Human Cytomegalovirus pp28 (UL99) Localizes to a Cytoplasmic Compartment Which Overlaps the Endoplasmic Reticulum-Golgi-Intermediate Compartment

Although the assembly of herpesviruses has remained an active area of investigation, considerable controversy continues to surround the cellular location of tegument and envelope acquisition. This controversy is particularly evident when the proposed pathways for α- and β-herpesvirus assembly are compared. We have approached this aspect of human cytomegalovirus (HCMV) assembly, specifically, envelopment, by investigating the intracellular trafficking of viral tegument proteins which localize in the cytoplasms of infected cells. In this study we have demonstrated that the virion tegument protein pp28 (UL99), a true late protein, was membrane associated as a result of myristoylation. A mutation in this protein which prevented incorporation of [(3)H]myristic acid also altered the detergent solubility and intracellular distribution of the protein when it was expressed in transfected cells. Using a panel of markers for intracellular compartments, we could localize the expression of wild-type pp28 to an intracellular compartment which colocalized with the endoplasmic reticulum-Golgi-intermediate compartment (ERGIC), a dynamic compartment of the secretory pathway which interfaces with both the ER and Golgi apparatus. The localization of this viral tegument protein within an early secretory compartment of the cell provided further evidence that the assembly of the HCMV tegument likely includes a cytoplasmic phase. Because pp28 has been shown to be localized to a cytoplasmic assembly compartment in HCMV-infected cells, our findings also suggested that viral tegument protein interactions within the secretory pathway may have an important role in the assembly of the virion.

尽管疱疹病毒的组装始终是研究热点领域，但围绕皮层（tegument）的细胞定位以及囊膜（envelope）获取的相关争议仍持续存在。当对比α疱疹病毒与β疱疹病毒的组装通路假说时，这类争议表现得尤为突出。本研究以人类巨细胞病毒（human cytomegalovirus, HCMV）的组装过程（具体为囊膜获取环节）为研究对象，通过追踪受感染细胞胞质内病毒皮层蛋白的细胞内运输过程展开相关探究。本研究证实，作为真晚期蛋白的病毒体（virion）皮层蛋白pp28（UL99）可通过肉豆蔻酰化（myristoylation）过程与膜结构结合。当该蛋白在转染细胞中表达时，若其发生突变从而阻断[³H]肉豆蔻酸的掺入，还会改变该蛋白的去污剂溶解性与细胞内分布特征。通过使用一系列细胞内区室的标志物组合，我们可将野生型pp28的表达定位至与内质网-高尔基体中间区室（endoplasmic reticulum-Golgi intermediate compartment, ERGIC）共定位的细胞内区室；ERGIC是分泌通路中连接内质网（ER）与高尔基体（Golgi apparatus）的动态区室。该病毒皮层蛋白定位于细胞早期分泌区室这一发现，进一步佐证了HCMV皮层的组装过程可能包含胞质阶段。鉴于已有研究证实pp28可定位于HCMV感染细胞的胞质组装区室，本研究结果还表明，分泌通路内的病毒皮层蛋白相互作用可能在病毒体组装过程中发挥重要作用。