RNAi screening identifies host determinants associated with susceptibility to respiratory virus infection
Item statusRestricted Access
Embargo end date15/05/2024
Acute respiratory infections, especially those that lead to lower respiratory tract (LRI) complications, are a large public health concern worldwide. It is estimated that adults obtain a common cold or similar up to three times a year. This is more than double for children under the age of 16, leading to annual costs for respiratory viral infections, excluding those related to influenza, to over $22 billion. Recently, there has been a move towards precision medicine in many different fields, using a patient-specific approach incorporating all relevant clinical, genetic, and biological information for each individual to maximise the therapeutic benefit of treatments. This can be applied to respiratory infections where there is a large disparity between the severity of symptoms in different patients. To aid this, and to gain greater insight into viral interactions with the host cell, RNAi human druggable genome screens were performed on two highly relevant RNA viruses; human Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Globally, RSV (family Paramyxovirus) causes approximately 45% of the hospitalizations and deaths in children over 6 months. Symptoms include, but are not limited to; fever, sore throat, cough, and shortness of breath, with some children developing bronchiolitis. The frequency of asthma and allergic sensitization is significantly higher in children who develop severe LRI, re-infection is a common occurrence and there are currently no licenced vaccines or effective antivirals against RSV. Here, we completed an RNAi screen using the Dharmacon Human Druggable Genome library on A549 cells with an RSV-A2-GFP reporter virus and subsequently compared this to two other published whole genome RNAi screens on RSV. Little overlap was seen between the proviral and antiviral hit tails for all three screens, but by looking at pathways and protein complexes members of the ADAM metalloprotease family were identified as proviral across all screens. Further investigations were not yet able to confirm their exact role in RSV infection, but hints towards a steric interaction with the cellular and extracellular matrix to facilitate successful syncytia formation and passage of virus into uninfected cells. As there are no effective antiviral treatments, which can be utilised against RSV we also investigated the antiviral activity of remdesivir, both on its own and in combination with ribavirin (a drug that has been clinically used to treat RSV), to determine if this would have a greater effect at lower concentrations and therefore be a more clinically viable antiviral for children and the immunocompromised. We found that remdesivir can achieve a similar level of viral inhibition at a concentration 2 log10’s lower than ribavirin and, interestingly, a combination of the two at low concentrations can work against RSV with a small synergistic effect. SARS-CoV-2 (family Coronaviridae), the causative agent of COVID-19, which triggered a global pandemic after its emergence in December 2019, causes similar symptoms to RSV; fatigue, headaches, and shortness of breath which can last for several months (long COVID). Since the beginning of the pandemic many vaccines have been developed against SARS-CoV-2, mainly targeting an immune response to the viral spike protein. But our knowledge about how the virus interacts with the host’s innate immune response and other proteins in the cell is still limited. Identification of a suitable, naturally permissive, human submerged cell line for SARS-CoV-2 infection was crucial to be able to perform the RNAi screen, as previously only Vero E6 (African green monkey) and heterogeneous human cell lines had been identified. We identified the Caki-1 cell line, derived from an epithelial kidney clear cell carcinoma, to be naturally permissive to SARS-CoV-2 and, very importantly, also other respiratory viruses (MERS-CoV, CoV-229E, IAV and RSV). By performing an RNAi screen on a clinical isolate of SARS-CoV-2 in this cell line, using released virus as the readout, we were able to demonstrate high clinical relevance and entry receptors previously reported to be involved in SARS-CoV-2 entry (ACE2, KREMEN1). We also managed to identify viral release and trafficking pathways, which have not been implicated with SARS-CoV-2 before and have not been found by CRISPR-based screens. Collectively, we have identified many host-factors involved in the replication of RSV and SARS-CoV-2, and managed to verify some of these hits, aiding greater understanding of how these who viruses replicate in the respiratory tract. We have also identified drugs for both RSV and SARS-CoV-2 which can be used with greater effect that the ones currently used in the clinic.
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