Molecular pathogenesis of human coronaviruses

National Institute Of Allergy And Infectious Diseases

Investigators

Abstract

Since early 2020, the COVID-19 pandemic, caused the SARS-CoV-2, has ravaged the world. 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 apply pre-existing expertise against this recently emerged pathogen. 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 and his collaborators are applying genome-wide screening strategies to better describe the host factors of this virus family. The identification and characterization of proviral host factors through genome-wide screens as described for TBFVs, above, has also been applied to Middle East Respiratory Syndrome (MERS) coronavirus. Collectively, the group has defined proviral host factors for six of the seven known human coronaviruses with the goal to identify pan-coronavirus host factors that could be used to develop treatments for novel coronaviruses. Specifically for the MERS screen, initial attempts to perform screens failed due to the extensive amount of syncytia formed by this virus in the liver-derived Huh7.5.1 cell line that was used for all other coronavirus screens. As a result, there was not enough cell survival to draw conclusions about possible proviral factors. However, after incorporating a proprotein convertase inhibitor that significantly reduced syncytia formation, into our approach, we were able to select for cells that survived MERS infection. Importantly, the use of the proprotein convertase inhibitors did not have a negative impact on the production of infectious MERS but drastically reduced the formation of syncytia at low micromolar concentrations. To this point, our validation studies have identified a single pan-coronavirus host factor called Transmembrane Protein 41B (TMEM41B). Interestingly, other recently published MERS genome-wide screens have shown very limited overlap with the results from our screen, suggesting that the identification of factors shared across screens using different viruses may not reveal obvious shared host factors. Indeed, aside from TMEM41B, no factor was consistently determined to be proviral for the six coronaviruses that we and our collaborators have tested. However, through performing pharmacological inhibition studies we have begun to identify cellular pathways that are commonly targeted by a wide range of coronaviruses. This suggests that focusing on the protein-level to identify similarities in coronavirus replication may offer too narrow a focus and that considering protein functionalities more broadly, as parts of complexes or metabolic pathways, offers more biological relevance. Finally, we have also performed genome-wide screens in a more physiologically relevant lung-derived cell line and are currently analyzing that data in an effort to reveal cell type dependent and cell type independent MERS host dependency factors. Immunotoxins as SARS-CoV-2 Therapeutics. In collaboration with Dr. Seth Pincus at Montana State University, Ms. Offerdahl has begun experiments to examine the potential of immunotoxins as a treatment for SARS-CoV-2 infections. Dr. Pincuss lab has produced several SARS-CoV-2 antibody-ricin A chain conjugations with the intent to discretely kill SARS-CoV-2 infected cells. We have been using an immunofluorescence assay to look for a reduction in the number of infected cells in immunotoxin treated cells. Modest effects have been seen thus far but we are still optimizing the assay.