UC Davis

Most virus families produce double-stranded (ds) RNA during their lifecycles, which can be sensed by multiple dsRNA-dependent innate immune sensors, including two potent host restriction factors OAS/RNase L and PKR. PKR mainly restricts replication of viruses that are dependent on eIF2, and RNase L inhibits viral replication via degradation of host and viral RNAs. However, while PKR is directly activated by dsRNA, RNase L activation depends on the OAS to both bind dsRNA and synthesize 2’-5’ oligoadenylate (2-5A). We hypothesized that engineering proteins to combine the dsRNA sensor domain of PKR with the effector domain of RNase L would bypass the need for OAS activation, making them less susceptible to inhibition by viral molecules, and preserve their potent antiviral activity. To test this, we generated Recombinant Enhanced Antiviral Restrictors (REAVRs) by combining dsRNA-binding domains of PKR from different species with the effector domain of human RNase L. We show that REAVRs led to RNA degradation and decreased the activity of a luciferase reporter, suggesting the REAVRs are functionally active. To investigate whether REAVRs could restrict viral replication, we generated T-REx 293 cells containing a single REAVR copy under the control of a doxycycline-inducible promoter. Some REAVRs exerted potent antiviral activities against five tested viruses: vaccinia virus, dengue virus, Zika virus, SARS-CoV-2, and vesicular stomatitis virus. Importantly, these REAVRs were also effective against viruses that are resistant to PKR activation, for example, SARS CoV-2 and flaviviruses. This study provides proof-of-concept that REAVRs are active in vitro and exhibit antiviral effects on various families of viruses. We envision that REAVRs can be used to generate transgenic organisms with increased viral resistance and represent promising candidates as therapeutics for viral infections.