Nuclear Translocation and Regulation of Intranuclear Distribution of Cytoplasmic Poly(A)-Binding Protein Are Distinct Processes Mediated by Two Epstein Barr Virus Proteins

Many viruses target cytoplasmic polyA binding protein (PABPC) to effect widespread inhibition of host gene expression, a process termed viral host-shutoff (vhs). During lytic replication of Epstein Barr Virus (EBV) we observed that PABPC was efficiently translocated from the cytoplasm to the nucleus. Translocated PABPC was diffusely distributed but was excluded from viral replication compartments. Vhs during EBV infection is regulated by the viral alkaline nuclease, BGLF5. Transfection of BGLF5 alone into BGLF5-KO cells or uninfected 293 cells promoted translocation of PAPBC that was distributed in clumps in the nucleus. ZEBRA, a viral bZIP protein, performs essential functions in the lytic program of EBV, including activation or repression of downstream viral genes. ZEBRA is also an essential replication protein that binds to viral oriLyt and interacts with other viral replication proteins. We report that ZEBRA also functions as a regulator of vhs. ZEBRA translocated PABPC to the nucleus, controlled the intranuclear distribution of PABPC, and caused global shutoff of host gene expression. Transfection of ZEBRA alone into 293 cells caused nuclear translocation of PABPC in the majority of cells in which ZEBRA was expressed. Co-transfection of ZEBRA with BGLF5 into BGLF5-KO cells or uninfected 293 cells rescued the diffuse intranuclear pattern of PABPC seen during lytic replication. ZEBRA mutants defective for DNA-binding were capable of regulating the intranuclear distribution of PABPC, and caused PABPC to co-localize with ZEBRA. One ZEBRA mutant, Z(S186E), was deficient in translocation yet was capable of altering the intranuclear distribution of PABPC. Therefore ZEBRA-mediated nuclear translocation of PABPC and regulation of intranuclear PABPC distribution are distinct events. Using a click chemistry-based assay for new protein synthesis, we show that ZEBRA and BGLF5 each function as viral host shutoff factors.

多种病毒以细胞质多聚腺苷酸结合蛋白（cytoplasmic polyA binding protein, PABPC）为靶标，以实现宿主基因表达的广泛抑制，这一过程被称为病毒宿主关闭（viral host-shutoff, vhs）。在EB病毒（Epstein Barr Virus, EBV）的裂解性复制过程中，我们观察到PABPC可被高效地从细胞质转运至细胞核。转运至细胞核的PABPC呈弥散性分布，但被排除于病毒复制区室之外。EBV感染期间的vhs过程由病毒碱性核酸酶BGLF5调控。单独将BGLF5转染至BGLF5基因敲除细胞或未感染的293细胞后，可促进PABPC的核转运，此时PABPC在细胞核内呈团簇状分布。ZEBRA是一种病毒bZIP蛋白，在EBV裂解程序中发挥核心功能，包括激活或抑制下游病毒基因的转录；同时它也是一种必需的病毒复制蛋白，可结合病毒裂解性复制起点（oriLyt），并与其他病毒复制蛋白发生相互作用。本研究发现，ZEBRA同时可作为vhs的调控因子：它能够介导PABPC向细胞核转运，调控PABPC的核内分布，并引发宿主基因表达的全局性关闭。将ZEBRA单独转染至293细胞后，在表达ZEBRA的大多数细胞中均可诱导PABPC的核转运。将ZEBRA与BGLF5共转染至BGLF5基因敲除细胞或未感染的293细胞后，可恢复EBV裂解性复制期间观察到的PABPC弥散性核内分布模式。DNA结合功能缺陷的ZEBRA突变体仍可调控PABPC的核内分布，并使PABPC与ZEBRA发生共定位。其中一株ZEBRA突变体Z(S186E)存在核转运缺陷，但仍可改变PABPC的核内分布，这表明ZEBRA介导的PABPC核转运与调控PABPC核内分布是两个相互独立的过程。通过基于点击化学的新蛋白合成检测实验，我们证实ZEBRA与BGLF5均可作为病毒宿主关闭因子发挥功能。