Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells.

Here, we present a gene regulation strategy enabling programmable control over eukaryotic translational initiation. By excising the natural poly-adenylation (poly-A) signal of target genes and replacing it with a synthetic control region harboring RNA-binding protein (RBP)-specific aptamers, cap-dependent translation is rendered exclusively dependent on synthetic translation initiation factors (STIFs) containing different RBPs engineered to conditionally associate with different eIF4F-binding proteins (eIFBPs). This modular design framework facilitates the engineering of various gene switches and intracellular sensors responding to many user-defined trigger signals of interest, demonstrating tightly controlled, rapid and reversible regulation of transgene expression in mammalian cells as well as compatibility with various clinically applicable delivery routes of in vivo gene therapy. Therapeutic efficacy was demonstrated in two animal models. To exemplify disease treatments that require on-demand drug secretion, we show that a custom-designed gene switch triggered by the FDA-approved drug grazoprevir can effectively control insulin expression and restore glucose homeostasis in diabetic mice. For diseases that require instantaneous sense-and-response treatment programs, we create highly specific sensors for various subcellularly (mis)localized protein markers (such as cancer-related fusion proteins) and show that translation-based protein sensors can be used either alone or in combination with other cell-state classification strategies to create therapeutic biocomputers driving self-sufficient elimination of tumor cells in mice. This design strategy demonstrates unprecedented flexibility for translational regulation and could form the basis for a novel class of programmable gene therapies in vivo.

Cryo-EM analysis of the HCoV-229E spike glycoprotein reveals dynamic prefusion conformational changes.

Coronaviruses spike (S) glycoproteins mediate viral entry into host cells by binding to host receptors. However, how the S1 subunit undergoes conformational changes for receptor recognition has not been elucidated in Alphacoronavirus. Here, we report the cryo-EM structures of the HCoV-229E S trimer in prefusion state with two conformations. The activated conformation may pose the potential exposure of the S1-RBDs by decreasing of the interaction area between the S1-RBDs and the surrounding S1-NTDs and S1-RBDs compared to the closed conformation. Furthermore, structural comparison of our structures with the previously reported HCoV-229E S structure showed that the S trimers trended to open the S2 subunit from the closed conformation to open conformation, which could promote the transition from pre- to postfusion. Our results provide insights into the mechanisms involved in S glycoprotein-mediated Alphacoronavirus entry and have implications for vaccine and therapeutic antibody design.

Insight into the evolution of nidovirus endoribonuclease based on the finding that nsp15 from porcine Deltacoronavirus functions as a dimer.

Nidovirus endoribonucleases (NendoUs) include nonstructural protein 15 (nsp15) from coronaviruses and nsp11 from arteriviruses, both of which have been reported to participate in the viral replication process and in the evasion of the host immune system. Results from a previous study of coronaviruses SARS-CoV, HCoV-229E, and MHV nsp15 indicate that it mainly forms a functional hexamer, whereas nsp11 from the arterivirus PRRSV is a dimer. Here, we found that porcine Deltacoronavirus (PDCoV) nsp15 primarily exists as dimers and monomers in vitro Biological experiments reveal that a PDCoV nsp15 mutant lacking the first 27 amino acids of the N-terminal domain (Asn-1-Asn-27) forms more monomers and displays decreased enzymatic activity, indicating that this region is important for its dimerization. Moreover, multiple sequence alignments and three-dimensional structural analysis indicated that the C-terminal region (His-251-Val-261) of PDCoV nsp15 is 10 amino acids shorter and forms a shorter loop than that formed by the equivalent sequence (Gln-259-Phe-279) of SARS-CoV nsp15. This result may explain why PDCoV nsp15 failed to form hexamers. We speculate that NendoUs may have originated from XendoU endoribonucleases (XendoUs) forming monomers in eukaryotic cells, that NendoU from arterivirus gained the ability to form dimers, and that the coronavirus variants then evolved the capacity to assemble into hexamers. We further propose that PDCoV nsp15 may be an intermediate in this evolutionary process. Our findings provide a theoretical basis for improving our understanding of NendoU evolution and offer useful clues for designing drugs and vaccines against nidoviruses.