A New Inhibitor of Apoptosis from Vaccinia Virus and Eukaryotes

A new apoptosis inhibitor is described from vaccinia virus, camelpox virus, and eukaryotic cells. The inhibitor is a hydrophobic, multiple transmembrane protein that is resident in the Golgi and is named GAAP (Golgi anti-apoptotic protein). Stable expression of both viral GAAP (v-GAAP) and human GAAP (h-GAAP), which is expressed in all human tissues tested, inhibited apoptosis induced by intrinsic and extrinsic apoptotic stimuli. Conversely, knockout of h-GAAP by siRNA induced cell death by apoptosis. v-GAAP and h-GAAP display overlapping functions as shown by the ability of v-GAAP to complement for the loss of h-GAAP. Lastly, deletion of the v-GAAP gene from vaccinia virus did not affect virus replication in cell culture, but affected virus virulence in a murine infection model. This study identifies a new regulator of cell death that is highly conserved in evolution from plants to insects, amphibians, mammals, and poxviruses

Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence.

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection

Functional analysis of viral schlafen from camelpox virus

This thesis concerns gene 176R from camelpox virus (CMLV) that encodes a protein known as viral schlafen (v-slfn). v-slfn has an N-terminal domain related to the p26 protein from baculovirus and a C-terminal domain related to mammalian schlafen proteins. A full length v-slfn is expressed by all sequenced orthopoxviruses except vaccinia virus (VACV) and variola virus. The baculovirus p26 proteins are poorly characterised, with no known function. In contrast, murine schlafen (m-slfn) proteins are upregulated in response to infection and the promoter for m-slfn2 has NF-κB and AP-1 binding sites. The prototypic slfn, m-slfn1, halts cellular proliferation by inhibition of cyclin D1 expression in vitro and both m-slfn1 and m-slfn8 reduce thymocyte proliferation in vivo. v-slfn is a predominantly cytoplasmic protein of 57 kDa that is expressed both early and late during CMLV infection. Expression of v-slfn reverses the growth arrest resulting from m-slfn1 expression, and this is a result of a reversal of the inhibition of cyclin D1 expression. This effect can be seen following overexpression of various transcription factors that upregulate cyclin D1 expression. Recombinant VACV expressing enhanced levels of v-slfn replicated and spread at a comparable rate to control viruses in vitro, but was less virulent than controls in the intranasal model of infection in vivo. A group of viruses based on VACV WR were constructed, which lack the gene fragments (B2R and B3R) corresponding to CMLV 176R. The undisrupted sequence for 176R was also re-inserted at this locus, resulting in a virus that expresses v-slfn from its natural promoter. In vitro characterisation showed no differences in replication or spread when compared to controls. Thus, v-slfn is an orthologue of mammalian slfn proteins, and may exert its effect by reversing their inhibition of cellular proliferation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Exploring Glucocorticoid Receptor Agonists Mechanism of Action Through Mass Cytometry and Radial Visualizations

Recent advances in combining flow cytometry and mass spectrometry have led to the development of mass cytometry, allowing for the interrogation of complex cell populations on an unprecedented scale. The volumes and high dimensionality of mass cytometry data pose significant challenges in terms of analysis and visualization. We implement a method called Radviz, where multidimensional single cell data can be visualized as a projection that maintains the original dimensions and data complexity whilst facilitating analysis and visualization. This enables identification of changes in populations, focusing the analysis on the most relevant aspect of large multidimensional datasets. To highlight the potential of Radviz, we profiled peripheral mononuclear blood cells (PBMCs) from three healthy donors and showed donor-specific differences in the number and composition of cell populations. In a second study, we explored the anti-inflammatory effects of two glucocorticoid receptor (GR) ligands (cpd6 and cpd11) compared to dexamethasone (Dex) on human primary macrophages. Standard analysis at the population level showed that cpd6 and cpd11 have an overall anti-inflammatory profile similar to that of Dex. CyTOF profiling and Radviz-driven analysis at the single cell level confirmed this observation, and identified a concentration-dependent effect of cpd6 that was not detected at the population level. Altogether, Radviz combines the strengths of a projection method, reducing the dimensionality of datasets, with that of a scatter plot, where the identity of each point can be inferred from the distance to the axis. This enables the visual exploration, analysis, and interpretation of complex, high dimensional data

Intradermal immune response after infection with Vaccinia virus.

Although Vaccinia virus (VACV) was used to eradicate smallpox by dermal vaccination, there is little information available about the immune response induced at the vaccination site. Previously, an intradermal murine model that mimics smallpox vaccination was established. Here, this model was used to investigate which leukocytes are recruited to the infected lesion and what are the kinetics of recruitment. Data presented show that VACV infection induced the infiltration of macrophages, followed by granulocytes and lymphocytes. Up to 4 days post-infection, the major lymphocyte population was TCRgammadelta T cells, but thereafter, there was a large recruitment of CD4(+) and CD8(+) T cells. Interestingly, the majority of T cells expressed the natural killer-cell marker DX5. This report is the first to characterize the local immune response sequence to VACV infection and represents a benchmark against which the responses induced by genetically modified VACVs may be compared

Identification of Small Molecules Which Induce Skeletal Muscle Differentiation in Embryonic Stem Cells via Activation of the Wnt and Inhibition of Smad2/3 and Sonic Hedgehog Pathways

The multi-lineage differentiation capacity of mouse and human embryonic stem (ES) cells offers a testing platform for small molecules that mediate mammalian lineage determination and cellular specialization. Here we report the identification of a small molecule which drives mouse ES cell differentiation to skeletal muscle with high efficiency without any genetic modification. Mouse embryoid bodies (EBs) were used to screen a library of 1,000 small molecules to identify compounds capable of inducing high levels of Pax3 mRNA. Stimulation of EBs with SMIs (Skeletal Muscle Inducer, SMI1 and SMI2) from the screen resulted in a high percentage of intensively twitching skeletal muscle fibers three weeks after induction. Gene expression profiling studies that were carried out for Mode of Actions (MoA) analysis showed that SMIs activated genes regulated by the Wnt pathway, and inhibited expression of Smad2/3 and Sonic Hedgehog target genes. A combination of three small molecules known to modulate these three pathways acted similarly to the SMIs found here, driving ES cells to skeletal muscle. Taken together, these data demonstrate that the SMI drives ES cells to skeletal muscle via concerted activation of the Wnt pathway, and inhibition of Smad2/3 signaling and Sonic Hedgehog (Shh) pathways. This provides important developmental biological information about skeletal muscle differentiation from embryonic stem cells and may lead to the development of new therapeutics for muscle disease

v-GAAP Is an Immunomodulator

<div><p>Groups of four BALB/c mice aged between 6 and 8 wk were mock-infected with PBS (•) or infected with 10⁷ pfu of v-GAAP WT (♦), v-ΔGAAP (), or v-GAAP Rev (▴).</p><p>(A) Mice were weighed daily, and results are the mean percentage weight change of each group ± standard error of the mean (SEM) compared with the weight on the day of infection.</p><p>(B) Animals from (A) were monitored daily for signs of illness, which was scored as described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030017#ppat-0030017-b027" target="_blank">27</a>]. Data are expressed as the mean ± SEM. <i>p</i>-Values were determined by using Student's <i>t</i>-test and indicate mean weight loss (A) or mean signs of illness. *, <i>p</i>-values of mice infected with v-ΔGAAP that were significantly different from those of mice infected with v-GAAP WT and v-GAAP Rev (B).</p><p>(C) Mice infected intranasally with 10⁷ pfu of v-GAAP WT, v-ΔGAAP, or v-GAAP Rev were sacrificed at various days p.i. as indicated. BAL cells were recovered and counted. Columns represent the mean cell yield per mouse ± SEM. Those marked with an asterisk indicate mean cell numbers recovered with v-ΔGAAP that were significantly different (<i>p</i> < 0.05) from those recovered from v-GAAP WT and v-GAAP Rev (three independent experiments with groups of four to five mice).</p></div