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      A New Inhibitor of Apoptosis from Vaccinia Virus and Eukaryotes

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          Abstract

          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.

          Author Summary

          Apoptosis is a conserved and strictly regulated process of cell suicide that, among other things, can remove virus-infected cells. In turn, many viruses, including poxviruses, have evolved strategies to block apoptosis to keep cells alive until virus replication is completed. Here, a novel viral anti-apoptotic protein from vaccinia virus and camelpox virus, viral Golgi anti-apoptotic protein (v-GAAP), and its human counterpart, h-GAAP, are described. Evolutionarily, GAAPs are extremely well conserved with closely related proteins in plants, insects, amphibia, and mammals, the viral and human counterparts sharing a striking 73% sequence identity. GAAPs are resident in the Golgi and inhibit apoptosis induced by a wide range of apoptotic stimuli. Knockout of h-GAAP, which is expressed in every tissue tested, induced cell death by apoptosis. The close relationship between the viral and the human proteins was confirmed in that v-GAAP could complement for the loss of h-GAAP and promote cell survival. Deletion of the v-GAAP gene from vaccinia virus affected virus virulence. Thus, this study identifies a new regulator of cell death that is highly conserved in evolution and has been hijacked by poxviruses. These data support a role for the Golgi complex in sensing pro-apoptotic stimuli.

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          Most cited references41

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          Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension.

          Gene splicing by overlap extension is a new approach for recombining DNA molecules at precise junctions irrespective of nucleotide sequences at the recombination site and without the use of restriction endonucleases or ligase. Fragments from the genes that are to be recombined are generated in separate polymerase chain reactions (PCRs). The primers are designed so that the ends of the products contain complementary sequences. When these PCR products are mixed, denatured, and reannealed, the strands having the matching sequences at their 3' ends overlap and act as primers for each other. Extension of this overlap by DNA polymerase produces a molecule in which the original sequences are 'spliced' together. This technique is used to construct a gene encoding a mosaic fusion protein comprised of parts of two different class-I major histocompatibility genes. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques.
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            Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference.

            In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.
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              Organelle-specific initiation of cell death pathways.

              Nuclear DNA damage and ligation of plasma-membrane death receptors have long been recognized as initial triggers of apoptosis that induce mitochondrial membrane permeabilization (MMP) and/or the direct activation of caspases. Accumulating evidence suggests that other organelles, including the endoplasmic reticulum (ER), lysosomes and the Golgi apparatus, are also major points of integration of pro-apoptotic signalling or damage sensing. Each organelle possesses sensors that detect specific alterations, locally activates signal transduction pathways and emits signals that ensure inter-organellar cross-talk. The ER senses local stress through chaperones, Ca2+-binding proteins and Ca2+ release channels, which might transmit ER Ca2+ responses to mitochondria. The ER also contains several Bcl-2-binding proteins, and Bcl-2 has been reported to exert part of its cytoprotective effect within the ER. Upon membrane destabilization, lysosomes release cathepsins that are endowed with the capacity of triggering MMP. The Golgi apparatus constitutes a privileged site for the generation of the pro-apoptotic mediator ganglioside GD3, facilitates local caspase-2 activation and might serve as a storage organelle for latent death receptors. Intriguingly, most organelle-specific death responses finally lead to either MMP or caspase activation, both of which might function as central integrators of the death pathway, thereby streamlining lysosome-, Golgi- or ER-elicited responses into a common pathway.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                ppat
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                February 2007
                23 February 2007
                : 3
                : 2
                : e17
                Affiliations
                [1 ] Department of Virology, Faculty of Medicine, Imperial College London, London, United Kingdom
                [2 ] Ludwig Institute for Cancer Research, Faculty of Medicine, Imperial College London, London, United Kingdom
                [3 ] Department of Medical Microbiology, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
                University of Texas Southwestern Medical Center, United States of America
                Author notes
                * To whom correspondence should be addressed. E-mail: c.gubser@ 123456imperial.ac.uk (CG); glsmith@ 123456imperial.ac.uk (GLS)
                Article
                06-PLPA-RA-0269R3 plpa-03-02-09
                10.1371/journal.ppat.0030017
                1803007
                17319741
                5e7c773f-0ced-4a83-8b9a-f77d757fdfad
                Copyright: © 2007 Gubser et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 11 July 2006
                : 21 December 2006
                Page count
                Pages: 14
                Categories
                Research Article
                Cell Biology
                Evolutionary Biology
                Genetics and Genomics
                Infectious Diseases
                Pathology
                Virology
                Viruses
                Homo (Human)
                Drosophila
                Xenopus
                Arabidopsis
                Custom metadata
                Gubser C, Bergamaschi D, Hollinshead M, Lu X, van Kuppeveld FJM, et al. (2007) A new inhibitor of apoptosis from vaccinia virus and eukaryotes. PLoS Pathog 3(2): e17. doi: 10.1371/journal.ppat.0030017

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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