The ongoing COVID-19 pandemic is caused by SARS-CoV-2, which is rapidly evolving with better transmissibility. Understanding the molecular basis of the SARS-CoV-2 interaction with host cells is of paramount significance, and development of antiviral agents provides new avenues to prevent and treat COVID-19 diseases. This study describes a molecular characterization of innate immune evasion mediated by the SARS-CoV-2 Nsp5 main protease and subsequent development of a small-molecule inhibitor.

Our studies offered simple and efficient methods to manipulate PRV.

Alphaherpesviruses that establish persistent infections rely partly on their ability to evade host antiviral responses, notably the type I interferon (IFN) response. However, the mechanisms employed by alphaherpesviruses to avoid this response are not well understood. Pseudorabies virus (PRV) is an economically important pathogen and a useful model system for studying alphaherpesvirus biology. To identify PRV proteins that antagonize type I IFN signaling, we performed a screen by using an IFN-stimulated response element reporter in the swine cell line CRL. Unexpectedly, we identified the dUTPase UL50 as a strong inhibitor. We confirmed that UL50 has the ability to inhibit type I IFN signaling by performing ectopic expression of UL50 in cells and deletion of UL50 in PRV. Mechanistically, UL50 impeded type I IFN-induced STAT1 phosphorylation, likely by accelerating lysosomal degradation of IFN receptor 1 (IFNAR1). In addition, this UL50 activity was independent of its dUTPase activity and required amino acids 225 to 253 in the C-terminal region. The UL50 encoded by herpes simplex virus 1 (HSV-1) also possessed similar activity. Moreover, UL50-deleted PRV was more susceptible to IFN than UL50-proficient PRV. Our results suggest that in addition to its dUTPase activity, the UL50 protein of alphaherpesviruses possesses the ability to suppress type I IFN signaling by promoting lysosomal degradation of IFNAR1, thereby contributing to immune evasion. This finding reveals UL50 as a potential antiviral target. Alphaherpesviruses can establish lifelong infections and cause many diseases in humans and animals. Pseudorabies virus (PRV) is a swine alphaherpesvirus that threatens pig production. Using PRV as a model, we found that this alphaherpesvirus could utilize its encoded dUTPase UL50 to induce IFNAR1 degradation and inhibit type I IFN signaling in an enzymatic activity-independent manner. Our finding reveals a mechanism employed by an alphaherpesvirus to evade the immune response and indicates that UL50 is an important viral protein in pathogenesis and is a potential target for antiviral drug development.

Type I interferon response plays a prominent role against viral infection, which is frequently disrupted by viruses. Here, we report Bcl-2 associated transcription factor 1 (Bclaf1) is degraded during the alphaherpesvirus Pseudorabies virus (PRV) and Herpes simplex virus type 1 (HSV-1) infections through the viral protein US3. We further reveal that Bclaf1 functions critically in type I interferon signaling. Knockdown or knockout of Bclaf1 in cells significantly impairs interferon-α (IFNα) -mediated gene transcription and viral inhibition against US3 deficient PRV and HSV-1. Mechanistically, Bclaf1 maintains a mechanism allowing STAT1 and STAT2 to be efficiently phosphorylated in response to IFNα, and more importantly, facilitates IFN-stimulated gene factor 3 (ISGF3) binding with IFN-stimulated response elements (ISRE) for efficient gene transcription by directly interacting with ISRE and STAT2. Our studies establish the importance of Bclaf1 in IFNα-induced antiviral immunity and in the control of viral infections.

During COVID-19 pandemic, mutations of SARS-CoV-2 produce new strains that can be more infectious or evade vaccines. Viral RNA mutations can arise from misincorporation by RNA-polymerases and modification by host factors. Analysis of SARS-CoV-2 sequence from patients showed a strong bias toward C-to-U mutation, suggesting a potential mutational role by host APOBEC cytosine deaminases that possess broad anti-viral activity. We report the first experimental evidence demonstrating that APOBEC3A, APOBEC1, and APOBEC3G can edit on specific sites of SARS-CoV-2 RNA to produce C-to-U mutations. However, SARS-CoV-2 replication and viral progeny production in Caco-2 cells are not inhibited by the expression of these APOBECs. Instead, expression of wild-type APOBEC3 greatly promotes viral replication/propagation, suggesting that SARS-CoV-2 utilizes the APOBEC-mediated mutations for fitness and evolution. Unlike the random mutations, this study suggests the predictability of all possible viral genome mutations by these APOBECs based on the UC/AC motifs and the viral genomic RNA structure.