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Mayuri Sharma Jeremy P. Kamil Margaret Coughlin Natalia I. Reim Donald M. Coen 《Journal of virology》2014,88(1):249-262
Herpesvirus nucleocapsids traverse the nuclear envelope into the cytoplasm in a process called nuclear egress that includes disruption of the nuclear lamina. In several herpesviruses, a key player in nuclear egress is a complex of two proteins, whose homologs in human cytomegalovirus (HCMV) are UL50 and UL53. However, their roles in nuclear egress during HCMV infection have not been shown. Based largely on transfection studies, UL50 and UL53 have been proposed to facilitate disruption of the nuclear lamina by recruiting cellular protein kinase C (PKC), as occurs with certain other herpesviruses, and/or the viral protein kinase UL97 to phosphorylate lamins. To investigate these issues during HCMV infection, we generated viral mutants null for UL50 or UL53. Correlative light electron microscopic analysis of null mutant-infected cells showed the presence of intranuclear nucleocapsids and the absence of cytoplasmic nucleocapsids. Confocal immunofluorescence microscopy revealed that UL50 and UL53 are required for disruption of the nuclear lamina. A subpopulation of UL97 colocalized with the nuclear rim, and this was dependent on UL50 and, to a lesser extent, UL53. However, PKC was not recruited to the nuclear rim, and its localization was not affected by the absence of UL50 or UL53. Immunoprecipitation from cells infected with HCMV expressing tagged UL53 detected UL97 but not PKC. In summary, HCMV UL50 and UL53 are required for nuclear egress and disruption of nuclear lamina during HCMV infection, and they recruit UL97, not PKC, for these processes. Thus, despite the strong conservation of herpesvirus nuclear egress complexes, a key function can differ among them. 相似文献
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根据GenBank已发表的PrVul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP。酶切鉴定,测序及WesternBlot验证重组质粒。ul24基因序列测定结果已提交GenBank,登录号DQ226544。Westernblot分析结果表明UL24-GFP融合蛋白为45KD。将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核。 相似文献
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根据GenBank已发表的PrV ul24基因序列(NC006151),设计并合成一对引物,PCR扩增出ul24基因编码区,克隆于pEGFP-N1载体,得到重组质粒pUL24-GFP.酶切鉴定,测序及Western Blot验证重组质粒.ul24基因序列测定结果已提交GenBank,登录号DQ226544.Western blot分析结果表明UL24-GFP融合蛋白为45KD.将pUL24-GFP转染真核细胞,激光共聚焦显微镜观察融合蛋白的细胞内定位,结果表明UL24-GFP融合蛋白定位于细胞核. 相似文献
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从人胎脑c DNA文库中筛选和鉴定出与人巨细胞病毒(Human cytomegalovirus,HCMV)UL55编码蛋白结合的蛋白。将UL55基因编码区克隆到诱饵载体p GBKT7中,在证实UL55蛋白不具有自激活作用的前提下,采用Match-maker GAL酵母双杂交系统筛选人胎脑c DNA文库中与UL55蛋白结合的宿主蛋白,用酵母双杂交回转实验验证UL55蛋白与获得的蛋白结合的可靠性。将酵母双杂交筛选出的文库蛋白烯醇化酶1(enolase1,ENO 1)构建到p GEX-4T-2载体上,利用GST pull-down技术体外验证ENO 1与HCMV UL55蛋白的结合。并依据所筛选出蛋白的生物学功能分析UL55蛋白可能的生物学功能。结果显示有10种蛋白与HCMV UL55编码蛋白结合。应用GST pull-down技术检测到ENO 1与HCMV UL55相互结合的蛋白条带。成功地筛选出10种与UL55蛋白相互结合的宿主蛋白,GST pull-down实验进一步表明ENO 1可以与HCMV UL55蛋白直接结合,为进一步研究UL55蛋白的功能提供了新的线索。 相似文献
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Lei GUO Ying ZHANG Yan-chun CHE Wen-juan WU Wei-zhong LI Li-chun WANG Yun LIAO Long-ding LIU Qi-han LI 《Virologica Sinica》2008,23(3):211-217
An interaction between the HSV-1 UL25 capsid protein and cellular microtubule-associated protein was found using a yeast two-hybrid screen and β-D-galactosidase activity assays. Immunofluorescence microscopy of the UL25 protein demonstrated its co-localization with cellular microtubule-associated protein in the plasma membrane. Further investigations with deletion mutants suggest that UL25 is likely to have a function in the nucleus. 相似文献
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The tegument of all herpesviruses contains a high-molecular-weight protein homologous to herpes simplex virus (HSV) UL36. This large (3,164 amino acids), essential, and multifunctional polypeptide is located on the capsid surface and present at 100 to 150 copies per virion. We have been testing the idea that UL36 is important for the structural organization of the tegument. UL36 is proposed to bind directly to the capsid with other tegument proteins bound indirectly by way of UL36. Here we report the results of studies carried out with HSV type 1-derived structures containing the capsid but lacking a membrane and depleted of all tegument proteins except UL36 and a second high-molecular-weight protein, UL37. Electron microscopic analysis demonstrated that, compared to capsids lacking a tegument, these capsids (called T36 capsids) had tufts of protein located at the vertices. Projecting from the tufts were thin, variably curved strands with lengths (15 to 70 nm) in some cases sufficient to extend across the entire thickness of the tegument (∼50 nm). Strands were sensitive to removal from the capsid by brief sonication, which also removed UL36 and UL37. The findings are interpreted to indicate that UL36 and UL37 are the components of the tufts and of the thin strands that extend from them. The strand lengths support the view that they could serve as organizing features for the tegument, as they have the potential to reach all parts of the tegument. The variably curved structure of the strands suggests they may be flexible, a property that could contribute to the deformable nature of the tegument.All herpesviruses have a tegument, a layer of protein located between the virus capsid and membrane. The tegument accounts for a substantial proportion of the overall virus structure. Its thickness (30 to 50 nm), for example, may be comparable to the capsid radius, and tegument proteins can account for 40% or more of the total virion protein. Herpesvirus tegument proteins are thought to function promptly after initiation of infection, before expression of virus genes can take place (11, 13, 14, 21, 33, 37).Electron microscopic analysis of virions has demonstrated that the tegument is not highly structured (9, 22). It does not have icosahedral symmetry like the capsid, and it may be uniformly or asymmetrically arranged around the capsid (26). Tegument structure is described as fibrous or granular, and its morphology is found to change as the virus matures. Studies with herpes simplex virus type 1 (HSV-1), for example, indicate that the tegument structure is altered in cell-associated compared to extracellular virus (26).The tegument has been most thoroughly studied in HSV-1, where biochemical analyses indicate that it is composed of approximately 20 distinct, virus-encoded protein species. The predominant components are the products of the genes UL47, UL48, and UL49, with each protein present in 800 or more copies per virion (12, 40). Other tegument proteins can occur in 100 or fewer copies, and trace amounts of cell-encoded proteins are also present (17). Tegument proteins are classified as inner or outer components based on their association with the capsid after it enters the host cell cytoplasm. The inner tegument proteins (UL36, UL37, and US3) are those that remain bound to the capsid after entry, while the others (the outer tegument proteins) become detached (7, 18).The HSV-1 UL36 protein has the potential to play a central role in organizing the overall structure of the tegument. With a length of 3,164 amino acids, UL36 could span the thickness of the tegument multiple times. One hundred to 150 UL36 molecules are present in the tegument (12), and they are bound to the capsid by way of an essential C-terminal domain (2, 16). UL36 is able to bind the major tegument components by way of documented direct (UL37 and UL48) and indirect (UL46, UL47, and UL49) contacts (6, 15, 24, 38).Here we describe the results of studies designed to test the idea that UL36 serves to organize the tegument structure. Beginning with infectious virus, a novel method has been used to isolate capsids that contain UL36 and UL37 but lack the virus membrane and are depleted of all other tegument proteins. These capsids (T36 capsids) were examined by electron microscopy to clarify the structure of UL36 and UL37 molecules and their location on the capsid surface. 相似文献
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In addition to eleven glycoproteins, the herpes simplex virus type 2 (HSV-2) genome encodes several proteins with potential membrane-spanning segments but no asparagine-linked carbohydrates. One of these is UL45. Fractionation of infected cells showed that HSV-2 UL45 is an integral membrane protein, and analysis of UL45 mutants with potential glycosylation sites showed that it has a type II membrane orientation, the first HSV protein known to have this orientation. Furthermore, it is detectable in infected cells at a time similar to that when glycoproteins gB and gD are detected, consistent with a role in cell-cell fusion, which has previously been found for HSV-1 UL45. 相似文献
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Ying Zhang Yan-mei Li Long-ding Liu Li Jiang Ma Ji Rui-ju Jiang Lei Guo Yun Liao Qi-han Li 《Virologica Sinica》2011,(3)
In light of the scarcity of reports on the interaction between HSV-1 nucleocapsid protein UL25 and its host cell proteins,the purpose of this study is to use yeast two-hybrid screening to search for cellular proteins that can interact with the UL25 protein.C9orf69,a protein of unknown function was identified.The interaction between the two proteins under physiological conditions was also confirmed by biological experiments including co-localization by fluorescence and immunoprecipitation.A preliminary study... 相似文献
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The function of the herpes simplex virus type 1(HSV-1)UL4 protein is still elusive. Our objective is to investigate the subcellular transport mechanism of the UL4 protein. In this study,fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells. By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein(EYFP),the nuclear export signals(NES)of UL4 were for the first time mapped to amino acid residues 178 to 186. In addition,the N-terminal 19 amino acids are identified to be required for the granule-like cytoplasmic pattern of UL4.Furthermore,the UL4 protein was demonstrated to be exported to the cytoplasm through the NES in a chromosomal region maintenance 1(CRM l)-dependent manner involving RanGTP hydrolysis. 相似文献
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Consistent with earlier analyses of human cytomegalovirus UL36 mRNA, we find that the UL36 protein is present throughout infection. In fact, it is delivered to the infected cell as a constituent of the virion. Curiously, much less UL36 protein accumulated in cells infected with the AD169 strain of human cytomegalovirus than in cells infected with the Towne or Toledo strain, and localization of the protein in cells infected with AD169 is strikingly different from that in cell infected with the Towne or Toledo strain. The variation in steady-state level of the proteins results from different stabilities of the proteins. The UL36 proteins from the three viral strains differ by several amino acid substitutions. However, this variability is not responsible for the different half-lives because the AD169 and Towne proteins, which exhibit very different half-lives within infected cells, exhibit the same half-life when introduced into uninfected cells by transfection with expression plasmids. We demonstrate that the UL36 protein is nonessential for growth in cultured cells, and we propose that the ability of the virus to replicate in the absence of UL36 function likely explains the striking strain-specific variation in the half-life and intracellular localization of the protein. 相似文献
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The function of the herpes simplex virus type 1 (HSV-1) UL4 protein is still elusive.Our objective is to investigate the subcellular transport mechanism of the UL4 protein.In this study,fluorescence microscopy was employed to investigate the subcellular localization of UL4 and characterize the transport mechanism in living cells.By constructing a series of deletion mutants fused with enhanced yellow fluorescent protein (EYFP),the nuclear export signals (NES) of UL4 were for the first time mapped to amino ac... 相似文献