Antiviral responses are shaped by heterogeneity in viral replication dynamics

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells

Viruses, when entering their host cells, are met by a fierce intracellular immune defense. One prominent antiviral pathway is the integrated stress response (ISR). Upon activation of the ISR - typically though not exclusively upon detection of dsRNA - translation-initiation factor eukaryotic initiation factor 2 (eIF2) becomes phosphorylated to act as an inhibitor of guanine nucleotide-exchange factor eIF2B. Thus, with the production of ternary complex blocked, a global translational arrest ensues. Successful virus replication hinges on effective countermeasures. Here, we review ISR antagonists and antagonistic mechanisms employed by picorna- and coronaviruses. Special attention will be given to a recently discovered class of viral antagonists that inhibit the ISR by targeting eIF2B, thereby allowing unabated translation initiation even at exceedingly high levels of phosphorylated eIF2

Antiviral responses are shaped by heterogeneity in viral replication dynamics

Antiviral signalling, which can be activated in host cells upon virus infection, restricts virus replication and communicates infection status to neighbouring cells. The antiviral response is heterogeneous, both quantitatively (efficiency of response activation) and qualitatively (transcribed antiviral gene set). To investigate the basis of this heterogeneity, we combined Virus Infection Real-time IMaging (VIRIM), a live-cell single-molecule imaging method, with real-time readouts of the dsRNA sensing pathway to analyse the response of human cells to encephalomyocarditis virus (EMCV) infection. We find that cell-to-cell heterogeneity in viral replication rates early in infection affect the efficiency of antiviral response activation, with lower replication rates leading to more antiviral response activation. Furthermore, we show that qualitatively distinct antiviral responses can be linked to the strength of the antiviral signalling pathway. Our analyses identify variation in early viral replication rates as an important parameter contributing to heterogeneity in antiviral response activation

SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera

Nationwide comprehensive gastro-intestinal cancer cohorts: the 3P initiative

Background: The increasing sub-classification of cancer patients due to more detailed molecular classification of tumors, and limitations of current trial designs, require innovative research designs. We present the design, governance and current standing of three comprehensive nationwide cohorts including pancreatic, esophageal/gastric, and colorectal cancer patients (NCT02070146). Multidisciplinary collection of clinical data, tumor tissue, blood samples, and patient-reported outcome (PRO) measures with a nationwide coverage, provides the infrastructure for future and novel trial designs and facilitates research to improve outcomes of gastrointestinal cancer patients. Material and methods: All patients aged ≥18 years with pancreatic, esophageal/gastric or colorectal cancer are eligible. Patients provide informed consent for: (1) reuse of clinical data; (2) biobanking of primary tumor tissue; (3) collection of blood samples; (4) to be informed about relevant newly identified genomic aberrations; (5) collection of longitudinal PROs; and (6) to receive information on new interventional studies and possible participation in cohort multiple randomized controlled trials (cmRCT) in the future. Results: In 2015, clinical data of 21,758 newly diagnosed patients were collected in the Netherlands Cancer Registry. Additional clinical data on the surgical procedures were registered in surgical audits for 13,845 patients. Within the first two years, tumor tissue and blood samples were obtained from 1507 patients; during this period, 1180 patients were included in the PRO registry. Response rate for PROs was 90%. The consent rate to receive information on new interventional studies and possible participation in cmRCTs in the future was >85%. The number of hospitals participating in the cohorts is steadily increasing. Conclusion: A comprehensive nationwide multidisciplinary gastrointestinal cancer cohort is feasible and surpasses the limitations of classical study designs. With this initiative, novel and innovative studies can be performed in an efficient, safe, and comprehensive setting

Nationwide comprehensive gastro-intestinal cancer cohorts: the 3P initiative

Background: The increasing sub-classification of cancer patients due to more detailed molecular classification of tumors, and limitations of current trial designs, require innovative research designs. We present the design, governance and current standing of three comprehensive nationwide cohorts including pancreatic, esophageal/gastric, and colorectal cancer patients (NCT02070146). Multidisciplinary collection of clinical data, tumor tissue, blood samples, and patient-reported outcome (PRO) measures with a nationwide coverage, provides the infrastructure for future and novel trial designs and facilitates research to improve outcomes of gastrointestinal cancer patients. Material and methods: All patients aged ≥18 years with pancreatic, esophageal/gastric or colorectal cancer are eligible. Patients provide informed consent for: (1) reuse of clinical data; (2) biobanking of primary tumor tissue; (3) collection of blood samples; (4) to be informed about relevant newly identified genomic aberrations; (5) collection of longitudinal PROs; and (6) to receive information on new interventional studies and possible participation in cohort multiple randomized controlled trials (cmRCT) in the future. Results: In 2015, clinical data of 21,758 newly diagnosed patients were collected in the Netherlands Cancer Registry. Additional clinical data on the surgical procedures were registered in surgical audits for 13,845 patients. Within the first two years, tumor tissue and blood samples were obtained from 1507 patients; during this period, 1180 patients were included in the PRO registry. Response rate for PROs was 90%. The consent rate to receive information on new interventional studies and possible participation in cmRCTs in the future was >85%. The number of hospitals participating in the cohorts is steadily increasing. Conclusion: A comprehensive nationwide multidisciplinary gastrointestinal cancer cohort is feasible and surpasses the limitations of classical study designs. With this initiative, novel and innovative studies can be performed in an efficient, safe, and comprehensive setting

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells

Viruses, when entering their host cells, are met by a fierce intracellular immune defense. One prominent antiviral pathway is the integrated stress response (ISR). Upon activation of the ISR - typically though not exclusively upon detection of dsRNA - translation-initiation factor eukaryotic initiation factor 2 (eIF2) becomes phosphorylated to act as an inhibitor of guanine nucleotide-exchange factor eIF2B. Thus, with the production of ternary complex blocked, a global translational arrest ensues. Successful virus replication hinges on effective countermeasures. Here, we review ISR antagonists and antagonistic mechanisms employed by picorna- and coronaviruses. Special attention will be given to a recently discovered class of viral antagonists that inhibit the ISR by targeting eIF2B, thereby allowing unabated translation initiation even at exceedingly high levels of phosphorylated eIF2

SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b.

The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding-prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Similar observations were made for the N protein of human coronavirus 229E, suggesting that this may be a general trait conserved among members of other orthocoronavirus (sub)genera

Sentinel node biopsy during thoracolaparoscopic esophagectomy for advanced esophageal cancer

BACKGROUND: Omitting extensive lymph node dissection could reduce esophagectomy morbidity in patients without lymph node metastases. Sentinel node biopsy may identify abdominal or thoracic lymph node metastases, thereby differentiating treatment. Feasibility of this approach was investigated in Western European esophageal cancer patients with advanced disease, without lymph node metastases at diagnostic work-up. METHODS: The sentinel node biopsy was performed in eight esophageal cancer patients with cT1-3N0 disease. One day pre-operatively, Tc-99m-labeled nanocolloid was endoscopically injected around the tumor. Lymphoscintigraphy was performed 1 and 3 h after injection. All patients underwent robotic thoracolaparoscopic esophagectomy with two-field lymph node dissection. Intraoperatively, sentinel nodes were detected by gamma probe. The resection specimen was analyzed for remaining activity by scintigraphy and gamma probe. RESULTS: Visualization rates of lymphoscintigraphy 1 and 3 h after tracer injection were 88 and 100 %, respectively. Intraoperative identification rate was 38 %. Postoperative identification was possible in all patients using the gamma probe to analyze the resection specimen. In 5/8 patients, lymph node metastases were found at histopathology, none of which was detected by the sentinel node biopsy. No adverse events related to the sentinel node biopsy were observed. CONCLUSIONS: In our advanced esophageal cancer patients who underwent thoracolaparoscopic esophagectomy, the sentinel node biopsy did not predict lymph node status. Probably the real sentinel node could not be identified due to localization adjacent to the primary tumor or bypassing due to metastatic tumor involvement. Therefore, we consider the sentinel node biopsy not feasible in advanced esophageal cancer