Translation of in vitro-transcribed RNA therapeutics.

In vitro transcribed, modified messenger RNAs (IVTmRNAs) have been used to vaccinate billions of individuals against the SARS-CoV-2 virus, and are currently being developed for many additional therapeutic applications. IVTmRNAs must be translated into proteins with therapeutic activity by the same cellular machinery that also translates native endogenous transcripts. However, different genesis pathways and routes of entry into target cells as well as the presence of modified nucleotides mean that the way in which IVTmRNAs engage with the translational machinery, and the efficiency with which they are being translated, differs from native mRNAs. This review summarises our current knowledge of commonalities and differences in translation between IVTmRNAs and cellular mRNAs, which is key for the development of future design strategies that can generate IVTmRNAs with improved activity in therapeutic applications.

Abracadabra, One Becomes Two: The Importance of Context in Viral -1 Programmed Ribosomal Frameshifting.

The constrained nature of viral genomes has allowed a translational sleight of hand known as -1 Programmed Ribosomal Frameshifting (-1 PRF) to flourish. Numerous studies have sought to tease apart the mechanisms and implications of -1PRF utilizing a few techniques. The dual-luciferase assay and ribosomal profiling have driven the PRF field to make great advances; however, the use of these assays means that the full impact of the genomic and cellular context on -1 PRF is often lost. Here, we discuss how the Minimal Frameshifting Element (MFE) and its constraints can hide contextual effects on -1 PRF. We review how sequence elements proximal to the traditionally defined MFE, such as the coronavirus attenuator sequence, can affect the observed rates of -1 PRF. Further, the MFE-based approach fully obscured -1 PRF in Barley yellow dwarf virus and would render the exploration of -1 PRF difficult in Porcine reproductive and respiratory syndrome virus, Encephalomyocarditis virus, Theiler's murine encephalomyelitis virus, and Sindbis virus. Finally, we examine how the cellular context of tRNA abundance, miRNAs, and immune response elements can affect -1 PRF. The use of MFE was instrumental in establishing the basic foundations of PRF; however, it has become clear that the contextual impact on -1 PRF is no longer the exception so much as it is the rule and argues for new approaches to study -1PRF that embrace context. We therefore urge our field to expand the strategies and methods used to explore -1 PRF.

A Structurally Conserved RNA Element within SARS-CoV-2 ORF1a RNA and S mRNA Regulates Translation in Response to Viral S Protein-Induced Signaling in Human Lung Cells.

The positive-sense, single-stranded RNA genome SARS-CoV-2 harbors functionally important cis-acting elements governing critical aspects of viral gene expression. However, insights on how these elements sense various signals from the host cell and regulate viral protein synthesis are lacking. Here, we identified two novel cis-regulatory elements in SARS-CoV-2 ORF1a and S RNAs and describe their role in translational control of SARS-CoV-2. These elements are sequence-unrelated but form conserved hairpin structures (validated by NMR) resembling gamma activated inhibitor of translation (GAIT) elements that are found in a cohort of human mRNAs directing translational suppression in myeloid cells in response to IFN-γ. Our studies show that treatment of human lung cells with receptor-binding S1 subunit, S protein pseudotyped lentivirus, and S protein-containing virus-like particles triggers a signaling pathway involving DAP-kinase1 that leads to phosphorylation and release of the ribosomal protein L13a from the large ribosomal subunit. Released L13a forms a virus activated inhibitor of translation (VAIT) complex that binds to ORF1a and S VAIT elements, causing translational silencing. Translational silencing requires extracellular S protein (and its interaction with host ACE2 receptor), but not its intracellular synthesis. RNA-protein interaction analyses and in vitro translation experiments showed that GAIT and VAIT elements do not compete with each other, highlighting differences between the two pathways. Sequence alignments of SARS-CoV-2 genomes showed a high level of conservation of VAIT elements, suggesting their functional importance. This VAIT-mediated translational control mechanism of SARS-CoV-2 may provide novel targets for small molecule intervention and/or facilitate development of more effective mRNA vaccines. IMPORTANCE Specific RNA elements in the genomes of RNA viruses play important roles in host-virus interaction. For SARS-CoV-2, the mechanistic insights on how these RNA elements could sense the signals from the host cell are lacking. Here we report a novel relationship between the GAIT-like SARS-CoV-2 RNA element (called VAITs) and the signal generated from the host cell. We show that for SARS-CoV-2, the interaction of spike protein with ACE2 not only serves the purpose for viral entry into the host cell, but also transduces signals that culminate into the phosphorylation and the release of L13a from the large ribosomal subunit. We also show that this event leads to the translational arrest of ORF1a and S mRNAs in a manner dependent on the structure of the RNA elements. Translational control of viral mRNA by a host-cell generated signal triggered by viral protein is a new paradigm in the host-virus relationship.

SARS-CoV-2 Codon Usage Bias Downregulates Host Expressed Genes With Similar Codon Usage.

Severe acute respiratory syndrome has spread quickly throughout the world and was declared a pandemic by the World Health Organization (WHO). The pathogenic agent is a new coronavirus (SARS-CoV-2) that infects pulmonary cells with great effectiveness. In this study we focus on the codon composition for the viral protein synthesis and its relationship with the protein synthesis of the host. Our analysis reveals that SARS-CoV-2 preferred codons have poor representation of G or C nucleotides in the third position, a characteristic which could result in an unbalance in the tRNAs pools of the infected cells with serious implications in host protein synthesis. By integrating this observation with proteomic data from infected cells, we observe a reduced translation rate of host proteins associated with highly expressed genes and that they share the codon usage bias of the virus. The functional analysis of these genes suggests that this mechanism of epistasis can contribute to understanding how this virus evades the immune response and the etiology of some deleterious collateral effect as a result of the viral replication. In this manner, our finding contributes to the understanding of the SARS-CoV-2 pathogeny and could be useful for the design of a vaccine based on the live attenuated strategy.