Project description:The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development.
Project description:The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development.
Project description:The continuous evolution of SARS-CoV-2 poses global health challenges. A safe, rapid, and versatile method for assessing functions of Spike protein mutations in ACE2 receptor binding and immune evasion would be highly valuable. To address this, we engineered a transcription- and replication-competent virus-like particle (trVLP) derived from the Sindbis virus, pseudotyped with the SARS-CoV-2 receptor binding domain (RBD). This trVLP exclusively propagates in BHK-21 cell engineered to express both RNA replicase and human ACE2, providing a controllable, safe model of SARS-CoV-2 RBD-ACE2 interaction mediated virus entry. The system enables characterization of RBD interactions with ACE2 from various mammalian hosts, demonstrating its utility for studying host-virus interactions. By leveraging the evolutionary capability of trVLP mediated by error-prone RNA replication, we screened for RBD variants that evade the antibody-mediated inhibition of cell entry. Together, these findings underscore the utility of the trVLP as a safe, rapid, and flexible platform for dissecting SARS-CoV-2 RBD evolution and identifying key adaptive mutations with implications for surveillance and countermeasure development.
Project description:To determine whether SUD2core influences the cellular response to SARS-CoV-2 trVLP, bulk RNA sequencing on control (empty vector expressed) and SUD2core-expression of dHL-60 cells following SARS-CoV-2 trVLP infection were performed.
Project description:To determine whether HEBP2 influences the cellular response to SARS-CoV-2 trVLP, bulk RNA sequencing (RNA-seq) on control (scramble gRNA-transfected) and HEBP2-knockout of dHL-60 cells following SARS-CoV-2 trVLP infection were performed.
Project description:Deep mutational scanning (DMS) makes it possible to perform massively parallel quantification of the relationship between genetic variants and phenotypes of interest. However, the difficulties in introducing large variant libraries into mammalian cells greatly hinder DMS under physiological states. Here we developed two novel strategies for DMS library construction in mammalian cells, namely ‘piggyBac-in-vitro ligation’ and ‘piggyBac-in-vitro ligation-PCR’. For the first strategy, we took the ‘in-vitro ligation’ approach to prepare high-diversity linear dsDNAs, and integrate them into the mammalian genome with a piggyBac transposon system. For the second strategy, we further added a PCR step using the in-vitro ligation dsDNAs as templates, for the construction of high-content genome-integrated libraries via large-scale transfection. Both strategies could successfully establish genome-integrated EGFP-chromophore randomized libraries in HEK293T cells and enrich the green fluorescence-chromophore amino acid sequences. And we further identified a novel transcriptional activator peptide with the ‘piggyBac-in-vitro ligation-PCR’ strategy. Our novel strategies greatly facilitate the construction of large variant DMS library in mammalian cells, and may have great application potential in the future.
Project description:To delineate the native structure of SF3A3 5'UTR, RNA was harvested from IMR90 human fibroblasts. Using specific primers and DMS-MaPSeq pipeline, we validated individual base pairing probabilities within the endogenous 5'UTR of SF3A3 (samples described as 'in vivo' transcribed). DMS-MaP-Seq is based on the principle that DMS is highly reactive to solvent-accessible, unpaired adenine (A) and cytosine (C) residues, but remains inert toward base-paired A and C engaged in Watson-Crick interactions (Rouskin et al., 2014). Using this methodology, we identify stable stem-loop structure (SL3) positioned within SF3A3 5'UTR. To further validate the functional importance of SL3, the structural point mutant (SF3A3 5'UTR mut: A55C and U95A) and rescue (SF3A3 5'UTR res: A55C and U95A and rescuing point mutations G61U and U100G) sequences of SF3A3 5'UTR were cloned into the reporter plasmid. For the validation of these mutate-and-rescue constructs, plasmids were in vitro transcribed and either used directly (samples described as 'in vitro') for DMS-MaP-Seq probing.
Project description:Dimethyl sulfate (DMS) is a methylating reagent that has long been used to detect footprints of DNA-bound proteins in vitro as well as in vivo. Here we describe DMS-seq for in vivo genome-wide mapping of protein-DNA interactions. DMS-seq exploits the cell-permeable nature of DMS to obviate the need for nuclear isolation, thereby simplifying the process to detect binding sites of transcription factors. Furthermore, we found that DMS preferentially attacks nucleosome centers in vivo, evidencing for DMS-seq as a first method that locates them without using genetically-modified histones and is hence applicable to any eukaryote. DMS-seq should be a simple and unique method in epigenomics.