Skip to content

Instantly share code, notes, and snippets.

@michael

michael/broken.json

Created October 4, 2019 14:53
Show Gist options
  • Star 0 You must be signed in to star a gist
  • Fork 0 You must be signed in to fork a gist
  • Save michael/5ce916ba778d1d131915bcf52e4dbdef to your computer and use it in GitHub Desktop.
Save michael/5ce916ba778d1d131915bcf52e4dbdef to your computer and use it in GitHub Desktop.
Download ZIP
Raw
broken.json
[
{
"id": "id",
"owner": "id",
"name": "string",
"title": "string",
"authors": "string[]",
"assets": "id[]",
"createdAt": "date",
"updatedAt": "date",
"data": "string",
"changeId": "number"
},
{
"id": 1,
"owner": 1,
"name": "lorem-ipsum",
"title": "Lorem Ipsum",
"assets": [
2,
3
],
"createdAt": 1567017261598,
"updatedAt": 1570026650319,
"data": "<?xml version=\"1.0\" encoding=\"UTF-8\"?>\n<!DOCTYPE essay PUBLIC \"-//SUBSTANCE//DTD Essay v1\" \"Essay-1.dtd\">\n<essay id=\"essay-1\"><title>Lorem Ipsum</title><body><paragraph id=\"p-1\">Lorem <bold id=\"b-1\">ipsum</bold> dolor sit <italic id=\"i-1\">amet</italic>, consectetur <link id=\"link-1\" href=\"http://substance.io\" linkType=\"\">adipiscing</link> elit. Donec convallis augue ut orci finibus laoreet. Aliquam venenatis ante scelerisque lectus malesuada, ut blandit leo facilisis. Integer at egestas urna. Nulla facilisi. Quisque imperdiet fermentum euismod. Donec vestibulum semper lorem id accumsan. In ut ligula at enim tempus dictum in nec sapien. Sed su<bold id=\"bold-d929eaf45ed7747c3d3e530823162888\">scipit</bold> aliquam sapien sit amet cursus. Integer tincidunt est nulla, sit amet bibendum sem condimentum in. Nulla bibendum non lorem eget feugiat. Mauris metus libero, euismod sed ante vitae, euismod commodo leo. Aliquam cursus, tellus at rhoncus mdfolestie, </paragraph><paragraph id=\"paragraph-23183017d29e7d672c288c0f8f9a9216\"/><paragraph id=\"paragraph-78e882f1c2177061a163a3fdda2f7216\">orci sapien iaculis massa, vitae venenatis justo quam eget dolor. Integer mollis imperdiet nunc, vel condimentum magna euismod nec. Mauris ornare, tortor vitae suscipit hendrerit, nunc sem congue ante, nec elementum lorem lorem lacinia magna. Donec vehicula, nisi eget facilisis egestas, dolor urna fringilla nisl, blandit vulputate quam ante vel magna. Nulla vitae maximus</paragraph><figure id=\"figure-41a9eab7cbbe237375d1586de7f52187\" image=\"Screenshot 2019-09-13 at 20.47.01.png\"><legend><paragraph id=\"paragraph-1ff81e13beb668278a2f663ca7e30cb1\">adsfasdfasdfas</paragraph></legend></figure><list id=\"list-1\" listType=\"\"><list-item id=\"list-item-1\" level=\"1\">Foo</list-item><list-item id=\"list-item-2\" level=\"1\">Bar</list-item><list-item id=\"list-item-8c56a1034af61ce6c414193a8723bd5e\" level=\"3\"/></list></body></essay>",
"changeId": 2
},
{
"id": 2,
"owner": 1,
"name": "norovirus-stress-granule-subversion",
"title": "Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation",
"assets": [
4
],
"createdAt": 1569233914510,
"updatedAt": 1570026943734,
"data": "<?xml version=\"1.0\" encoding=\"UTF-8\"?>\n<!DOCTYPE essay PUBLIC \"-//SUBSTANCE//DTD Essay v1\" \"Essay-1.dtd\">\n<essay id=\"essay-1\"><title>Noroviruses subvert the core stress granule component G3BP1 to promote viral VPg-dependent translation</title><body><heading id=\"heading-bc76046799054ea131dbc0b74e06be08\" level=\"1\">Abstract</heading><paragraph id=\"paragraph-1\">Knowledge of the host factors required for norovirus replication has been hindered by the challenges associated with culturing human noroviruses. We have combined proteomic analysis of the viral translation and replication complexes with a CRISPR screen, to identify host factors required for norovirus infection. The core stress granule component <bold id=\"bold-93bc0ab4786c4a3224a2b84f4dd013d5\">G3BP1 was identified as a host factor essential for efficient human and murine norovirus infection, demonstrating a conserved function across the Norovirus genus. Furthermore, we show that G3BP1 functions</bold> in the novel paradigm of viral VPg-dependent translation initiation, contributing to the assembly of translation complexes on the VPg-linked viral positive sense RNA genome by facilitating 40S recruitment. Our data suggest that G3BP1 functions by providing viral RNA a competitive advantage over capped cellular RNAs, uncovering a novel function for G3BP1 in the life cycle of positive sense RNA viruses and identifying the first host factor with pan-norovirus pro-viral activity.</paragraph><figure id=\"figure-d18dbd84057bad56642a94a12c0b97e3\" image=\"2931.jpg\"><legend><paragraph id=\"paragraph-205b3f11684290e937a4c312542f36de\"/></legend></figure><heading id=\"heading-f0ab61a0ec936244e17d407c914ddf5e\" level=\"1\">Introduction</heading><paragraph id=\"paragraph-f1909d42c825c12f381e357a19f48859\">Positive sense RNA viruses rely heavily on host cell factors for all aspects of their life cycle. They replicate on host derived membranous vesicles that are induced following viral infection, the formation of which requires the activity of key membrane bound viral enzymes (Altan-Bonnet, 2017). Within the membrane bound viral replication complex, translation of the viral genome and the synthesis of new viral RNA occurs in a highly coordinated process. Positive sense RNA viruses have evolved novel gene expression mechanisms that enable them to overcome the genome size limitations that accompany error-prone replication and which might restrict their overall coding capacity (Firth and Brierley, 2012). In addition, viral modification of the host cell translation machinery often provides a competitive advantage allowing for the efficient translation of viral RNA in an environment where competing cellular RNAs are in abundance (McCormick and Khaperskyy, 2017). This ability to compete with cellular RNAs is particularly important for the initiation of infection where the incoming viral genome may be present at only a single copy per cell.</paragraph><paragraph id=\"paragraph-2\">We have previously described a novel paradigm of viral translation that relies on the interaction of host translation initiation factors with a virus-encoded protein (VPg), covalently linked to the 5’ end of the genome of members of the Caliciviridae family of positive sense RNA viruses (Chaudhry et al., 2006; Chung et al., 2014; Goodfellow et al., 2005; Hosmillo et al., 2014; Leen et al., 2016). Unlike the 22-amino acid VPg peptides from picornaviruses, the VPg protein linked to the genomes of caliciviruses is significantly larger and is essential for the translation of viral RNA and viral RNA infectivity (Goodfellow, 2011).</paragraph><paragraph id=\"paragraph-3\">Human noroviruses (HuNoV) and sapoviruses (HuSaV) are enteropathogenic members of the Caliciviridae family of positive sense RNA viruses, and together cause &gt;20% of all cases of gastroenteritis (GE). They are also a significant cause of morbidity and mortality in the immunocompromised; individuals with genetic immune-deficiencies, cancer patients undergoing treatment and transplant recipients often experience chronic norovirus infections lasting months to years (van Beek et al., 2016). The economic impact of HuNoV is estimated to be at least ~$4.2 billion in direct health care costs, with wider societal costs of ~$60 billion (Bartsch et al., 2016). Despite their socioeconomic impact, we have, until very recently lacked a detailed understanding of much of the norovirus life cycle and many significant questions remain unanswered. HuNoV replicons (Chang et al., 2006), a murine norovirus that replicates in cell culture (Karst et al., 2003; Wobus et al., 2004) and the recent B cell (Jones et al., 2014), stem-cell derived organoid (Ettayebi et al., 2016) and zebrafish larvae infection models (Van et al., 2019), have all provided invaluable tools to dissect the norovirus life cycle. However, due to the technical limitations associated with many of these experimental systems, in comparison to other positive sense RNA viruses, our knowledge of the intracellular life of noroviruses is significantly lacking (reviewed in Thorne and Goodfellow, 2014).</paragraph><paragraph id=\"paragraph-4\">In the current study, we have combined three independent unbiased approaches to identify host factors involved in the norovirus life cycle. Combining experimental systems that incorporated both murine and human noroviruses, allowed the identification of cellular factors for which the function is likely conserved across the Norovirus genus. By combining three complimentary approaches, we identify the host protein G3BP1 as a critical host factor required for norovirus VPg-dependent translation, identifying a new role for G3BP1 in virus-specific translation.</paragraph><heading id=\"heading-c2900fc98307dcec8607e3e884b3d04f\" level=\"1\">Results</heading><paragraph id=\"paragraph-adce1fa63044dc4647e16c675c1a258c\">The MNV and the prototype HuNoV Norwalk virus (NV) VPg proteins contain a highly conserved C-terminal domain (Fig 1A) which we have previously shown to be necessary and sufficient for binding to the translation initiation factor eIF4G (Chung et al., 2014; Leen et al., 2016). Using affinity purification on m7-GTP sepharose, we confirmed that the NV VPg protein, as produced during authentic virus replication in a NV replicon bearing cell line, interacts with the cap-binding complex eIF4F (Fig 1B). Components of the eIF4F complex, namely the eIF4E cap-binding protein, the eIF4A helicase and the eIF4GI scaffold protein, along with poly-A binding protein (PABP) and eIF3 subunits, were readily purified on m7-GTP sepharose, whereas GAPDH was not. In NV-replicon containing cells, mature VPg was also enriched on m7-GTP sepharose but the NS3 protein, known to have RNA binding and helicase activity (Li et al., 2018), was not. Furthermore, we demonstrated that transfection of GFP-tagged versions of either the MNV or NV VPg proteins into 293T cells allowed for the affinity purification of eIF4F components and that mutations in the eIF4G binding domain of VPg reduced this association (Fig 1C).</paragraph><heading id=\"heading-6524fc4e4538c16a86fddf1fdad4af9b\" level=\"1\">Discussion</heading><paragraph id=\"paragraph-bfb1bd5a50e8910278c52a90d020c1f4\">In this study, we have used a combination of biochemical and genetic approaches to identify host factors involved in the norovirus life cycle. Our combined approaches resulted in the identification of the core stress granule component G3BP1 as a host protein critical for the replication of both murine and human noroviruses in cell culture. Furthermore, we determined that G3BP1 plays a key role in the processes of norovirus VPg-dependent protein synthesis, uncovering a new function for G3BP1 in facilitating RNA virus genome translation.</paragraph><paragraph id=\"paragraph-e3dad6fe278c3ce5020a6c18b6518eb5\">The orthogonal approaches used in the current study provide an unprecedented insight into the identity of host factors with potential roles in the norovirus life cycle. The detailed proteomic analysis of the viral replication and translation complexes formed during MNV infection (Fig 2) resulted in the identification of several host factors with previously identified roles in the MNV life cycle. We focused our efforts on G3BP1 as it was identified in all three approaches and was also identified in a CRISPR screen published during this study (Orchard et al., 2016). Furthermore, we have previously shown that feline calicivirus (FCV), a relative of noroviruses within the Vesivirus genus, cleaves G3BP1 to inhibit stress granule formation (Humoud et al., 2016). In contrast, MNV infection does not result in G3BP1 cleavage and instead forms cytoplasmic foci the composition of which is distinct from canonical stress granules (Brocard et al., 2018).</paragraph></body></essay>",
"changeId": 6
}
]
Sign up for free to join this conversation on GitHub. Already have an account? Sign in to comment