Advances in high-throughput methods for the identification of virus receptors

Fig. 1. Schematic representation of RNAi silencing mechanism. For shRNA LOF screening, a lentiviral vector or plasmid encoding the shRNA (blue) is first integrated into the cellular DNA (green). The DNA is then transcribed by RNA polymerase III into shRNA and the shRNA exported into the cytoplasm by Exportin 5. For dsRNA and siRNA LOF screening, the interfering RNA is directly transfected into the cells. Both shRNAs and dsRNAs are processed in the cytoplasm by Dicer into siRNAs. The siRNAs are then loaded into the RISC and the passenger strand removed from the guide strand. The guide strand can then direct the RISC to the complementary mRNA target, where RISC cleaves the mRNA, leading to its degradation

Fig. 2. Schematic representation of CRISPR/Cas9, CRISPR/Cas9 nickase, and CRISPR/Cas9 FOKI genetic knockout mechanism. CRISPR/Cas9 genetic knockout: the CRISPR/Cas9 complex is first formed by combining an sgRNA and a Cas9 endonuclease. The sgRNA then guides the CRISPR/Cas9 complex to a specific region of the genomic DNA, and a double-stranded DNA break is induced by the Cas9 endonuclease. The double-stranded DNA break is then repaired non-homologously by the host cell, leading to the formation of insertions or deletions, which disrupt the open reading frame of the gene. CRISPR/Cas9 nickase genetic knockout: two CRISPR/Cas9 nickase complexes are formed with two separated sgRNAs and mutated Cas9 nickase endonucleases. The sgRNAs then guide the CRISPR/Cas9 complexes to specific regions of the genomic DNA, and two single-stranded DNA breaks are induced by the Cas9 endonucleases, forming a staggered double-stranded break. The double-stranded DNA break is then repaired non-homologously by the host cell. CRISPR/Cas9 FOKI genetic knockout: Two CRISPR/Cas9 FOKI complexes are formed with two separated sgRNAs and mutated dead Cas9 fused with the endonuclease FOKI. The sgRNAs then guide the…

Fig. 3. AP–MS and XL-MS workflow comparison. AP–MS workflow: cells are first infected with the virus of interest, harvested, and lysed. The virus–receptor complexes are then isolated using affinity purification, and the purified proteins enzymatically digested and identified using LC–MS/MS and database searching. In XL-MS, the cells are also first infected with virus. However, a chemical cross-linking reaction is then performed, before the harvesting and lysis of the host cells. Next, the proteins are enzymatically digested and identified using LC–MS/MS and database searching