Messenger RNA translation enhancement by immune evasion proteins: a comparative study between EKB (vaccinia virus) and NS1 (influenza A virus)

Yi Liu,Kyle K. L. Phua,Jas Min Chin,En Lin Choo

doi:10.1038/s41598-019-48559-6

Yi Liu, Kyle K. L. Phua + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-48559-6

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Abstract

In this study, we compared vaccinia virus derived monofunctional E3, K3 and B18R (also known as EKB) with influenza A virus derived multifunctional non-structural protein 1 (NS1) based on their ability to enhance mRNA translation. EKB and NS1-TX91 were all found to enhance mRNA translation and suppress interferon production, yet level of enhancement by EKB was much lower than NS1-TX91. Similarly, greater luciferase expression was mediated by co-delivery of unmodified luciferase with NS1 mRNA, compared to co-delivery of unmodified luciferase with either E3, K3 or B18R mRNA, respectively. Different combinations of E3, K3 and/or B18R mRNA were mixed with NS1-TX91 mRNA at varying ratios and co-delivered with luciferase mRNA. However, no synergism was observed as mRNA translation enhancement mediated by NS1-TX91 could not be improved by the inclusion EKB in all tested combinations. Lastly, it was found that E3 was able to rescue mRNA translation enhancement mediated by NS1 PKR knockout mutant (PR8PKR−), suggesting that one of NS1’s multiple immune evasion mechanisms overlapped with E3. Altogether, our data validated mRNA translation enhancement mediated by immune evasion proteins (EKB and NS1) and showed that the multifunctional nature of NS1 accounted for its superior performance.

Highlights

MRNA therapeutics has demonstrated great potential in recent clinical trials and preclinical studies[1,2]
In order to confirm that mRNA translation could be enhanced by EKB, as well as to compare their enhancement with that led by non-structural protein 1 (NS1)-TX91, human foreskin fibroblasts (BJ fibroblasts) and human hepatocellular carcinoma cells (HepG2) were pre-transfected with pseudouridine (ψ) modified mRNA encoding E3, K3, B18R, NS1-TX91 or green florescence protein (GFP) as a control, followed by transfection with unmodified luciferase mRNA 6 h later
To evaluate the capability of EKB and NS1-TX91 to enhance mRNA translation by co-delivery, mRNA encoding each of these immune evasion proteins was transfected together with luciferase mRNA at three different ratios to HepG2 cells

Summary

Introduction

MRNA therapeutics has demonstrated great potential in recent clinical trials and preclinical studies[1,2]. Type I interferon (IFN) is produced, leading to strengthened antiviral responses and detrimental effects including RNA degradation, global protein synthesis shutdown and cytotoxicity, all of which greatly affect mRNA’s translation capacity and reduce its biosafety[9]. By co-delivery of mRNA encoding immune evasion proteins and mRNA-of-interest, immune responses triggered during transfection are effectively suppressed, and translation of mRNA-of-interest is greatly enhanced. Such applications have been reported using vaccinia virus derived E3, K3, and B18R (EKB)[10] and influenza A virus derived NS111,12. It inhibits immune-related proteins such as PKR, OAS, interferon regulatory factor 3 (IRF3) and NF-κB, www.nature.com/scientificreports/. NS1’s binding and inhibition of CPSF30 leads to accumulation of unprocessed pre-mRNA inside nucleus and disrupts global host gene expression, including expression of hundreds of IFN-stimulated genes (ISGs)[15]

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