|dc.description.abstract||Bovine herpesvirus-1 (BoHV-1) infects bovine species causing respiratory and genital syndromes. VP8, encoded by the ul47 gene, is a tegument protein essential for BoHV-1 to infect host animals. This protein is abundantly incorporated into the virus and is extensively expressed in BoHV-1-infected cells. VP8 is phosphorylated by a viral kinase, unique short protein 3 (US3), and a cellular kinase, casein kinase 2 (CK2). With the aim to investigate the function of VP8 in the life cycle of BoHV-1, we hypothesized that US3- and CK2-mediated phosphorylation changes the functions of VP8, which in turn affects the replication of BoHV-1.
Using site-directed mutagenesis and liquid chromatography-mass spectrometry we identified minimal amino-acid residues of VP8 required for phosphorylation by US3 and CK2, respectively. US3 phosphorylated VP8 at two residues, serine 16 (S16) and serine 32 (S32). Phosphorylation at S16 was essential for subsequent phosphorylation at S32. CK2 phosphorylated at least eleven threonines (T) and serines (S) in VP8. Mutating seven of them (T65, S66, S79, S80, S82, S88 and T107) blocked the whole protein being phosphorylated by CK2. By replacing the critical phosphorylation sites for US3 and CK2 in VP8, we generated a mutant VP8 that was not phosphorylated by US3 and CK2. This non-phosphorylated VP8 was studied in parallel with wild-type VP8. Promyelocytic leukemia (PML) protein was redistributed by wild-type VP8 but not by non-phosphorylated VP8 in transfected cells. This implicates that VP8 may be involved in counteracting PML-related host antiviral defenses by redistributing PML protein, and is dependent upon its phosphorylation. VP8-transfected cells developed nuclear lipid droplets (LDs), suggesting that VP8 causes lipid accumulation in host cells. Wild-type VP8 was more abundant around the surface of the droplets compared with the mutant VP8.
We next investigated the roles of phosphorylated VP8 in the life cycle of BoHV-1. A mutant virus (BoHV-1-YmVP8) with non-phosphorylated VP8 was generated by using homologous recombination in mammalian cells. The results showed that the DNA encapsidation was reduced in mutant virus-infected cells, resulting in a lower virus titer, when compared with wild-type virus-infected cells. Capsids without DNA cores were more frequently found in the nuclei and perinuclear spaces of mutant virus-infected cells than that of wild-type virus-infected cells. However, we did not observe obvious restriction of virus egress during the infection with BoHV-1-YmVP8. Virions lacking DNA core were released into the extracellular medium. Therefore, we conclude that phosphorylated VP8 promotes DNA encapsidation of BoHV-1 in the nuclei of infected cells. VP8 appeared in the Golgi apparatus of cells infected with wild-type virus late during infection. The non-phosphorylated VP8 did not leave the nuclei of BoHV-1-YmVP8-infected cells, suggesting that phosphorylation is critical for the cytoplasmic transport of VP8. By measuring the amount of mature virion-incorporated VP8, we found that mutant BoHV-1 contained lower amounts of VP8 than wild-type virus. Together with the finding that non-phosphorylated VP8 was not able to localize in the Golgi, it implies that phosphorylation is important to place VP8 into a proper cellular location.
Based on the finding that cytoplasmic localization of VP8 was inhibited by mutating phosphorylation residues, we hypothesized that the cellular localization of VP8 is regulated by US3- and/or CK2-dependent phosphorylation. We verified that VP8 was initially localized into the nucleus and then transported into the cytoplasm when BoHV-1 infection progressed to late stages. The cytoplasmic VP8 subsequently accumulated in the Golgi apparatus. We also found that the kinase activity of US3 was critical for VP8 to be exported from the nucleus. Deletion of US3 in BoHV-1 or mutating the essential residue for US3-mediated phosphorylation in VP8 blocked the nuclear-cytoplasmic transport of VP8. In the amino-acid sequence of VP8, US3-phosphorylated residues are closely adjacent to nuclear localization signals (NLSs), providing the possibility that addition of phosphoryl groups to these residues may affect the binding activity of the NLSs, blocking VP8 entering or re-entering the nucleus. The US3-phosphorylated VP8 is transported to the cytoplasm through more than two pathways that require different types of nuclear export signals (NESs). In the cytoplasm, VP8 might experience virus-induced modifications or protein interactions, which led the protein to localize in the cis-Golgi cisternae. VP8 was less likely to present in the trans-Golgi network (TGN). The cis-Golgi localization of VP8 and Golgi accumulation of viral particles suggest that VP8 localizes in the Golgi for incorporation into the virions, and that it may not directly contribute to viral egress.
Altogether, this research described the importance of phosphorylation in regulating the functions of VP8 during BoHV-1 infection. Nuclear VP8 phosphorylated through CK2 is responsible for DNA encapsidation of BoHV-1 and remodeling the cellular antiviral factor PML. When nuclear VP8 is phosphorylated by US3, the cytoplasmic translocation of VP8 is activated possibly by inactivating the NLSs. Subsequently, VP8 aggregates towards the cis-Golgi apparatus where VP8 is abundantly incorporated into virions such that the mature virus contains enough VP8 to benefit future infection.||