Abstract
Bluetongue virus (BTV) causes infections in wild and domesticated ruminants with high morbidity and mortality and is responsible for significant economic losses in both developing and developed countries. BTV serves as a model for the study of other members of the Orbivirus genus. Previously, the importance of casein kinase 2 for BTV replication was demonstrated. To identify intracellular signaling pathways and novel host-cell kinases involved during BTV infection, the phosphoproteome of BTV infected cells was analyzed. Over 1000 phosphosites were identified using mass spectrometry, which were then used to determine the corresponding kinases involved during BTV infection. This analysis yielded protein kinase A (PKA) as a novel kinase activated during BTV infection. Subsequently, the importance of PKA for BTV infection was validated using a PKA inhibitor and activator. Our data confirmed that PKA was essential for efficient viral growth. Further, we showed that PKA is also required for infection of equid cells by African horse sickness virus, another member of the Orbivirus genus. Thus, despite their preference in specific host species, orbiviruses may utilize the same host signaling pathways during their replication.
Highlights
From the ‡Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK; §University of Cambridge, Division of Virology, Department of Pathology, Lab block level 5, Box 237, Addenbrookes Hospital, Cambridge, UK
Casein kinase 2 (CK2) has been reported to be involved for phosphorylation of one of the non-structural proteins, NSP5, of Rotavirus, a Reoviridae family member, whereas CK1␣ was essential for NSP5 hyperphosphorylation [15, 16]
We chose one of these kinases, protein kinase A (PKA), which constituted a novel host factor that had not been previously associated with Bluetongue virus (BTV)
Summary
To mock infected cells, BTV infected cells treated with the Dibutyryl-cAMP activator showed a significant increase in phosphorylated PKA substrates compared with DMSO-only treated cells, increasing by ϳ26% (Ϯ 5%) in HeLa cells and by ϳ33% (Ϯ 4%) in sheep PT cells (Fig. 3A). At 18 h.p.i there is a significant increase of phosphorylated PKA substrates in infected HeLa and sheep PT cells when compared with mock infected cells (Fig. 3B), this observation agrees with the phosphoproteome data obtained (supplemental Table S3–S5).
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