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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - COV RESTRIC (Unravelling species barriers of coronaviruses)

Teaser

Summary

Coronavirus (CoV) infections can pose a significant global health threat due to the emergence of highly pathogenic variants, like the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and more recently the Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which emerged during cross-species transmissions from animals to humans. One hallmark of these two highly pathogenic viruses, SARS-CoV and MERS-CoV, is that they arose through zoonotic transmissions. Although the first cross-species transmission of SARS-CoV to the human population was mediated by civet cats from live animal markets in Guangdong, China in 2002, the viral reservoir has later been traced back to horseshoe bats. A sustained human-to-human transmission was the primary cause for the human epidemic, which led to total of 8437 infected people and 813 SARS-related deaths worldwide. Immediate and effective public health measures contained the outbreak by 2003 and, as of today, the virus has been declared eradicated. In contrast, MERS-CoV emerged in Middle Eastern countries in 2012 and has so far caused 2458 infections with a fatality rate of approximately 35 % of reported patients. Dromedary camels have been identified as the primary host for transmission of the virus to humans, however bats are speculated as the ancestral host reservoir. In comparison to SARS-CoV, MERS-CoV spillover from dromedary camels to humans is constantly ongoing as the virus is circulating in this camelid population.

The precise mechanisms that allow coronaviruses to jump across species barriers still remain elusive. Therefore, studies addressing viral control mechanisms precluding transmission across species barriers are of utmost importance to ultimately contribute to the prevention of zoonotic infections.
We hypothesized that conserved restriction factors exists between different species which limit viral replication and which need to be overcome via viral evasion and adaptation strategies in order to establish a zoonotic infection. We proposed a genetic screening approach in combination with state-of-the-art technologies to identify unknown coronavirus restriction factors, restricting replication of different coronavirus variants originating from different hosts, including the highly pathogenic human SARS-CoV and MERS-CoV. We specifically aimed to identify interferon stimulated genes (ISGs) that are conserved between different species. The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of ISGs, some of which encode direct antiviral effectors that could act as viral restriction factors.

Upon application of an ISG screening approach we could identify a novel ISG, namely lymphocyte antigen 6 complex, locus E (LY6E) that potently restricts human CoVs (Figure 1). We analyzed its mechanism of action and further characterized the role of this ISG in the CoV replication cycle. In conclusion, we provide novel knowledge about innate CoV restriction factors. A deeper understanding of viral restriction could in the end lead to novel therapeutic options against emerging CoVs.

Work performed

We started this project with the utilization of an ISG screening approach. To this end we collaborated with Prof. Charles Rice from the Rockefeller University in New York, USA. We screened an ISG library (Schoggins et. al. Nature 2011, Schoggins et. al. Nature 2014) against the human CoV 229E (HCoV-229E). Interestingly we found one outstanding, very potent restriction factor, namely the ISG LY6E (Figure 1). We confirmed the antiviral activity of LY6E against the highly pathogenic MERS-CoV and SARS-CoV. In addition, a viral inhibition could be observed against HCoV-OC43 as well as murine hepatitis virus (MHV), confirming a broad antiviral role of this ISG against CoV infections. In order to analyze LY6E as innate species barrier, we analyzed its conservation and tested orthologous genes from rhesus macaque, mouse, bat and camel. Interestingly, all orthologous variants of this gene were able to inhibit human CoV, indicating that it is conserved across species. In addition, we tested CoV originating from camelid species (dromedary camel-229E-like CoV ACNO4 and JC50) and could show that they could be inhibited as well. We narrowed down its mechanism of action to virus entry, however, the precise mechanism is still elusive. In conclusion, we identified a novel ISG that is able to control pan-species coronavirus infections.
We disseminated the results of our research through different channels. We are currently preparing a manuscript for publication (Pfaender et. al. manuscript in preparation) in a high impact scientific journal. Furthermore, data has been shared at international conferences including the 29th Annual Meeting of the Society for Virology (DÃ¼sseldorf, Germany, oral presentation, abstract ID 132), the Positive Strand RNA Virus Meeting (Keystone Meeting, Killarney, Ireland; poster presentation, abstract ID 3058) and the European Congress of Virology Meeting 2019 (Rotterdam, Netherlands, poster presentation, abstract ID. 139). In addition, dissemination has been realized upon presentation of the data in internal seminars including regular progress reports and internal symposia.
With respect to Article 28 of the H2020 annotated model grant agreement, measures were taken to ensure exploitation of the research results. On the one hand, results will be exploited for future research. A â€œhit listâ€ of possible candidate genes restricting coronavirus replication has been obtained which can be used for diverse follow up projects. Due to time limitations, we were not able to conduct a detailed analysis of the mechanism of action for our top hit but aim to address this question in future studies.

Final results

We were able to identify a novel ISG that is potently able to inhibit CoVs across various species, including the highly pathogenic MERS-CoV and SARS-CoV. We provide novel knowledge about intrinsic host defense mechanisms that are conserved across species and that could be exploited for therapeutic intervention. Given that no therapeutic options exits for CoV infections, our results could pave the way for the development of novel therapeutic options.