Kim Young-Eui, Oh Se Eun, Kwon Ki Mun, Lee Chan Hee, Ahn Jin-Hyun
Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Republic of Korea.
Division of Life Sciences, Chungbuk National University, Cheongju, Republic of Korea.
J Virol. 2016 Jan 13;90(6):3229-42. doi: 10.1128/JVI.02766-15.
Human cytomegalovirus (HCMV) protein pUL48 is closely associated with the capsid and has a deubiquitinating protease (DUB) activity in its N-terminal region. Although this DUB activity moderately increases virus replication in cultured fibroblast cells, the requirements of the N-terminal region of pUL48 in the viral replication cycle are not fully understood. In this study, we characterized the recombinant viruses encoding UL48(ΔDUB/NLS), which lacks the DUB domain and the adjacent nuclear localization signal (NLS), UL48(ΔDUB), which lacks only the DUB, and UL48(Δ360-1200), which lacks the internal region (amino acids 360 to 1200) downstream of the DUB/NLS. While ΔDUB/NLS and Δ360-1200 mutant viruses did not grow in fibroblasts, the ΔDUB virus replicated to titers 100-fold lower than those for wild-type virus and showed substantially reduced viral gene expression at low multiplicities of infection. The DUB domain contained ubiquitination sites, and DUB activity reduced its own proteasomal degradation in trans. Deletion of the DUB domain did not affect the nuclear and cytoplasmic localization of pUL48, whereas the internal region (360-1200) was necessary for cytoplasmic distribution. In coimmunoprecipitation assays, pUL48 interacted with three tegument proteins (pUL47, pUL45, and pUL88) and two capsid proteins (pUL77 and pUL85) but the DUB domain contributed to only pUL85 binding. Furthermore, we found that the ΔDUB virus showed reduced virion stability and less efficiently delivered its genome into the cell than the wild-type virus. Collectively, our results demonstrate that the N-terminal DUB domain of pUL48 contributes to efficient viral growth by regulating its own stability and promoting virion stabilization and virus entry.
HCMV pUL48 and its herpesvirus homologs play key roles in virus entry, regulation of immune signaling pathways, and virion assembly. The N terminus of pUL48 contains the DUB domain, which is well conserved among all herpesviruses. Although studies using the active-site mutant viruses revealed that the DUB activity promotes viral growth, the exact role of this region in the viral life cycle is not fully understood. In this study, using the mutant virus lacking the entire DUB domain, we demonstrate that the DUB domain of pUL48 contributes to viral growth by regulating its own stability via autodeubiquitination and promoting virion stability and virus entry. This report is the first to demonstrate the characteristics of the mutant virus with the entire DUB domain deleted, which, along with information on the functions of this region, is useful in dissecting the functions associated with pUL48.
人巨细胞病毒（HCMV）蛋白pUL48与衣壳紧密相关，并且在其N端区域具有去泛素化蛋白酶（DUB）活性。尽管这种DUB活性适度增加了病毒在培养的成纤维细胞中的复制，但pUL48的N端区域在病毒复制周期中的需求尚未完全了解。在本研究中，我们对编码UL48(ΔDUB/NLS)、UL48(ΔDUB)和UL48(Δ360 - 1200)的重组病毒进行了特性分析，其中UL48(ΔDUB/NLS)缺失DUB结构域和相邻的核定位信号（NLS），UL48(ΔDUB)仅缺失DUB，UL48(Δ360 - 1200)缺失DUB/NLS下游的内部区域（氨基酸360至1200）。虽然ΔDUB/NLS和Δ360 - 1200突变病毒在成纤维细胞中不生长，但ΔDUB病毒的复制滴度比野生型病毒低100倍，并且在低感染复数时病毒基因表达大幅降低。DUB结构域包含泛素化位点，并且DUB活性在反式作用中降低了其自身的蛋白酶体降解。DUB结构域的缺失不影响pUL48的核定位和胞质定位，而内部区域（360 - 1200）对于胞质分布是必需的。在免疫共沉淀实验中，pUL48与三种包膜蛋白（pUL47、pUL45和pUL88）以及两种衣壳蛋白（pUL77和pUL85）相互作用，但DUB结构域仅有助于与pUL85的结合。此外，我们发现ΔDUB病毒显示出病毒体稳定性降低，并且与野生型病毒相比，将其基因组传递到细胞中的效率较低。总体而言，我们的结果表明，pUL48的N端DUB结构域通过调节其自身稳定性、促进病毒体稳定和病毒进入来促进病毒的有效生长。
HCMV pUL48及其疱疹病毒同源物在病毒进入、免疫信号通路调节和病毒体组装中起关键作用。pUL48的N端包含DUB结构域，该结构域在所有疱疹病毒中高度保守。尽管使用活性位点突变病毒的研究表明DUB活性促进病毒生长，但该区域在病毒生命周期中的确切作用尚未完全了解。在本研究中，使用缺失整个DUB结构域的突变病毒，我们证明pUL48的DUB结构域通过自去泛素化调节其自身稳定性、促进病毒体稳定和病毒进入来促进病毒生长。本报告首次展示了缺失整个DUB结构域的突变病毒的特性，连同该区域功能的信息，有助于剖析与pUL48相关的功能。
