ABSTRACT
Nonstructural protein 1 (nsp1) of severe acute respiratory syndrome coronavirus (SARS-CoV), inhibits host translation thorough cleaving host mRNA and blocking the translation initiation site on the 40S ribosome. Stem-Loop-1 (SL-1) of the viral RNA leader sequence has been identified to bind to nsp1, allowing viral RNA to escape translation repression. However, the specific residues on nsp1 and the specific sequences on SL-1 important to binding have not been experimentally verified. To investigate this binding, we used gel-shift assay and RNA pull-down to verify binding between nsp1 and SL-1. By mutating SL- 1, we seek to identify the nucleotides of SL-1 that bind to nsp1. Based on recent literature, we hypothesized that disrupting the stem region of SL-1 will decrease binding between nsp1 and SL-1. Moreover, we seek to identify the residues important to binding to SL-1 by mutating specific amino acids of nsp1. Interestingly, nsp1 is a small protein (180 amino acids) with intrinsically unstructured regions at both C- and N-terminal ends of the protein. Based on recent literature we hypothesize that disrupting the R124 and K125 residues will decrease binding to SL-1. The results of this study will increase the knowledge of how viral RNA is able to escape suppression of host gene expression. To investigate the binding of nsp1 to SL1, we used nsp1 purified from bacterial lysate using glutathione beads followed by precision protease cleavage of GST-nsp1, and biotinylated RNA. LightShift Chemiluminescence RNA EMSA Kit (Promega) was used to detect the RNA in complex with nsp1 using a gel shift assay. Contrary to our hypothesis, we found an increase in nsp1 binding to the RNA carrying stem mutation, and a decrease in nsp1 binding to the RNA with the loop mutation. Moreover, we observed two distinct bands in the stem mutant indicating two possible binding sites on SL-1. Using an electrophoretic mobility shift assay, the loop region of SL-1 has been determined to be vital for binding to nsp1 in vitro. We hypothesize when the stem was mutated, we created a new binding site for nsp1. Currently we are further investigating several mutations in SL-1 to identify the actual binding site. This project was supported by the DRP award from SC INBRE (NIGMS, P20GM103499).Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
RATIONALE: Treatments for the coronavirus disease of 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), are urgently needed but remain limited. SARS-CoV-2 infects cells through the interactions of its spike (S) protein with ACE2 and TMPRSS2 on host cells. Multiple cells and organs are targeted, particularly airway epithelial cells. OM-85, a standardized lysate of human airway bacteria with strong immunomodulating properties and an impeccable safety profile, is widely used to prevent recurrent respiratory infections. Our finding that the airway administration of OM-85 inhibits Ace2 and Tmprss2 transcription in mouse lungs prompted us to investigate whether and how OM-85 may protect non-human primate and human epithelial cells against SARS-CoV-2 infection. METHODS: ACE2 and TMPRSS2 mRNA and protein expression, cell binding of SARS-CoV-2 S1 protein, cell entry of SARS-CoV-2 S protein-pseudotyped lentiviral particles, and SARS-CoV-2 cell infection were measured in kidney, lung and intestinal epithelial cell lines, primary human bronchial epithelial cells, and ACE2- transfected HEK293T cells treated with OM-85 in vitro. RESULTS: OM-85 significantly downregulated ACE2 and TMPRSS2 mRNA in epithelial cell lines and primary bronchial epithelial cells, and strongly inhibited SARS-CoV-2 S protein binding to, SARS-CoV-2 S proteinpseudotyped lentivirus entry into, and SARS-CoV-2 infection of epithelial cells. These effects of OM-85 appeared to depend on the downregulation of SARS-CoV-2 receptor expression. CONCLUSIONS: OM-85 inhibits SARS-CoV-2 epithelial cell infection in vitro by downregulating SARS-CoV-2 receptor expression. Further studies are warranted to assess whether OM-85 may prevent and/or reduce the severity of COVID-19.
ABSTRACT
Background: There is a good chance to soon control the COVID-19 pandemic but other respiratory tract infections (RTIs) will continue to impact public health. Viral lower RTIs in children are associated with hospitalization, wheezing and asthma inception, while upper RTIs are less severe but have a higher prevalence. Vaccination against prevalent viruses like RSV and RV are still neither available currently nor in the near future. Non-specific immunomodulation for RTI prophylaxis is an interesting treatment alternative. For example, the oral bacterial lysate Broncho-Vaxom (OM-85) has demonstrated efficacy in prevention of recurrent RTIs, specifically in at-risk pediatric populations. For targeting other populations and RTI-indications, robust efficacy data need to be generated, but clinical trials are strongly impacted by the pandemic. Globally, all trials other than dedicated to COVID-19 are experiencing delays or even halts, e.g. due to patient recruitment issues. At the same time, RTI burden changes -through lockdown or social distancing -with an uncertain trajectory. The feasibility of RTI prophylaxis clinical trials in this context thus remains an open question. Method: As a step towards feasibility forecasting, we have implemented a dedicated in silico approach. A mechanistic pharmacokinetics/ pharmacodynamics and within-host viral infection disease model is interfaced with a population-scale (between-host) SIRS disease burden model -thereby accounting for seasonality and extrinsic factors through time-dependent transmission. On the back of this model and a Virtual Population, we conduct in silico clinical trials with variations in observational periods, eligibility criteria (defining the included at-risk population) and follow-up giving us efficacy metrics and sample size estimates as outputs. Results: We demonstrate how the SIRS model can be used to reproduce disease burden data under lockdown and social distancing measures with the example of RCGP 2019-2020 data and how we can translate this data into instantaneous control group prevalence and efficacy dependent on this modulation. We also show how and why different containment scenarios vary in their impact of demonstrated efficacy, recruitment needs and difficulty through analyses of the predicted outcome distributions. Conclusion: We are in the position to forecast probable scenarios of containment strategies with their impact on RTI prophylaxis trials that can serve as to inform go-no/ go decisions in clinical development.