Eluding innate immune responses by IBV: role of non-structural protein 15

Upon virus infection, cells of the host develop various anti-viral defenses while viruses in turn have evolved strategies to antagonize the host defenses. This arms race between the viral antagonistic strategies and the host anti-viral defenses determines the final outcome of a virus infection. Knowledge on this virus-host arms race is crucial to understand the viral pathogenicity, and to develop anti-viral therapeutics. Coronaviruses (CoVs) are a group of RNA viruses that encode structural, non-structural and accessory proteins. The non-structural and accessory proteins, although not being part of the viral particles, usually play important roles in the aforementioned virus-host arms race. Infectious Bronchitis, caused by γ-CoV Infectious Bronchitis Virus (IBV), is an economically important disease of chickens and other fowl. IBV targets the upper respiratory tract causing near complete morbidity and high rates of mortality. Type I interferon plays a crucial role in fighting the virus infection and regulating the immune system of the host. It was previously reported that IBV resists the activity of type I interferon and suggests an important role of the four accessory proteins (3a, 3b, 5a, 5b). However, the roles of the non-structural proteins of IBV still remained to be addressed.   

Non-structural protein (nsp)15 of CoVs is an endoribonuclease (EndoU) that catalyzes the breakdown of RNAs. The role of the nsp15 EndoU of IBV in the IBV-host arms race was not investigated before. In this thesis, we describe for the first time the role of nsp15 EndoU in IBV’s antagonism against interferon-β (IFN-β) activity, as well as the activation of the protein kinase R (PKR)-pathway and the formation of stress granules (SGs). Following transfection of a plasmid encoding IBV nsp15, we demonstrate that the overexpressed IBV nsp15 inhibits global protein synthesis in the transfected cells. The EndoU activity of IBV nsp15 appears indispensable for the above-described inhibitory effect, implying that nsp15 EndoU targets cellular substrates. Suppression of host translation machinery is usually fine-tuned by the virus to avoid interference with the synthesis of viral proteins. In this thesis we also investigate how the role of nsp15 EndoU is fine-tuned during IBV infection to the advantage of IBV replication.

In chapter 1, we generally introduce CoVs and particularly IBV, the induction of host anti-viral defenses, and the viral strategies to antagonize these host defenses. In chapter 2, we describe an improved protocol of CoV reverse genetics, via which a nsp15 EndoU-deficient recombinant mutant IBV was successfully retrieved. In chapter 3, we demonstrate that, compared to the wild type IBV, the EndoU-deficient rIBV-nsp15H238A generated more double stranded RNA (dsRNA), a molecule generated during IBV infection that is able to induce host defenses. Accordingly, the rIBV-nsp15H238A induced stronger dsRNA-induced anti-viral responses including IFN-β induction and PKR phosphorylation-mediated stress granules (SGs) formation. In chapter 4, we show that the overexpressed IBV nsp15 obtained by transfection inhibits the global protein synthesis in the transfected cells, for which the EndoU activity is indispensable. We discovered that the above-mentioned inhibition of global protein synthesis is via a cytoplasmic poly (A) binding protein (PABPC1) nuclear localization-involved mechanism. In addition, the above inhibition of global protein synthesis is conserved across nsp15 EndoU of α-CoVs (PEDV, TGEV) and β-CoVs (SARS-CoV-1, MERS-CoV, SARS-CoV-2). Together, via overexpressing nsp15 EndoU alone, we demonstrate that nsp15 EndoU of γ-CoV IBV, α-CoVs and β-CoVs, inhibits protein synthesis in the transfected cells. This suggests, for the first time, that nsp15 EndoU of CoVs targets cellular substrates of the host. In chapter 5, we show that both the wild type IBV and the EndoU-deficient rIBV-nsp15H238A induce host shutoff, albeit via different mechanisms. The rIBV-nsp15H238A-induced host shutoff does not benefit viral replication as it does not restrict the capacity for SGs formation or production of IFN-β. In chapter 4, we showed that IBV nsp15 EndoU inhibits protein synthesis in cells via a mechanism related to a nuclear localization of PABPC1. The IBV nsp15 EndoU-induced nuclear localization of PABPC1 was not detected after the wild type IBV infection, suggesting that IBV nsp15’s inhibitory effect of global protein synthesis in the host cells may be fine-tuned during IBV infection. Previous reports showed that PABPC1 is likely involved in CoV replication, and we therefore hypothesize that fine-tuning nsp15’s EndoU activity is a viral tactic to avoid interference with the viral replication. In addition, we describe two possible mechanisms how IBV nsp15’s EndoU activity is fine-tuned during IBV infection. One mechanism is related to the subcellular localization of nsp15 EndoU during IBV infection, which might separate nsp15 EndoU from its cellular substrates. Double membrane vesicles (DMVs), concentrated in the perinuclear area, are central hubs for the replication and transcription complex (RTC)-mediated synthesis of viral RNA. CoV nsp15 is likely associated with RTC as MHV nsp15 was previously reported to strongly colocalize with two RTC proteins nsp8, nsp12. We observed a perinuclear aggregation of nsp15 EndoU during IBV infection, implying that IBV nsp15 EndoU is located inside DMVs and separated from its cellular substrates. The other mechanism is mediated by the interaction between nsp15 EndoU and other IBV nsps. We demonstrated that the overexpressed nsp2, nsp7 or nsp16 of IBV restored nsp15 EndoU’s inhibition of global protein synthesis in the transfected cells.  In chapter 6, I put the findings in this thesis on the nsp15 EndoU of IBV into the context of nsp15 EndoU of CoVs in general.

In conclusion, the work described in this thesis demonstrates for the first time how nsp15 EndoU of IBV antagonizes dsRNA-induced anti-viral defenses. Also, this thesis demonstrates the regulation mechanisms of the role of nsp15 EndoU to avoid reduced IBV replication. In addition, we analyze the inhibitory effect on global protein synthesis by nsp15 EndoU of α- and β-CoVs. This thesis thereby provides novel insights into nsp15 EndoU’s role in the infection with IBV but also other coronaviruses, which can promote the development of novel vaccines for IBV or novel anti-viral drugs for coronaviruses in general.