ABSTRACT
Infectious Bronchitis Virus (1BV) belongs to the y coronavirus, however, the function of IBV encoded endoribonuclease (non - structural protein 15, nsp15) has not been determined yet. To explore the function of nsp15 in the process of IBV replication, we mutated the IBV nsp15 endonuclease core residue His238 to Ala, constructed the nsp15-defective recombinant virus rIBV-nsp15-H238A, via in vitro ligation and recombination technology. Plaque assay and TCID50 were performed to measure virus titer, virus plaque size and growth curve. The IBV Beaudette-R genome was cloned as 5 fragments in vectors, BsaI or BsmBI restriction sites were added to the end of each fragment. After plasmid amplification, the cDNA fragments were obtained by enzymatic digestion, followed with in vitro ligation and transcription. Full - length genomic RNA was electroporated into Vero cells, together with N transcript, to rescue the recombinant viruses rIBV and rIBV - nsp15- H238A. Plaque assay was performed to detect and compare the viral titer and plaque size of these two recombinant viruses. Results showed that the virus titer of rIBV -nsp15 -H238A was 2.71x106PFU/mL, 3 times lower than that of rIBV (9.4x106PFU/mL). The plaque size of rIBV-nsp15-H238A was much smaller than that of rIBV, indicating that rIBV-nsp15-11238A replicates and spreads slower than rIBV. The growth curve of rIBV-nsp15-H238A was slower than that of rIBV. Our study demonstrates that nsp15 I-1238 is the key amino acid and plays an important role in the replication and spread of IBV. The construction of nsp15 defective recombinant virus provides a powerful tool for the study of the function of nsp15.
ABSTRACT
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.