Molecular pathogenesis of human coronaviruses

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

Since early 2020, the COVID-19 pandemic, caused the novel -coronavirus SARS-CoV-2, has ravaged the world and has disrupted much biomedical research. Consequently, we decided to focus a portion of our efforts on human coronaviruses, like SARS-CoV-2 and the Middle East Respiratory Syndrome coronavirus or MERS-CoV. Both MERS-CoV and SARS-CoV-2 are high consequence viral pathogens with the capacity to cause severe human disease, and SARS-CoV-2 has established a raging pandemic. This project will leverage pre-existing expertise and novel approaches against this new pathogen. Suppression of SARS-CoV-2 and MERS-CoV replication by FDA-approved drugs. The recent emergence of SARS-CoV-2 in 2019 and the emergence of MERS-CoV and SARS-CoV-1 in the past 20 years highlight the necessity of broad-spectrum antivirals that suppress coronaviruses and can be deployed against currently circulating coronaviruses and coronaviruses that emerge in the future. Additionally, understanding the evolutionary factors that influence or contribute to coronavirus emergence, such as quasispecies dynamics, is of the utmost importance. Dr. Stacey Scroggs evaluated the potency of enoxacin, ciprofloxacin, levofloxacin, and moxifloxacin to suppress SARS-CoV-2 and MERS-CoV replication based on our previous work 1,2 and the work of other suggesting anti-SARS activity 36. We demonstrated that at micromolar concentrations over 150M all four drugs suppress SARS-CoV-2 replication in Vero and A549/ACE2 cells. The effective concentration 50 values (EC50) for SARS-CoV-2 were all higher than 100 M and therefore these drugs are unlikely to elicit a clinical benefit alone. In order to assess the role of quasispecies dynamics in the emergence of zoonotic coronaviruses, Dr. Scroggs is currently establishing a combination ex vivo and in vitro tissue culture system to study the evolution of zoonotic coronaviruses before and after emergence into a new host. For the ex vivo culture portion we are growing bovine airway epithelial cell cultures at an air-liquid interface (ALI). These cultures will be infected with bovine coronavirus (bCoV), isolated from an experimentally infected calf, for multiple rounds of replication before the resulting passaged virus will be used to serially infect human cells. The viruses pre and post host switch will be deep sequenced and their interhost viral populations and fitness will be analyzed. Cellular mechanisms modulating replication of high consequence human coronaviruses. As a result of the SARS-CoV-2 pandemic and a necessary shift in research focus to coronaviruses, Dr. Dylan Flather is applying genome-wide screening strategies to better describe the host factors of this virus family. With our collaborator Dr. Andreas Puschnik of the Chan-Zuckerberg Biohub, we set out to identify pan-coronavirus host factors that could be targeted by host-targeting antivirals. Dr. Puschniks lab and secondary collaborators began this work with SARS-CoV-2 and seasonally-circulating coronaviruses, while we focused our efforts on Middle East Respiratory Syndrome (MERS) coronavirus. Because MERS infection of the Huh-derived cell line used in these protocols resulted in extensive syncytia formation, comprehensive optimization work with small molecule proprotein convertase inhibitors was required before the full screen could be performed. The formation of syncytia was problematic because cells resistant to MERS infection could be incorporated into growing syncytium, precluding the identification of proviral candidates from cells refractory to infection. Importantly, the use of these inhibitors did not have an impact on the production of infectious MERS but significantly reduced the formation of syncytia at low micromolar concentrations. Our MERS screens revealed only a single host factor shared with other coronaviruses, illustrating the unique replication strategies that even closely-related viruses employ. Candidate proteins from our screen had significant overlap with one published report and one preprint that used different cell types in their screening strategies. We have validated the top hits from our screens with single-gene knockout cell lines as well as small molecule inhibitors. We are currently preparing for another MERS host factor screen in airway epithelial cells, a medically relevant cell type, using the protocol we established with Huh-derived cells. Fully defining the repertoire of host factors utilized by coronaviruses could prove to be invaluable for future coronavirus pandemics. SARS-CoV-2 and the Liver X Receptor Pathway. The liver X receptor (LXR) pathway plays an integral role in cholesterol metabolism and the inflammatory response. Like many viruses, SARS-CoV-2 hijacks cell host machinery. Lipid metabolism is a frequent target of viruses as lipids are crucial for the viral lifecycle. Previous work by our lab and others have shown perturbation of the LXR pathway can have a negative effect on viral replication. The work investigating the potential effects of LXR agonists and antagonists on SARS-CoV-2 replication will continue. Anti-CD47 and anti-IL6 as SARS-CoV-2 Therapeutics. In collaboration with Dr. Kim Hasenkrugs group in LPVD/RML, we examined the role of CD47 during SARS-CoV-2 infection. Building upon Dr. Hasenkrug labs previous work demonstrating anti-CD47 can decrease antiviral immune response, limit inflammation, & increase infection clearance rate, we hoped to be able to use anti-CD47 antibodies as therapeutics. Unfortunately, with the cell lines we utilized, we didnt detect any upregulation of CD47 with SARS-CoV-2 infection and, therefore, have ended this project.