on 03-05-201506:33 AM - edited on 10-15-202111:35 AM by AnalyteGuru
Zhang K, Afroz S, Brownlie R, Snider M, van Drunen Littel-van den Hurk S. J Virol. 2015 Feb 11. pii: JVI.03180-14. The major tegument protein of bovine herpesvirus-1 (BoHV-1), VP8, is essential for viral replication in cattle. VP8 is phosphorylated in vitro by casein kinase 2 (CK2) and BoHV-1 unique short protein 3 (US3). In this study VP8 was found to be phosphorylated in both transfected and infected cells, but was detected as a non-phosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral lifecycle. The regulation and function of VP8 phosphorylation by US3 and CK2 was further analyzed. An in vitro kinase assay, site-directed mutagenesis and liquid chromatography-mass spectrometry were used to identify the active sites for US3 and CK2. The two kinases phosphorylate VP8 at different sites, resulting in distinct phosphopeptide patterns. S16 is a primary phosphoreceptor for US3 and it subsequently triggers phosphorylation at S32. CK2 has multiple active sites, among which T107 appears to be the preferred residue. Additionally, CK2 consensus motifs in the N-terminus of VP8 are essential for the phosphorylation. Based on these results, a non-phosphorylated VP8 mutant was constructed, and used for further studies. In transfected cells phosphorylation was not required for nuclear localization of VP8. Phosphorylated VP8 appeared to recruit promyelocytic leukemia (PML) protein, and to remodel the distribution of PML in the nucleus; however, PML protein did not show an association with non-phosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by re-distributing PML protein, and that this function depends on the phosphorylation of VP8.
The progression of VP8 phosphorylation over time and its function in BoHV-1 replication have not been characterized. This study demonstrates that activation of S16 initiates further phosphorylation at S32 by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T107 having the highest level of phosphorylation. Evidence for a difference in phosphorylation status of VP8 in host cells and mature virus is presented for the first time. The phosphorylation was found to be a critical modification, which enables VP8 to attract and to redistribute PML protein in the nucleus. This might promote viral replication through interference with PML-mediated antiviral defense. This study provides new insights into the regulation of VP8 phosphorylation and suggests a novel, phosphorylation-dependent function for VP8 in the lifecycle of BoHV-1, which is important in view of the fact that VP8 is essential for viral replication in vivo.