The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation
The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation
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DOI：
10.1128/jvi.00384-16
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发表时间：
2016-07-01
影响因子：
5.4
通讯作者：
Messerle, Martin
中科院分区：
文献类型：
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作者：
Borst, Eva Maria;Bauerfeind, Rudolf;Messerle, Martin
几种必需的病毒蛋白被认为参与了人巨细胞病毒（HCMV）的基因组扩增，其中包括pUL 77和pUL 93，它们在很大程度上仍未被表征。为了深入了解它们的特性，我们产生了表达pUL 77单体增强型绿色荧光蛋白（mGFP）融合蛋白和pUL 93特异性抗体的HCMV突变体。免疫印迹表明，这两种蛋白质被纳入衣壳和病毒粒子。相反的数据表明内部翻译起始位点内的UL 93开放阅读框（ORF），我们提供的证据表明，pUL 93的合成开始于第一个起始密码子。在感染的细胞中，pUL 77-mGFP被发现在核复制区室和点样结构中，与衣壳蛋白共定位。核衣壳的免疫金标记显示pUL 77存在于A、B和C衣壳上。pUL 77-mGFP的下拉揭示了pUL 93的共纯化，表明这些蛋白质之间的相互作用，当衣壳形成被阻止时仍然发生。pUL 77-mGFP的正确亚核分布需要pUL 93以及主要衣壳蛋白（因此可能存在衣壳），但不需要被膜蛋白pp 150或衣壳化蛋白pUL 52，这表明pUL 77核靶向的发生独立于DNA填充的衣壳的形成。当pUL 77或pUL 93缺失时，未观察到单位长度基因组的产生，并且仅产生空的B衣壳。总之，这些结果表明pUL 77和pUL 93是HCMV基因组衣壳化所需的衣壳组分。因此，pUL 77的任务似乎不同于其α疱疹病毒直向同源物pUL 25的任务，pUL 25在基因组切割包装后发挥其功能。重要提示：必需的HCMV蛋白质pUL 77和pUL 93被认为参与病毒基因组切割包装，但在生化和功能上的特征都很差。通过产生针对pUL 93的单克隆抗体并产生其中pUL 77与荧光蛋白融合的HCMV突变体，我们表明pUL 77和pUL 93是衣壳成分，其中pUL 77在所有衣壳类型上类似地丰富。每种蛋白质都是基因组衣壳化所必需的，因为pUL 77或pUL 93的缺失导致基因组包装缺陷，仅形成空衣壳。这将pUL 77与其α疱疹病毒直向同源物pUL 25区分开来，后者富集在DNA填充的衣壳上，并在病毒DNA包装后发挥其功能。我们的数据首次描述了一个荧光衣壳的HCMV突变体，并提供了对pUL 77和pUL 93的作用的深入了解，从而有助于更好地理解HCMV衣壳化网络。
Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus ( HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein ( and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging.IMPORTANCEThe essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.