A viral replicon system suited to the stable replication of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), without cytopathic effects, has been reported recently in a study appearing on the bioRxiv* preprint server.
Study: Stable Cell Clones Harboring Self-Replicating SARS-CoV-2 RNAs for Drug Screen. Image Credit: Corona Borealis Studio/ Shutterstock
Several cellular systems have been reported for the replication of SARS-CoV-2 without harming the cells, but the cell lines produced have been unstable and unsuited to a large-scale screening of large libraries of potential inhibitory compounds.
The current study reports a SARS-CoV-2 replicon system within stable cell lines that can be cultured with ease in a standard biosafety level 2 (BSL2) laboratory. This means they are amenable to high-throughput screening of large compound libraries.
This achievement represents a ground-breaking discovery that will greatly accelerate the pace of developing treatments for COVID-19.”
The virus in question has a 30 kb ribonucleic acid (RNA) genome, with a set of untranslated regions (3’ and 5’ UTR), 13 open reading frames, and a polyadenine (polyA) tail. It translates subgenomic RNAs (sgRNAs) to make its own ORFs, which encode the polyproteins, giving rise to the virus's structural and accessory proteins.
Importantly, there are also 16 non-structural proteins (NSPs) that make up the replication complex for the virus, allowing productive infection and escape from the host immune response.
The main viral protease (NSP5) and the RNA-dependent RNA-polymerase (RdRp, NSP 12) are among the top targets for antiviral drug development because they mediate cleavage of replicase polyproteins and virus replication, respectively. It would be very helpful to have a cell-based system that contains only viral replication and translation machinery to screen multiple viral inhibitors in parallel in a BSL2 setting.
To achieve this, researchers use subgenomic viral RNA molecules called replicons that replicate by themselves within cells but cannot give rise to infectious virions. In the case of SARS-CoV-2, this has not been accomplished hitherto because of the inherent cytotoxicity of the replicons of this virus.
The need for rapid detection of replication, the failure to produce cell banks that can ensure consistency between different lots, and the difficulty of scaling up these events for industrial processes mean that it is not practical to use unstable replicon systems for high-throughput screening (HTS) of thousands of compounds in one or more libraries.
In the current study, the derivation and characteristics of stable cell clones used for this purpose are described, and the potential for using this system in drug screening.
What did the study show?
The scientists first introduced modifications into the SARS-CoV-2 replicon, replacing the spike, membrane, and envelope genes and introducing a nanoluciferase reporter. This did not show persistent replication in any mammalian cell line.
The NSP1 is associated with severe loss of viability in human lung cell lines. The C-terminal end of this protein inserts into the messenger RNA (mRNA) entrance channel on the ribosome and thus prevents host or viral mRNA access, suppressing translation.
During this study, the researchers found that introducing K164A/H165A mutations into NSP1 led to the production of stable cell clones containing SARS-CoV-2 replicons. This agrees with the known structure of NSP1, which predicts a reduced interaction between the C-terminal end and the ribosome in the presence of this mutation, thus opening up the ribosomes to host mRNA. The outcome would be a less cytopathic effect due to NSP1 activity.
Conversely, the presence of R124S/K125E mutations may partially block the binding of the NSP1 N-terminal end to the 5'-untranslated region (5’-UTR) of viral mRNA. In this scenario, the C-terminal end remains bound to the ribosome, inhibiting access to the ribosome by viral or host mRNA.
N128S/K129E is a mutation without a published structure, but it is known to weaken the inhibition of interferons by SARS-CoV NSP1. This mutation was incompatible with cell viability in the presence of the replicon.
The presence of K164A/H165A mutations allowed stable cells to be recovered only with the baby hamster kidney (BHK-21) cell line, causing cytopathic effects in other cell types.
This finding has not been replicated with another replicon carrying the same mutation but with some differences. The researchers question if the presence of other mutations, in addition, in the cell lines used in the current study, led to stable replication.
The replicons were used to screen over 270 compounds, of which three were found to be inhibitory in replicon cells and human cell lines.
What are the implications?
The researchers claim to have established a cellular replicon system, harboring autonomously replicating SARS-CoV-2 RNAs, that is stable and suitable for high-throughput screening of antiviral drugs. This is the first such system and allows cells to be cultured, stored in banks, qualified, and used in a laboratory of certified BSL-2 standard for HTS drug screening.
Hitherto, antiviral screening was hindered by the absence of an efficient cell-based system such as the above.
This work establishes a robust, cell-based system for genetic and functional analyses of SARS-CoV-2 replication and for the development of antiviral drugs. This innovation will undoubtedly accelerate the pace of developing treatments for COVID-19.”
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.