PIM1 Facilitates β-Coronavirus Replication Through BTRC-Mediated IFNAR1 Degradation

PIM1通過BTRC介導的IFNAR1降解促進β-冠狀病毒複製

Student thesis: Doctoral Thesis

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Author(s)

Detail(s)

Awarding Institution
Supervisors/Advisors
  • Mingliang HE (Supervisor)
  • Robert WEISS (External person) (External Co-Supervisor)
Award date28 Nov 2022

Abstract

Coronaviruses are single-stranded RNA viruses. Currently, seven coronaviruses can infect human beings. Before 2003, little attention was paid to coronaviruses because they can only cause mild illnesses, like the common cold. With the emergency of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and SARS-CoV-2 in 2003, 2012, and 2019, respectively, Coronaviruses have threatened the world repeatedly. SARS-CoV, MERS-CoV, and SARS-CoV-2 are highly pathogenic. Patients infected with SARS-CoV, MERS-CoV, or SARS-CoV-2 have a higher risk of developing severe symptoms such as acute respiratory distress syndrome (ARDS) and some extrapulmonary manifestations.

Human innate immunity should be the first line to protect humans from being harmed by infectious pathogens. The intact interferon induction and response are of great significance for anti-viral infection. However, clinical data show that patients of COVID-19, especially those with severe symptoms, often exhibit decreased/delayed IFN induction. Researchers have studied several mechanisms about how SARS-CoV-2 inhibits the induction of interferons. Worse, the administration of interferons to patients also has not been as effective as expected. This indicates that the downstream of the signalling is also dampened by viruses.

In my study, I found that the serine/threonine kinase PIM1 which plays a major role in viral infection, immunological response, and malignancy, was significantly elevated in response to coronavirus infection. Moreover, this finding was highly consistent with our previous discoveries that PIM1 is increased when cells are invaded by enterovirus-A71 (EV-A71) and zika virus (ZIKV) virus. Both of them are RNA viruses. Most importantly, I demonstrated that PIM1 was essential in facilitating coronavirus replication through loss- and gain-of-function experiments. I then determined whether PIM1 kinase activity was required for assisting coronavirus replication with three PIM1-specific inhibitors (CX-6258, AZD-1208, and SGI-1776). All these inhibitors highly potentially suppressed viral infectivity in a dose-dependent manner. These data together demonstrated the significance of PIM1 and its kinase activity in coronavirus infection.

Further, I employed interferon stimulation response elements (ISRE) and IFNα/β promoter luciferase assays to determine the effect of PIM1 on IFN response. I found that WT PIM1 dramatically attenuated interferon response signalling but PIM1 in an inactivated form could not reduce the IFN response signalling. In addition, I detected the activation of several factors in the IFN signalling pathways after ectopically overexpressing PIM1 or pharmaceutical inhibition of PIM1 with inhibitors. I found that PIM1 could inhibit all the activation of factors in IFN signalling pathways, evidenced by decreased phosphorylation levels of STAT1/STAT2/JAK1 and TYK2. Consistently, PIM1 inhibitors increased p-STAT1 and p-STAT2 levels in response to poly: IC or IFN α stimulation. Then, by gain- and loss-of-function experiments, I revealed that PIM1 down-regulated IFNAR1, not IFNAR2, to attenuate the IFN response. IFNAR1 and IFNAR2 are the type I interferon co-receptors that start the interferon response upon binding with interferon. The level of IFNAR1 expression on cell surfaces is essential for amplifying the interferon response.

The expression level of IFNAR1 is delicately regulated both transcriptionally and post-transcriptionally. From my results, the mRNA level of IFNAR1 was affected neither by coronavirus infection nor by PIM1 ectopic expression. Therefore, I detected the protein turnover rate of IFNAR1. The result showed depletion of PIM1 slowed down the degradation speed of IFNAR1. The turnover of IFNAR1 undergoes phosphorylation-dependent ubiquitination, internalization, and lysosomal degradation. I found that PIM1 did not phosphorylate IFNAR1, but the degradation of IFNAR1 by PIM1 proceeded indeed in a phosphorylation-dependent manner. Then, I found overexpression of WT PIM1 increased the ubiquitination of IFNAR1, but PIM1 in an inactivated form did not affect the ubiquitination level of IFNAR1, while depletion of PIM1 with PIM1-specific siRNA was found to decrease the ubiquitination level of IFANR1. Finally, I found that PIM1 modulated the ubiquitination and degradation of IFNAR1 through E3 ligase BTRC. Knocking down of BTRC abolished PIM1-induced IFNAR1 degradation.

My study found that the BTRC expression level was increased after PIM1 overexpression. Furthermore, depletion or inhibition of PIM1 decreased BRTC expression at the protein level. Moreover, I determined that BRTC was a potent substrate of PIM1. Both in-vivo and in-vitro experiments showed that PIM1 interacted with and phosphorylated BTRC. To identify specific residuals that may be phosphorylated by PIM1, I screened seven predicted sites using an ISRE-mini promoter luciferase assay. I found that a mutation of BTRC serine at sites 82 or 521 into alanine affected the function of BTRC decreasing ISRE luciferase stimulation. In addition, mutated BTRC at both S82 and S521 could not decrease IFNAR1, as well as the p-STAT1and p-STAT2 level compared with WT-BTRC. Consistently, mutated BTRC at S82 and S521 no longer promoted coronavirus replication. Further, I found that a mutation of S82A or S521A impaired IFNAR1 interaction ability but not its stability which was the reason for impaired BTRC function of degrading IFNAR1. Finally, I found that the S82A mutation of BTRC could not be phosphorylated by PIM1, which indicated that S82 was the phosphorylation site of PIM1 on BTRC, or at least S82 could affect PIM1’s phosphorylation of BTRC.