Cholesterol Alters the Orientation and Activity of the Influenza Virus M2 Amphipathic Helix in the Membrane

The influenza virus M2 amphipathic helix (M2AH) alters membrane curvature in a cholesterol-dependent manner, mediating viral membrane scission during influenza virus budding. Here, we have investigated the biophysical effects of cholesterol on the ability of an M2AH peptide to manipulate membrane properties. We see that the ability of the M2AH to interact with membranes and form an α-helix is independent of membrane cholesterol concentration; however, cholesterol affects the angle of the M2AH peptide within the membrane. This change in membrane orientation affects the ability of the M2AH to alter lipid order. In lowcholesterol membranes, the M2AH is inserted near the level of the lipid head groups, increasing lipid order, which may contribute to generation of the membrane curvature. As the cholesterol content increases, the M2AH insertion becomes flatter and slightly deeper in the membrane below the lipid headgroups, where the polar face can continue to interact with the headgroups while the hydrophobic face binds cholesterol. This changed orientation minimizes lipid packing defects and lipid order changes, likely reducing the generation of membrane curvature. Thus, cholesterol regulates M2 membrane scission by precisely modulating M2AH positioning within the membrane. This has implications for the understanding of many of amphipathic-helix-driven cellular budding processes that occur in specific lipid environments

Acquired resistance to oxaliplatin is not directly associated with increased resistance to DNA damage in SK-N-ASrOXALI4000, a newly established oxaliplatin-resistant sub-line of the neuroblastoma cell line SK-N-AS

The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin

The role of vaccinia virus K7R gene in evasion of innate immunity

This thesis reports an investigation of the role of Vaccinia virus (VACV) gene K7R in evasion of innate immunity. The K7 protein is an inhibitor of intracellular signalling pathways leading to the activation of the innate immune response and was shown to act by inhibiting TBK1/IKK-ε - mediated IRF signalling by targeting DDX3, a cellular RNA helicase (Schroder et al., 2008). To characterise K7 further and study its interaction with DDX3, K7 in complex with fragments of DDX3 was expressed in bacterial cells with the aim of obtaining co-crystals. The role of K7R in the context of VACV infection was also studied. Previous in vivo work using a recombinant virus lacking K7R had shown that K7 contributes to virulence. However, virulence was retained when a mutation of the initiating methionine codon abrogated K7 protein expression while preserving the nucleic acid sequence. It has been shown here that this is likely to be due to the presence of viral miRNA(s) encoded from the K7R sequence. Computational analysis identified putative pre-miRNAs within K7R and mutated versions of the K7R ORF which contained the predicted pre-miRNA sequences but did not make protein were still able to inhibit the IFN-β, NF-κB and ISRE-dependent intracellular signalling in vitro. Several of these mutated genes were introduced into the K7R deletion VACV and their virulence will be examined in a mouse model of infection. The presence of a miRNA also needs to be formally demonstrated using northern blot and/or qPCR analysis. Finally, there is a strong interest in using attenuated VACV strains such as Modified Vaccinia Ankara (MVA) as vaccine vectors against various pathogens. To investigate whether the removal of immunomodulators from MVA, such as the orthologue of K7R, can make the virus more immunogenic, a deletion mutant and its revertant counterpart were made and characterised in vitro

Representative fluorescence in situ hybridisation (FISH) images of chromosomes 2 (A, D and G), 12 (B, E and H) and 8 (C, F and I) in SK-N-AS (A-C), SK-N-ASrOALI4000(-) (D-F), and SK-N-ASrOXALI4000 (G-I) neuroblastoma cells.

<p>Scale bar represents 10μm.</p

Oxygen consumption by SK-N-AS and SK-N-AS^rOXALI⁴⁰⁰⁰ cells.

<p>Oxygen consumption was determined in intact cells in the absence of treatment (baseline), in response to oligomycin (8 μg/mL), an inhibitor of ATP synthase that causes a leak of protons resulting in inhibition of respiration (leak), and in response to FCCP (10 μM) that uncouples the electron transport chain resulting in maximum oxidative phosphorylation.</p

Phosphorylation status of 49 receptor tyrosine kinases.

<p>Receptor tyrosine kinase phosphorylation was determined by a commercial kit (Proteome Profiler Human Phospho-RTK Array Kit, R&D Systems, Abingdon, UK) with subsequent densitometric analysis using ImageJ software (<a href="http://imagej.nih.gov/ij/" target="_blank">http://imagej.nih.gov/ij/</a>). A) Receptor tyrosine kinase phosphorylation status expressed as fold change spot density relative to a control membrane area. Images of the membranes are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172140#pone.0172140.s001" target="_blank">S1 Fig</a>. B) Differential phosphorylation of receptor tyrosine kinases that were found phosphorylated in at least one cell line (as indicated by a fold change spot density relative to a control membrane area >2) in SK-N-AS^rOXALI⁴⁰⁰⁰ or SK-N-AS^rOXALI^4000(-) cells relative to SK-N-AS.</p

Effects of H1N1 influenza A virus infection on cell viability.

<p>Non-MYCN-amplified SK-N-AS neuroblastoma cells, SK-N-AS cells with acquired resistance to oxaliplatin (SK-N-AS^rOXALI⁴⁰⁰⁰), SK-N-AS^rOXALI⁴⁰⁰⁰ cells that were passaged for 10 passages in absence of oxaliplatin (SK-N-AS^rOXALI^4000(-)), or MYCN-amplified UKF-NB-3 neuroblastoma cells were infected with H1N1 influenza strain A/WSN/33 virus at different multiplicities of infection (MOIs) and cell viability was determined 48h post infection relative to non-treated control. The dotted line indicates the viability of non-infected control cells. * P < 0.05 relative to non-infected control cells.</p