Inducible CRISPR activation screen for interferon-stimulated genes identifies OAS1 as a SARS-CoV-2 restriction factor.

Interferons establish an antiviral state through the induction of hundreds of interferon-stimulated genes (ISGs). The mechanisms and viral specificities for most ISGs remain incompletely understood. To enable high-throughput interrogation of ISG antiviral functions in pooled genetic screens while mitigating potentially confounding effects of endogenous interferon and antiproliferative/proapoptotic ISG activities, we adapted a CRISPR-activation (CRISPRa) system for inducible ISG expression in isogenic cell lines with and without the capacity to respond to interferons. We used this platform to screen for ISGs that restrict SARS-CoV-2. Results included ISGs previously described to restrict SARS-CoV-2 and novel candidate antiviral factors. We validated a subset of these by complementary CRISPRa and cDNA expression experiments. OAS1, a top-ranked hit across multiple screens, exhibited strong antiviral effects against SARS-CoV-2, which required OAS1 catalytic activity. These studies demonstrate a high-throughput approach to assess antiviral functions within the ISG repertoire, exemplified by identification of multiple SARS-CoV-2 restriction factors.

Isofunctional Clustering and Conformational Analysis of the Arsenate Reductase Superfamily Reveals Nine Distinct Clusters.

Arsenate reductase (ArsC) is a superfamily of enzymes that reduce arsenate. Due to active site similarities, some ArsC can function as low-molecular weight protein tyrosine phosphatases (LMW-PTPs). Broad superfamily classifications align with redox partners (Trx- or Grx-linked). To understand this superfamily's mechanistic diversity, the ArsC superfamily is classified on the basis of active site features utilizing the tools TuLIP (two-level iterative clustering process) and autoMISST (automated multilevel iterative sequence searching technique). This approach identified nine functionally relevant (perhaps isofunctional) protein groups. Five groups exhibit distinct ArsC mechanisms. Three are Grx-linked: group 4AA (classical ArsC), group 3AAA (YffB-like), and group 5BAA. Two are Trx-linked: groups 6AAAAA and 7AAAAAAAA. One is an Spx-like transcriptional regulatory group, group 5AAA. Three are potential LMW-PTP groups: groups 7BAAAA, and 7AAAABAA, which have not been previously identified, and the well-studied LMW-PTP family group 8AAA. Molecular dynamics simulations were utilized to explore functional site details. In several families, we confirm and add detail to literature-based mechanistic information. Mechanistic roles are hypothesized for conserved active site residues in several families. In three families, simulations of the unliganded structure sample specific conformational ensembles, which are proposed to represent either a more ligand-binding-competent conformation or a pathway toward a more binding-competent state; these active sites may be designed to traverse high-energy barriers to the lower-energy conformations necessary to more readily bind ligands. This more detailed biochemical understanding of ArsC and ArsC-like PTP mechanisms opens possibilities for further understanding of arsenate bioremediation and the LMW-PTP mechanism.