The ESCRT Machinery Is Recruited by the Viral BFRF1 Protein to the Nucleus-Associated Membrane for the Maturation of Epstein-Barr Virus

The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.     

Subviral Dense Bodies of Human Cytomegalovirus Stimulate Maturation and Activation of Monocyte-Derived Immature Dendritic Cells

Dendritic cells play a central role in the immune control of human cytomegalovirus (HCMV) infection. This work aimed at investigating the impact of noninfectious, subviral dense bodies of HCMV on the maturation and activation of dendritic cells (DC). Treatment of immature DC with dense bodies led to the maturation of these cells and significantly increased their capacity for cytokine release and antigen presentation. Dense body-activated DC may thereby contribute to the development of antiviral immunity.     

A Septin Double Ring Controls the Spatiotemporal Organization of the ESCRT Machinery in Cytokinetic Abscission

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An Endothelial Cell-Specific Requirement for the UL133-UL138 Locus of Human Cytomegalovirus for Efficient Virus Maturation

Human cytomegalovirus (HCMV) infects a variety of cell types in humans, resulting in a varied pathogenesis in the immunocompromised host. Endothelial cells (ECs) are considered an important target of HCMV infection that may contribute to viral pathogenesis. Although the viral determinants important for entry into ECs are well defined, the molecular determinants regulating postentry tropism in ECs are not known. We previously identified the UL133-UL138 locus encoded within the clinical strain-specific ULb′ region of the HCMV genome as important for the latent infection in CD34⁺ hematopoietic progenitor cells (HPCs). Interestingly, this locus, while dispensable for replication in fibroblasts, was required for efficient replication in ECs infected with the TB40E or fusion-inducing factor X (FIX) HCMV strains. ECs infected with a virus lacking the entire locus (UL133-UL138_NULL virus) complete the immediate-early and early phases of infection but are defective for infectious progeny virus production. ECs infected with UL133-UL138_NULL virus exhibited striking differences in the organization of intracellular membranes and in the assembly of mature virions relative to ECs infected with wild-type (WT) virus. In UL133-UL138_NULL virus-infected ECs, Golgi stacks were disrupted, and the viral assembly compartment characteristic of HCMV infection failed to form. Further, progeny virions in UL133-UL138_NULL virus-infected ECs inefficiently acquired the virion tegument and secondary envelope. These defects were specific to infection in ECs and not observed in fibroblasts infected with UL133-UL138_NULL virus, suggesting an EC-specific requirement for the UL133-UL138 locus for late stages of replication. To our knowledge, the UL133-UL138 locus represents the first cell-type-dependent, postentry tropism determinant required for viral maturation.     

Characterization and Intracellular Trafficking of Epstein-Barr Virus BBLF1, a Protein Involved in Virion Maturation

Epstein-Barr virus (EBV) BBLF1 shares 13 to 15% amino acid sequence identities with the herpes simplex virus 1 UL11 and cytomegalovirus UL99 tegument proteins, which are involved in the final envelopment during viral maturation. This study demonstrates that BBLF1 is a myristoylated and palmitoylated protein, as are UL11 and UL99. Myristoylation of BBLF1 both facilitates its membrane anchoring and stabilizes it. BBLF1 is shown to localize to the trans-Golgi network (TGN) along with gp350/220, a site where final envelopment of EBV particles takes place. The localization of BBLF1 at the TGN requires myristoylation and two acidic clusters, which interact with PACS-1, a cytosolic protein, to mediate retrograde transport from the endosomes to the TGN. Knockdown of the expression of BBLF1 during EBV lytic replication reduces the production of virus particles, demonstrating the requirement of BBLF1 to achieve optimal production of virus particles. BBLF1 is hypothesized to facilitate the budding of tegumented capsid into glycoprotein-embedded membrane during viral maturation.     

HCMV感染抑制人海马神经干细胞分化

研究HCMV感染对体外培养的人海马源性神经干细胞(Neural stem cells,NSCs)分化的影响。体外分离、培养人海马NSCs,应用免疫荧光方法检测其NSCs标记物-巢蛋白(Nestin)的表达。10%胎牛血清诱导NSCs贴壁分化,同时用MOI为5的HCMV AD169株感染NSCs,7d后使用激光共聚焦显微镜免疫荧光方法检测Nestin、神经胶质纤维酸性蛋白(GFAP)和HCMV即刻早期蛋白(IE)的表达,计算阳性细胞比率。本实验所培养的细胞(4～6代)95±8%表达Nestin;分化诱导7d后,感染组86±12%细胞表达IE,未感染组和感染组Nestin阳性率分别为50±19%和93±10%(t=6.03,P<0.01),GFAP阳性细胞率分别为81±11%和55±17%(t=3.77,P<0.01)。以上结果表明分化过程中的NSCs是HCMV的容许细胞;HCMV感染可以抑制NSCs的分化。     

Crystal Structure of the Human Cytomegalovirus Glycoprotein B

Human cytomegalovirus (HCMV), a dsDNA, enveloped virus, is a ubiquitous pathogen that establishes lifelong latent infections and caused disease in persons with compromised immune systems, e.g., organ transplant recipients or AIDS patients. HCMV is also a leading cause of congenital viral infections in newborns. Entry of HCMV into cells requires the conserved glycoprotein B (gB), thought to function as a fusogen and reported to bind signaling receptors. gB also elicits a strong immune response in humans and induces the production of neutralizing antibodies although most anti-gB Abs are non-neutralizing. Here, we report the crystal structure of the HCMV gB ectodomain determined to 3.6-Å resolution, which is the first atomic-level structure of any betaherpesvirus glycoprotein. The structure of HCMV gB resembles the postfusion structures of HSV-1 and EBV homologs, establishing it as a new member of the class III viral fusogens. Despite structural similarities, each gB has a unique domain arrangement, demonstrating structural plasticity of gB that may accommodate virus-specific functional requirements. The structure illustrates how extensive glycosylation of the gB ectodomain influences antibody recognition. Antigenic sites that elicit neutralizing antibodies are more heavily glycosylated than those that elicit non-neutralizing antibodies, which suggest that HCMV gB uses glycans to shield neutralizing epitopes while exposing non-neutralizing epitopes. This glycosylation pattern may have evolved to direct the immune response towards generation of non-neutralizing antibodies thus helping HCMV to avoid clearance. HCMV gB structure provides a starting point for elucidation of its antigenic and immunogenic properties and aid in the design of recombinant vaccines and monoclonal antibody therapies.     

Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis

TSG101 and ALIX both function in HIV budding and in vesicle formation at the multivesicular body (MVB), where they interact with other Endosomal Sorting Complex Required for Transport (ESCRT) pathway factors required for release of viruses and vesicles. Proteomic analyses revealed that ALIX and TSG101/ESCRT-I also bind a series of proteins involved in cytokinesis, including CEP55, CD2AP, ROCK1, and IQGAP1. ALIX and TSG101 concentrate at centrosomes and are then recruited to the midbodies of dividing cells through direct interactions between the central CEP55 'hinge' region and GPP-based motifs within TSG101 and ALIX. ESCRT-III and VPS4 proteins are also recruited, indicating that much of the ESCRT pathway localizes to the midbody. Depletion of ALIX and TSG101/ESCRT-I inhibits the abscission step of HeLa cell cytokinesis, as does VPS4 overexpression, confirming a requirement for these proteins in cell division. Furthermore, ALIX point mutants that block CEP55 and CHMP4/ESCRT-III binding also inhibit abscission, indicating that both interactions are essential. These experiments suggest that the ESCRT pathway may be recruited to facilitate analogous membrane fission events during HIV budding, MVB vesicle formation, and the abscission stage of cytokinesis.     

人巨细胞病毒疫苗研究进展

人巨细胞病毒(Human cytomegalovirus, HC-MV)是一类在自然界普遍存在的具有严格种属特异性的病毒,属β疱疹病毒亚科。Zsofia Gyulai等[1]对HCMV感染作了流行病学调查,发现体内抗病毒体液免疫反应主要针对gB;而细胞免疫反应主要针对pp65、pp150。根据HCMV感染的特点和机制,以上     

人巨细胞病毒疫苗研究进展

人巨细胞病毒(Human cytomegalovirus,HCMV)是一类在自然界普遍存在的具有严格种属特异性的病毒,属β疱疹病毒亚科.Zsofia Gyulai等[1]对HCMV感染作了流行病学调查,发现体内抗病毒体液免疫反应主要针对gB;而细胞免疫反应主要针对pp65、pp150.根据HCMV感染的特点和机制,以上述几种结构糖蛋白设计候选基因工程疫苗,已得到了广泛的研究,包括基因工程亚单位疫苗、质粒载体DNA疫苗、病毒载体DNA疫苗、合成蛋白(肽)疫苗,另外还有Towne减毒活疫苗.在国外,有的疫苗已完成Ⅱ期临床试验研究[2].本文现就HCMV疫苗的种类及优缺点、相关佐剂、展望进行综述.     

人巨细胞病毒与肿瘤研究进展

人巨细胞病毒（HCMV）是目前已知最大的β疱疹病毒。HCMV感染具有持续性和潜在性,感染率在全世界范围内都很高,并随着年龄的增长而升高,女性感染率高于男性,其主要的传播途径有垂直传播和性传播等。近年来的研究显示在人类胶质瘤、结直肠癌、乳腺癌和前列腺癌及小细胞型肝癌等多种类型的肿瘤组织中都存在HCMV感染和病毒基因表达,并与肿瘤的恶性程度有相关性,这提示HCMV可能在人类某些类型肿瘤的形成和发展过程中扮演重要角色,有可能成为一种新的人类肿瘤相关病毒。HCMV基因产物可通过多种细胞信号通路抑制细胞凋亡、促进细胞增殖、侵袭、转移和血管生成,并形成独特的免疫逃避机制对抗机体免疫反应。深入研究HCMV与肿瘤的病因学关系及其作用机制,可为肿瘤的临床防治提供新思路。