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

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.

Videography supported adhesion, and proliferation behavior of MG-63 osteoblastic cells on 2.5D titania nanotube matrices.

Human osteosarcoma cells MG-63 were cultured on anodically etched titania nanotubes (TiO2 NT), with diameters ranging from 40-100 nm, to study the correlations between cell proliferation and adhesion on the 2.5 dimensional (2.5D) extracellular matrix (ECM). Unlike other reports, mostly based on mouse stem cells, and 2D cell culture, our studies indicate that the 2.5D NT promote higher proliferation and activity, but less 2D adhesion. Proliferation of the MG-63 cells was significantly higher in the NTs, the best being the 70 nm diameter sample, compared to planar titania (control). This is consistent with previous studies. However, cellular adhesion was stronger on TiO2 NT with increasing diameter, and highest on the control as obtained from shear stress measurement, paxilin imaging, and western blot measurements probing focal adhesion kinase, p130 CAS, and extracellular-regulated kinase, in addition to cell morphology imaging by fluorescence microscopy. We provide direct videography of cell migration, and cell speed data indicating faster filopodial activity on the TiO2 NT surfaces having lower adhesion. This evidence was not available previously. The NT matrices promote cells with smaller surface area, because of less 2D stretching. In contrast, on comparatively planar 2D-like surfaces uniaxial stretching of the cell body with strong anchoring of the filopodia, resulted in larger cell surface area, and demonstrated stronger adhesion. The difference in the results, with those previously published, may be generally attributed to, among others, the use of mouse stem cells (human osteosarcoma used here), and unannealed as-grown TiO2 NTs used previously (annealed ECMs used here).