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Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.

ABSTRACT: The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus 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 nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The regulation and function of VP8 phosphorylation by US3 and CK2 were 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. S(16) is a primary phosphoreceptor for US3, and it subsequently triggers phosphorylation at S(32). CK2 has multiple active sites, among which T(107) appears to be the preferred residue. Additionally, CK2 consensus motifs in the N terminus of VP8 are essential for phosphorylation. Based on these results, a nonphosphorylated 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 nonphosphorylated VP8. This suggests that VP8 plays a role in resisting PML-related host antiviral defenses by redistributing 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 S(16) initiates further phosphorylation at S(32) by US3. Additionally, VP8 is phosphorylated by CK2 at several residues, with T(107) having the highest level of phosphorylation. Evidence for a difference in the phosphorylation status of VP8 in host cells and mature virus is presented for the first time. 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 a 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 life cycle of BoHV-1, which is important in view of the fact that VP8 is essential for virus replication in vivo.

SUBMITTER: Zhang K

PROVIDER: S-EPMC4442349 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature

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Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.

Zhang Kuan K Afroz Sharmin S Brownlie Robert R Snider Marlene M van Drunen Littel-van den Hurk Sylvia S

Journal of virology 20150211 8

<h4>Unlabelled</h4>The major tegument protein of bovine herpesvirus 1 (BoHV-1), VP8, is essential for virus 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 nonphosphorylated form in mature virions. This suggests that phosphorylation of VP8 is strictly controlled during different stages of the viral life cycle. The r ...[more]

PMID: 25673708

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Project description:UnlabelledThe UL47 gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that a UL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulence in vitro and is avirulent in vivo In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without any de novo protein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells.ImportanceSince VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication. Indeed, while nonessential in vitro, it is critical for BoHV-1 replication in vivo In this study, we determined that VP8 plays a role in downregulation of the antiviral host response by inhibiting IFN-β signaling. VP8 interacted with and prevented nuclear accumulation of STAT1 at 2 h postinfection in the absence of de novo viral protein synthesis. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for this interaction. These results provide a new functional role for VP8 in BoHV-1 infection and a potential explanation for the lack of viral replication of the UL47 deletion mutant in cattle.

Project description: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 ΔUL47-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.

Project description:A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses.

Dataset Information

Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.

Publications

Regulation and function of phosphorylation on VP8, the major tegument protein of bovine herpesvirus 1.

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