Ebola virus VP35 hijacks the PKA-CREB1 pathway for replication and pathogenesis by AKIP1 association
Zhu L, Gao T, Huang Y, Jin J, Wang D, Zhang L, Jin Y, Li P, Hu Y, Wu Y, Liu H, Dong Q, Wang G, Zheng T, Song C, Bai Y, Zhang X, Liu Y, Yang W, Xu K, Zou G, Zhao L, Cao R, Zhong W, Xia X, Xiao G, Liu X, Cao C
Applications: WB
Reactive species: Ebola virus
"Abstract: Ebola virus (EBOV), one of the deadliest viruses, is the cause of fatal Ebola virus disease (EVD). The underlying mechanism of viral replication and EBOV-related hemorrhage is not fully understood. Here, we show that EBOV VP35, a cofactor of viral RNA-dependent RNA polymerase, binds human A kinase interacting protein (AKIP1), which consequently activates protein kinase A (PKA) and the PKA-downstream transcription factor CREB1. During EBOV infection, CREB1 is recruited into EBOV ribonucleoprotein complexes in viral inclusion bodies (VIBs) and employed for viral replication. AKIP1 depletion or PKA-CREB1 inhibition dramatically impairs EBOV replication. Meanwhile, the transcription of several coagulation-related genes, including THBD and SERPINB2, is substantially upregulated by VP35-dependent CREB1 activation, which may contribute to EBOV-related hemorrhage. The finding that EBOV VP35 hijacks the host PKA-CREB1 signal axis for viral replication and pathogenesis provides novel potential therapeutic approaches against EVD."
Article snippet: Immunoblotting was performed with anti-VP35 (Creative Diagnostics, CABT-B292 or CABT-B1099, 1:1000 dilution) and anti-L (Creative Diagnostics, CABT-B1095, 1:500 dilution) antibodies.
Ebola virus sequesters IRF3 in viral inclusion bodies to evade host antiviral immunity
Lin Zhu, Jing Jin, Tingting Wang, Yong Hu, Hainan Liu, Ting Gao, Qincai Dong, Yanwen Jin, Ping Li, Zijing Liu, Yi Huang, Xuan Liu, Cheng Cao
Applications: WB, IF
Reactive species: Ebola Virus
"Abstract: Viral inclusion bodies (IBs) commonly form during the replication of Ebola virus (EBOV) in infected cells, but their role in viral immune evasion has rarely been explored. Here, we found that interferon regulatory factor 3 (IRF3), but not TANK-binding kinase 1 (TBK1) or IκB kinase epsilon (IKKε), was recruited and sequestered in viral IBs when the cells were infected by EBOV transcription- and replication-competent virus-like particles (trVLPs). Nucleoprotein/virion protein 35 (VP35)-induced IBs formation was critical for IRF3 recruitment and sequestration, probably through interaction with STING. Consequently, the association of TBK1 and IRF3, which plays a vital role in type I interferon (IFN-I) induction, was blocked by EBOV trVLPs infection. Additionally, IRF3 phosphorylation and nuclear translocation induced by Sendai virus or poly(I:C) stimulation were suppressed by EBOV trVLPs. Furthermore, downregulation of STING significantly attenuated VP35-induced IRF3 accumulation in IBs. Coexpression of the viral proteins by which IB-like structures formed was much more potent in antagonizing IFN-I than expression of the IFN-I antagonist VP35 alone. These results suggested a novel immune evasion mechanism by which EBOV evades host innate immunity."
Article snippet: Antibody anti-Zaire Ebola virus VP35 antibody (Mouse monoclonal) Creative Diagnostics Cat# CABT-B292 IF (1:50); WB (1:1000) 
Figure 1. HepG2 cells infected with the EBOV trVLPs were immunostained with anti-VP35 (green) antibodies. 
Figure 2. IRF3 levels in HepG2 cells infected with EBOV trVLPs were analyzed by immunoblotting with an anti-IRF3 antibody at the indicated hours post infection (hpi).
Ebola virus replication is regulated by the phosphorylation of viral protein VP35
Lin Zhu, Ting Gao, Weihong Yang, Yaoning Liu, Xuan Liu, Yong Hu, Yanwen Jin, Ping Li, Ke Xu, Gang Zou, Lei Zhao, Ruiyuan Cao, Wu Zhong, Xianzhu Xia, Cheng Cao
Biochem Biophys Res Commun2020 JanPubMed ID: 31694758Read Article
Applications: WB, IP
Reactive species: Unspecified reactive species
"Abstract: Ebola virus (EBOV) is a zoonotic pathogen, the infection often results in severe, potentially fatal, systematic disease in human and nonhuman primates. VP35, an essential viral RNA-dependent RNA polymerase cofactor, is indispensable for Ebola viral replication and host innate immune escape. In this study, VP35 was demonstrated to be phosphorylated at Serine/Threonine by immunoblotting, and the major phosphorylation sites was S187, S205, T206, S208 and S317 as revealed by LC-MS/MS. By an EBOV minigenomic system, EBOV minigenome replication was shown to be significantly inhibited by the phosphorylation-defective mutant, VP35 S187A, but was potentiated by the phosphorylation mimic mutant VP35 S187D. Together, our findings demonstrate that EBOV VP35 is phosphorylated on multiple residues in host cells, especially on S187, which may contribute to efficient viral genomic replication and viral proliferation."
Article snippet: Soluble proteins were immunoprecipitated with anti-Flag M2 antibody (*), anti-VP35 antibody (Creative Diagnostics) or anti-mouse IgG antibody (*). 
Figure 1. EBOV VP35 S187A impairs VP35 phosphorylation in HepG2 cells
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