US3 Kinase-Mediated Phosphorylation of Tegument Protein VP8 Plays a Critical Role in the Cellular Localization of VP8 and Its Effect on the Lipid Metabolism of Bovine Herpesvirus 1-Infected Cells

Abstract

Bovine herpesvirus 1 (BoHV-1) infects bovine species, causing respiratory infections, genital disorders and abortions. VP8 is the most abundant tegument protein of BoHV-1 and is critical for virus replication in cattle. In this study, the cellular transport of VP8 in BoHV-1-infected cells and its ability to alter the cellular lipid metabolism were investigated. A viral kinase, US3, was found to be involved in regulating these processes. In the early stages of infection VP8 was localized in the nucleus. Subsequently, presumably after completion of its role in the nucleus, VP8 was translocated to the cytoplasm. When US3 was deleted or the essential US3 phosphorylation site of VP8 was mutated in BoHV-1, the majority of VP8 was localized in the nuclei of infected cells. This suggests that phosphorylation by US3 may be critical for cytoplasmic localization of VP8. Eventually, the cytoplasmic VP8 was accumulated in the cis-Golgi apparatus but not in the trans-Golgi network, implying that VP8 was not involved in virion transport toward and budding from the cell membrane. VP8 caused lipid droplet (LD) formation in the nuclei of transfected cells and increased cellular cholesterol levels. Lipid droplets were not found in the nuclei of BoHV-1-infected cells when VP8 was cytoplasmic in the presence of US3. However, when US3 was deleted or phosphorylation residues in VP8 were mutated, nuclear VP8 and LDs appeared in BoHV-1-infected cells. The total cholesterol level was increased in BoHV-1-infected cells but not in ΔU L 47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection. IMPORTANCE Nuclear localization signals (NLSs) and nuclear export signals (NESs) are important elements directing VP8 to the desired locations in the BoHV-1-infected cell. In this study, a critical regulator that switches the nuclear and cytoplasmic localization of VP8 in BoHV-1-infected cells was identified. BoHV-1 used viral kinase US3 to regulate the cellular localization of VP8. Early during BoHV-1 infection VP8 was localized in the nucleus, where it performs various functions; once US3 was expressed, phosphorylated VP8 was cytoplasmic and ultimately accumulated in the cis-Golgi apparatus, presumably to be incorporated into virions. The Golgi localization of VP8 was only observed in virus-infected cells and not in US3-cotransfected cells, suggesting that this is mediated by other viral factors. Interestingly, VP8 was shown to cause increased cholesterol levels, which is a novel function for VP8 and a potential strategy to supply lipid for viral replication. KEYWORDS US3, VP8, bovine herpesvirus 1, cellular localization, lipid metabolism, protein phosphorylation Citation Zhang K, Donovan T, Sucharita S, Brownlie R, Snider M, Tikoo SK, van Drunen Littel-van den Hurk S. 2019. US3 kinasemediated phosphorylation of tegument protein VP8 plays a critical role in the cellular localization of VP8 and its effect on the lipid metabolism of bovine herpesvirus 1-infected cells. J RESULTSNuclear VP8 i...

Duck plague virus US3 protein kinase phosphorylates UL47 and regulates the subcellular localization of UL47

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“…However, US3 was not the only protein kinase that phosphorylated UL47 protein in DPV, because other Ser sites were also detected to be phosphorylated in our phosphorylation MS analysis ( Table 2 ), such as Ser67, Ser120, Ser144, and Ser283, which were found in the enrichment of UL47 co-transfected with and without US3 expression. Among them, the phosphopeptides containing residues Ser67, Ser120, and Ser144 match the cellular casein kinase 2 (CK2) motif S/T-X-X-D/E ( Joughin et al, 2012 ; Zhang et al, 2015 ), which is universally expressed in almost every subcellular structure ( Zhang et al, 2019 ). Therefore, DPV UL47 protein may be also phosphorylated by CK2 protein, but its function of nucleocytoplasmic-shuttling is not affected by CK2-regulated phosphorylation.…”

Associating bovine herpesvirus 1 envelope glycoprotein gD with activated phospho-PLC-γ1(S1248)

“…It has been reported that virions of HSV-1 localize to the Golgi apparatus, which enables virus packaging, envelopment, and trafficking to the cytoplasm ( 25 ). Like HSV-1, Golgi apparatus is the essential site for BoHV-1 packaging into mature virions ( 26 ). Here, Western blotting analysis was performed to detect viral proteins in the isolated Golgi apparatus via using a monoclonal antibody against virus glycoprotein gD and a polyclonal antibody (pAb) against virion-associated proteins, respectively.…”

Duo in a Mystical Realm—Nuclear Lipid Droplets and the Inner Nuclear Membrane

“…Hepatitis C virus core protein, which distributes to LDs when expressed alone, binds PML-NBs and inactivates their apoptosis-inducing function (Herzer et al., 2005). Expression of VP8, a tegument protein of bovine herpesvirus 1, targets PML-NBs and increases nuclear LDs (Zhang et al., 2015, 2019). These results suggest that nuclear LDs may be utilized by viruses to evade host defense mechanisms.…”