VIDO-InterVac, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
Vaccinology and Immunotherapeutics, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.
J Virol. 2019 Mar 5;93(6). doi: 10.1128/JVI.02151-18. Print 2019 Mar 15.
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 -Golgi apparatus but not in the -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 ΔU47-BoHV-1-infected cells, further supporting a role for VP8 in altering the cellular lipid metabolism during infection. 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 -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.
牛疱疹病毒 1（BoHV-1）感染牛科动物，引起呼吸道感染、生殖器官疾病和流产。VP8 是 BoHV-1 中最丰富的被膜蛋白，对病毒在牛体内的复制至关重要。本研究探讨了 BoHV-1 感染细胞中 VP8 的细胞内运输及其改变细胞脂质代谢的能力。发现一种病毒激酶 US3 参与了这些过程的调节。在感染的早期，VP8 定位于细胞核内。随后，VP8 可能在完成其在核内的作用后，被转运到细胞质中。当 US3 缺失或 BoHV-1 中 VP8 的必需 US3 磷酸化位点发生突变时，大多数 VP8 定位于感染细胞的细胞核内。这表明 US3 的磷酸化可能对 VP8 的细胞质定位至关重要。最终，细胞质中的 VP8 积累在 -高尔基体中，但不在 -高尔基体网络中，这表明 VP8 不参与病毒颗粒向细胞膜运输和出芽。VP8 在转染细胞的细胞核中引起脂滴（LD）形成，并增加细胞内胆固醇水平。当 US3 存在时，VP8 在细胞质中，BoHV-1 感染细胞的细胞核中没有发现脂滴。然而，当 US3 缺失或 VP8 中的磷酸化残基发生突变时，BoHV-1 感染细胞中出现核内 VP8 和脂滴。BoHV-1 感染细胞中的总胆固醇水平升高，但在ΔU47-BoHV-1 感染细胞中没有升高，这进一步支持 VP8 在感染过程中改变细胞脂质代谢的作用。核定位信号（NLS）和核输出信号（NES）是指导 VP8 在 BoHV-1 感染细胞中到达所需位置的重要元件。本研究确定了一种在 BoHV-1 感染细胞中切换 VP8 核内和细胞质定位的关键调节因子。BoHV-1 使用病毒激酶 US3 来调节 VP8 的细胞内定位。在 BoHV-1 感染的早期，VP8 定位于细胞核内，在那里它执行各种功能；一旦表达 US3，磷酸化的 VP8 就位于细胞质中，并最终积累在 -高尔基体中，推测是为了整合到病毒颗粒中。VP8 在病毒感染细胞中的高尔基体定位仅在病毒感染细胞中观察到，而不在 US3 共转染细胞中观察到，这表明这是由其他病毒因子介导的。有趣的是，VP8 被证明会导致胆固醇水平升高，这是 VP8 的一个新功能，也是为病毒复制提供脂质的一种潜在策略。
