Porcine Epidemic Diarrhea Virus Infection Induces Caspase-8-Mediated G3BP1 Cleavage and Subverts Stress Granules To Promote Viral Replication.

Porcine epidemic diarrhea virus (PEDV) is an α-coronavirus causing severe diarrhea and high mortality rates in suckling piglets and posing significant economic impact. PEDV replication is completed and results in a large amount of RNA in the cytoplasm. Stress granules (SGs) are dynamic cytosolic RNA granules formed under various stress conditions, including viral infections. Several previous studies suggested that SGs were involved in the antiviral activity of host cells to limit viral propagation. However, the underlying mechanisms are poorly understood. This study aimed to delineate the molecular mechanisms regulating the SG response to PEDV infection. SG formation is induced early during PEDV infection, but as infection proceeds, this ability is lost and SGs disappear at late stages of infection (>18 h postinfection). PEDV infection resulted in the cleavage of Ras-GTPase-activating protein-binding protein 1 (G3BP1) mediated by caspase-8. Using mutational analysis, the PEDV-induced cleavage site within G3BP1 was identified, which differed from the 3C protease cleavage site previously identified. Furthermore, G3BP1 cleavage by caspase-8 at D168 and D169 was confirmed in vitro as well as in vivo The overexpression of cleavage-resistant G3BP1 conferred persistent SG formation and suppression of viral replication. Additionally, the knockdown of endogenous G3BP1 abolished SG formation and potentiated viral replication. Taken together, these data provide new insights into novel strategies in which PEDV limits the host stress response and antiviral responses and indicate that caspase-8-mediated G3BP1 cleavage is important in the failure of host defense against PEDV infection.IMPORTANCE Coronaviruses (CoVs) are drawing extensive attention again since the outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019. CoVs are prone to variation and own the transmission capability by crossing the species barrier resulting in reemergence. How CoVs manipulate the antiviral responses of their hosts needs to be explored. Overall, the study provides new insight into how porcine epidemic diarrhea virus (PEDV) impaired SG assembly by targeting G3BP1 via the host proteinase caspase-8. These findings enhanced the understanding of PEDV infection and might help identify new antiviral targets that could inhibit viral replication and limit the pathogenesis of PEDV.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.

Tightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and assemble within viral factories, dynamic compartments formed within the host cells associated with human stress granules. Here, we test the possibility that the multivalent RNA-binding nucleocapsid protein (N) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) condenses with RNA via liquid-liquid phase separation (LLPS) and that N protein can be recruited in phase-separated forms of human RNA-binding proteins associated with SG formation. Robust LLPS with RNA requires two intrinsically disordered regions (IDRs), the N-terminal IDR and central-linker IDR, as well as the folded C-terminal oligomerization domain, while the folded N-terminal domain and the C-terminal IDR are not required. N protein phase separation is induced by addition of non-specific RNA. In addition, N partitions in vitro into phase-separated forms of full-length human hnRNPs (TDP-43, FUS, hnRNPA2) and their low-complexity domains (LCs). These results provide a potential mechanism for the role of N in SARS-CoV-2 viral genome packing and in host-protein co-opting necessary for viral replication and infectivity.