation of the ATR-Chk1 pathway than the ATM-Chk2 pathway. Surprisingly, γH2AX induction was attenuated in shRNA knockdown cells exposed to a topoisomerase II inhibitor (etoposide) but not to other genotoxic agents including IR. However, this finding is compatible with recent studies linking SWI/SNF with TOP2A and TOP2BP1. Depletion of BRG1 and BRM did not result in genomic instability in a tumor-derived cell line but did result in nucleoplasmic bridges in normal human fibroblasts. Taken together, these results suggest that SWI/SNF tumor-suppressor activity involves a role in the DDR to attenuate replicative stress and genomic instability. These results may also help to inform the selection of chemotherapeutics for tumors deficient for SWI/SNF function

Nucleozin Targets Cytoplasmic Trafficking of Viral Ribonucleoprotein-Rab11 Complexes in Influenza A Virus Infection

Novel antivirals are needed to supplement existing control strategies for influenza A virus (IAV). A promising new class of drug, exemplified by the compound nucleozin, has recently been identified that targets the viral nucleoprotein (NP). These inhibitors are thought to act as "molecular staples" that stabilize interactions between NP monomers, promoting the formation of nonfunctional aggregates. Here we detail the inhibitory mechanism of nucleozin, finding that the drug has both early- and late-acting effects on the IAV life cycle. When present at the start of infection, it inhibited viral RNA and protein synthesis. However, when added at later time points, it still potently blocked the production of infectious progeny but without affecting viral macromolecular synthesis. Instead, nucleozin blocked the cytoplasmic trafficking of ribonucleoproteins (RNPs) that had undergone nuclear export, promoting the formation of large perinuclear aggregates of RNPs along with cellular Rab11. This effect led to the production of much reduced amounts of often markedly smaller virus particles. We conclude that the primary target of nucleozin is the viral RNP, not NP, and this work also provides proof of the principle that IAV replication can be effectively inhibited by blocking cytoplasmic trafficking of the viral genome.MRC grant: (G0700815), University of Cambridge/Trinity College grant: (Newton Trust), RGC Hong Kong grant: (GRF 768010 M)