Optimization of 5′UTR to evade SARS-CoV-2 Nonstructural protein 1-directed inhibition of protein synthesis in cells

Abstract

Maximizing the expression level of therapeutic proteins in cells is the general goal for DNA/mRNA therapies. It is particularly challenging to achieve efficient protein expression in the cellular contexts with inhibited translation machineries, such as in the presence of cellular Nonstructural protein 1 (Nsp1) of coronaviruses (CoVs) that has been reported to inhibit overall protein synthesis of host genes and exogenously delivered mRNAs/DNAs. In this study, we thoroughly examined the sequence and structure contexts of viral and non-viral 5′UTRs that determine the protein expression levels of exogenously delivered DNAs and mRNAs in cells expressing SARS-CoV-2 Nsp1. It was found that high 5′-proximal A/U content promotes an escape from Nsp1-directed inhibition of protein synthesis and results in selective protein expression. Furthermore, 5′-proximal Cs were found to significantly enhance the protein expression in an Nsp1-dependent manner, while Gs located at a specific window close to the 5′-end counteract such enhancement. The distinct protein expression levels resulted from different 5′UTRs were found correlated to Nsp1-induced mRNA degradations. These findings ultimately enabled rational designs for optimized 5′UTRs that lead to strong expression of exogenous proteins regardless of the translationally repressive Nsp1. On the other hand, we have also identified several 5′-proximal sequences derived from host genes that are capable of mediating the escapes. These results provided novel perspectives to the optimizations of 5′UTRs for DNA/mRNA therapies and/or vaccinations, as well as shedding light on the potential host escapees from Nsp1-directed translational shutoffs. Key points • The 5′-proximal SL1 and 5a/b derived from SARS-CoV-2 genomic RNA promote exogenous protein synthesis in cells expressing Nsp1 comparing with non-specific 5′UTRs. • Specific 5′-proximal sequence contexts are the key determinants of the escapes from Nsp1-directed translational repression and thereby enhance protein expressions. • Systematic mutagenesis identified optimized 5′UTRs that strongly enhance protein expression and promote resistance to Nsp1-induced translational repression and RNA degradation. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-023-12442-2.

The differential effect of SARS-COV-2 NSP1 on mRNA translation and stability reveals new insights linking ribosome recruitment, codon usage and virus evolution

“…Another unresolved question is how SARS-Cov-2 mRNAs escape the inhibitory activity of NSP1. The presence of a cap-proximal stem-loop (SL1) in the 5’ leader (5’ L) of genomic and subgenomic SARS-CoV-2 mRNAs has been reported as essential to escape the inhibitory effect of NSP1 25,34,35,37–39 . Although a proven mechanistic model for this resistance is still lacking, some authors have proposed that binding of SL1 to N-terminal domain would displace NSP1 from the mRNA entry channel, allowing the attachment of viral 5’ L to 43S-PIC 40 .…”

“…Another unresolved question is how SARS-Cov-2 mRNAs escape the inhibitory activity of NSP1. The presence of a cap-proximal stem-loop (SL1) in the 5' leader (5' L) of genomic and subgenomic SARS-CoV-2 mRNAs has been reported as essential to escape the inhibitory effect of NSP1 25,34,35,[37][38][39] .…”

Tertiary folds of the SL5 RNA from the 5′ proximal region of SARS-CoV-2 and related coronaviruses

“…The 5’ proximal region of the SARS-CoV-2 genome is a highly structured and functionally important genomic locus (27, 41, 45, 46). This region is divided into secondary structure domains termed “stem-loops,” with multiple stem-loops predicted to be present across the coronavirus family (47, 48) or within a specific genus (47, 49, 50).…”

“…On the other hand, to date, only a few small fragments of the RNA genomestem-loop 1 in complex with non-structural protein 1; stem-loop 2; and stem-loop 4 from the 5'UTR; the frameshift stimulation element; and the stem-loop 2 motif from the 3'UTRhave been characterized in three dimensions (28)(29)(30)(31)(32)(33)(34)(35). While current designs of RNA-targeting therapeutics are often based on the two-dimensional base-pairing pattern, known as RNA secondary structure (29,(36)(37)(38)(39)(40)(41), three-dimensional (3D) structures are necessary for structure-guided design of many classes of therapeutics (21,40,(42)(43)(44).…”

Tertiary folds of the SL5 RNA from the 5′ proximal region of SARS-CoV-2 and related coronaviruses

“…The 5′ proximal region of the SARS-CoV-2 genome is a highly structured and functionally important genomic locus ( 24 , 25 ). This region is divided into secondary structure domains termed “stem-loops,” with multiple stem-loops predicted to be present in the 5′ proximal region across the coronavirus family ( 25 – 27 ).…”