A study conducted by researchers at Ohio State University and the New York University Grossman School of Medicine in the USA, has revealed that optimization of codon for bacterial expression causes the production of an inactive enzyme non-structural protein 12 (Nsp12), a catalytic subunit of RNA-dependent RNA polymerase of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The study is currently available on the bioRxiv* preprint server.
SARS-CoV-2, the causative pathogen of the coronavirus disease 2019 (COVID-19) pandemic, is a single-stranded, positive-sense RNA virus with a genome size of about 30 kb. In addition to encoding immunogenic structural proteins such as spike, membrane, nucleocapsid, and envelop proteins, the genome of SARS-CoV-2 encodes several non-structural proteins (Nsps) that are required for viral replication and gene expression. In a minimally active condition, the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 comprises Nsp12, Nsp7, and Nsp8.
In the current study, the scientists conducted an in-depth structural and functional analysis of SARS-CoV-2 RdRp.
The scientists used an E. Coli expression platform to purify essential proteins and evaluate the mechanistic details of RdRp. They observed that RdRp assembled with purified Nsp12 remains mostly inactive. With further analysis, they recognized that recoding of Nsp12 is the primary reason for this low level of RdRp activity.
While investigating the reason for low activity level, they observed that the enzymatic activity of Nsp7-Nsp8-Nsp12 remains very low on different templates, such as the optimal hairpin scaffold. After thoroughly investigating the purification process and reaction conditions, they removed the His10 tag that is attached with Nsp12 for purification. However, the removal of His10 tag did not increase the enzyme activity.
Furthermore, they hypothesized that the reduction in RdRp activity level may occur due to recombinant protein misfolding. Any alteration in the coding mRNA could be a potential cause of low enzymatic activity. During the production of Nsp12 using an E. Coli expression platform, the codon sequence of Nsp12 is generally altered to match the codon usage of the host. It is a routine practice despite the fact that even a single codon substitution can significantly alter the protein function.
To verify their hypothesis, they conducted a series of experiments and observed that RdRp assembled with active Nsp12 has a significantly higher activity level than that assembled with purified Nsp12. Regarding protein misfolding, they observed significant structural difference between active and purified Nsp12 in terms of accessibility to several domains of RdRp.
The team’s comparative analysis has revealed that there are two regions with rare codon clusters in the active Nsp12, but not in purified Nsp12. Rare codons are necessary to pause the ribosome and ensure proper protein folding. By constructing chimeric proteins by replacing these regions of active Nsp12 with corresponding regions of purified Nsp12, they observed that a chimeric protein with 350-435 codons derived from purified Nsp12 is nonfunctional.
These observations suggest that proper translation of the 350-435 region is a prerequisite for optimal Nsp12 activity and Nsp7-Nsp12 interaction, which in turn is essential for the proper functioning of RdRp.
With further structural analysis, the scientists noticed a variation between active and purified Nsp12 in the folding of the NiRAN domain, which is required for nucleotide binding. The scientists have thus suggested that this variation in protein folding might be responsible for the difference in active and purified Nsp12 activities. Using E. Coli strain with mutated ribosomal protein S12, they have observed that an attenuated translation can potentially facilitate the proper folding of Nsp12.
The study reveals that codon frequency optimization (recoding) of Nsp12 mRNA to support bacterial protein expression can lead to protein misfolding, which in turn can significantly reduce the activity of SARS-CoV-2 RdRp. The presence of rare codons in mRNAs is essential for protein folding.
In other words, the study suggests that translation of an under-optimized mRNA can lead to the generation of a highly active RdRp, and these findings are particularly valuable for functional studies, for the development of appropriate bacterial expression platforms, and more importantly, for the identification of novel RdRp inhibitors.
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.