Transcriptional activation of endoplasmic reticulum chaperone GRP78 by HCMV IE1-72 protein

Glucose-regulated protein 78 (GRP78), a key regulator of endoplasmic reticulum (ER) stress, facilitates cancer cell growth and viral replication. The mechanism leading to grp78 gene activation during viral infection is largely unknown. In this study, we show that the immediate-early 1 (IE1-72) protein of the human cytomegalovirus (HCMV) is essential for HCMV-mediated GRP78 activation. IE1-72 upregulated grp78 gene expression depending on the ATP-binding site, the zinc-finger domain and the putative leucine-zipper motif of IE1-72, as well as the ER stress response elements (ERSEs) on the grp78 promoter. The purified IE1-72 protein bound to the CCAAT box within ERSE in vitro, whereas deletion mutants of IE1-72 deficient in grp78 promoter stimulation failed to do so. Moreover, IE1-72 binding to the grp78 promoter in infected cells accompanied the recruitment of TATA box-binding protein-associated factor 1 (TAF1), a histone acetyltransferase, and the increased level of acetylated histone H4, an indicator of active-state chromatin. These results provide evidence that HCMV IE1-72 activates grp78 gene expression through direct promoter binding and modulation of the local chromatin structure, indicating an active viral mechanism of cellular chaperone induction for viral growth.     

人巨细胞病毒IE1-72蛋白在胶质瘤中的表达及意义

目的:探讨人巨细胞病毒(HCMV)立刻早期基因1-72(IE1-72)蛋白在胶质瘤中的表达水平以及HCMV感染与胶质瘤发生的病因学关系。方法:采用免疫组化方法检测HCMV IE1-72蛋白在125例人脑胶质瘤组织及10例正常人脑组织中的表达,分析其表达水平与胶质瘤临床病理学特征的关系。结果:IE1-72蛋白在胶质瘤组织中的表达水平明显高于正常人脑组织(P=0.000);IE1-72蛋白免疫染色强度随胶质瘤病理级别的升高而明显增强(r=0.310,P=0.000),其在高恶性度胶质瘤中的染色强度明显强于低恶性度胶质瘤(P=0.004);IE1-72蛋白染色强度与胶质瘤患者的年龄存在正相关(r=0.234,P=0.009),而与胶质瘤患者的性别(r=0.038,P=0.675)以及肿瘤部位(r=0.086,P=0.341)无明显相关性。结论:HCMV感染及其蛋白IE1-72表达可能与人脑胶质瘤的发生和发展密切相关,但其确切的致瘤机制尚需进一步研究。     

The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells.

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.     

A protein kinase CK2 site flanking the nuclear targeting signal enhances nuclear transport of human cytomegalovirus ppUL44

The processivity factor of the human cytomegalovirus (HCMV) DNA polymerase phosphoprotein ppUL44 plays an essential role in viral replication, showing nuclear localization in infected cells. The present study examines ppUL44's nuclear import pathway for the first time, ectopic expression of ppUL44 revealing a strong nuclear localization in transfected COS-7 and other cell types, implying that no other HCMV proteins are required for nuclear transportation and retention. We show that of the two potential nuclear localization signals (NLSs) located at amino acids 162-168 (NLS1) and 425-431 (NLS2), NLS2 is necessary and sufficient to confer nuclear localization. Moreover, using enzyme-linked immunosorbent assays and gel mobility shift assays, we show that NLS2 is recognized with high affinity by the importin (IMP) alpha/beta heterodimer. Using gel mobility shift and transient transfection assays, we find that flanking sequences containing a cluster of potential phosphorylation sites, including a consensus site for protein kinase CK2 (CK2) at Ser413 upstream of the NLS, increase NLS2-dependent IMP binding and nuclear localization, suggesting a role for these sites in enhancing UL44 nuclear transport. Results from site-directed mutagenic analysis and live-cell imaging of green fluorescent protein (GFP)-UL44 fusion protein-expressing cells treated with the CK2-specific inhibitor 4,5,6,7-tetrabromobenzotriazole are consistent with phosphorylation of Ser413 enhancing ppUL44 nuclear transport.     

Dynamic nuclear localization of the baculovirus proteins IE2 and PE38 during the infection cycle: the promyelocytic leukemia protein colocalizes with IE2

The early gene products IE2 and PE38 of Autographa californica multicapsid nuclear polyhedrosis virus localize to distinct nuclear domains after transient expression. Here, the nuclear localization pattern and the putative association with cellular proteins have been determined during virus infection to shed light on the functional significance of the nuclear domains. IE2 was always localized to distinct nuclear structures while PE38 was partly present in nuclear dots. Confocal imaging indicated colocalization of PE38 and IE2 to common domains, prominently at 2 h p.i. The nuclear dot localization of PE38 in infected cells was different from that in transfected cells. Hence, we have performed cotransfection experiments that suggested that a viral factor influences the nuclear distribution. Since the promyelocytic leukemia protein (PML) that localizes to distinct nuclear multiprotein complexes termed ND10/PODs in mammalian cells functions as a target for some immediate early viral proteins, we have investigated whether baculovirus proteins act similarly. Transiently expressed IE2 and PE38 were found to be associated with endogenous PML in the mammalian cell line BHK21. Infection with a recombinant virus that expresses the human pml gene in insect cells reveals IE2 and PML to be colocalized during the early phase of infection followed by a redistribution of both proteins. Taken together our results provide first evidence that the early baculovirus protein IE2 associates at least with one component of mammalian PODs during virus infection, suggesting that POD-like structures can be formed in insect cells.     

Cell-cycle-dependent localization of human cytomegalovirus UL83 phosphoprotein in the nucleolus and modulation of viral gene expression in human embryo fibroblasts in vitro

The nucleolus is a multifunctional nuclear compartment widely known to be involved in several cellular processes, including mRNA maturation and shuttling to cytoplasmic sites, control of the cell cycle, cell proliferation, and apoptosis; thus, it is logical that many viruses, including herpesvirus, target the nucleolus in order to exploit at least one of the above-mentioned functions. Recent studies from our group demonstrated the early accumulation of the incoming ppUL83 (pp65), the major tegument protein of human cytomegalovirus (HCMV), in the nucleolus. The obtained results also suggested that a functional relationship might exist between the nucleolar localization of pp65, rRNA synthesis, and the development of the lytic program of viral gene expression. Here we present new data which support the hypothesis of a potentially relevant role of HCMV pp65 and its nucleolar localization for the control of the cell cycle by HCMV (arrest of cell proliferation in G1-G1/S), and for the promotion of viral infection. We demonstrated that, although the incoming pp65 amount in the infected cells appears to be constant irrespective of the cell-cycle phase, its nucleolar accumulation is prominent in G1 and G1/S, but very poor in S or G2/M. This correlates with the observation that only cells in G1 and G1/S support an efficient development of the HCMV lytic cycle. We propose that HCMV pp65 might be involved in regulatory/signaling pathways related to nucleolar functions, such as the cell-cycle control. Co-immunoprecipitation experiments have permitted to identify nucleolin as one of the nucleolar partners of pp65.     

Human cytomegalovirus infection dysregulates neural progenitor cell fate by disrupting Hes1 rhythm and down-regulating its expression

Human cytomegalovirus(HCMV) infection is a leading cause of birth defects, primarily affecting the central nervous system and causing its maldevelopment. As the essential downstream effector of Notch signaling pathway, Hes1, and its dynamic expression, plays an essential role on maintaining neural progenitor/stem cells(NPCs) cell fate and fetal brain development. In the present study, we reported the first observation of Hes1 oscillatory expression in human NPCs, with an approximately1.5 hour periodicity and a Hes1 protein half-life of about 17(17.6 ± 0.2) minutes. HCMV infection disrupts the Hes1 rhythm and down-regulates its expression. Furthermore, we discovered that depleting Hes1 protein disturbed NPCs cell fate by suppressing NPCs proliferation and neurosphere formation, and driving NPCs abnormal differentiation. These results suggested a novel mechanism linking disruption of Hes1 rhythm and down-regulation of Hes1 expression to neurodevelopmental disorders caused by congenital HCMV infection.     

PIAS1 enhances SUMO-1 modification and the transactivation activity of the major immediate-early IE2 protein of human cytomegalovirus

The protein inhibitor of activated STAT1 (PIAS1), known to be a small ubiquitin-like modifier (SUMO) E3 ligase, was found to interact with the human cytomegalovirus IE2 protein. We found that the sumoylation of IE2 was markedly enhanced by wild-type PIAS1 but not by a mutant containing a Cys to Ser substitution at position 351 (C351S) within the RING finger-like domain. In target reporter gene assays, wild-type PIAS1, but not the C351S mutant, enhanced the IE2-mediated transactivations of viral polymerase promoter and cellular cyclin E promoter and this augmentation required the intact sumoylation sites of IE2. Our results suggest that PIAS1 acts as a SUMO E3 ligase toward IE2 and that it may regulate the transactivation function of IE2. To our knowledge, IE2 is the first viral target found to be regulated by a SUMO E3 ligase.     

Spatial patterns of protein expression in focal infections of human cytomegalovirus

Human cytomegalovirus (HCMV) is a medically significant human pathogen that infects a wide range of cell and tissue types. During infection, HCMV activates a variety of signal transduction pathways that induce profound changes in cellular processes and dramatically affect cellular gene expression patterns. To better define how these virus-host interactions affect the local microenvironment and influence the spatial and temporal spread of HCMV, we initiated HCMV focal infections on normal human dermal fibroblast monolayers and monitored viral gene expression patterns and infection spread over 45 days. To establish baseline temporal measurements of HCMV infection and spread in cell monolayers, we characterized the influence of three experimental variables on viral gene expression: cell plating density, the presence of serum, and neutralization of cellular antiviral responses with an antibody against interferon-beta. We found that high cell plating density or the inclusion of serum correlated with enhanced HCMV infection spread. Dramatic differences in the expression pattern of the viral immediate early 2 (IE2) gene were observed under these conditions as compared to low plating density or the absence of serum. In the latter case round, uniform foci were observed with a clear wave of IE2 expression visible in advance of a late stage viral protein, envelope glycoprotein B. By contrast, larger irregular foci with arms of IE2 expression were observed in the presence of serum. Addition of the antibody had little effect on the rate of spread, which is consistent with the knowledge that HCMV represses antiviral responses during infection. This experimental system provides a useful means to visualize and quantify complex virus-host interactions.     

Cytoplasmic targeting of mutant poly(A)-binding protein nuclear 1 suppresses protein aggregation and toxicity in oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. The autosomal dominant form of this disease is caused by a polyalanine expansion from 10 to 12-17 residues, located at the N-terminus of the poly(A)-binding protein nuclear 1 (PABPN1). A distinct pathological hallmark of OPMD is the presence of filamentous intranuclear aggregates in patients' skeletal muscle cells. Wildtype PABPN1 protein is expressed ubiquitously and was shown to be mostly concentrated in discrete nuclear domains called 'speckles'. Using an established cell- culture model, we show that most mutant PABPN1- positive (alanine expanded form) intranuclear aggregates are structures distinct from intranuclear speckles. In contrast, the promyelocytic leukaemia protein, a major component of nuclear bodies, strongly colocalized to intranuclear aggregates of mutant PABPN1. Wildtype PABPN1 can freely shuttle between the nucleus and cytoplasm. We determined whether the nuclear environment is necessary for mutant PABPN1 inclusion formation and cellular toxicity. This was achieved by inactivating the mutant PABPN1 nuclear localization signal and by generating full-length mutant PABPN1 fused to a strong nuclear export sequence. A green fluorescence protein tag inserted at the N-terminus of both wildtype PABPN1 (ala10) and mutant PABPN1 (ala17) proteins allowed us to visualize their subcellular localization. Targeting mutant PABPN1 to the cytoplasm resulted in a significant suppression of both intranuclear aggregates formation and cellular toxicity, two histological consequences of OPMD. Our results indicate that the nuclear localization of mutant PABPN1 is crucial to OPMD pathogenesis.     

Optimization of CD4+ T lymphocyte response to human cytomegalovirus nuclear IE1 protein through modifications of both size and cellular localization

We have previously reported that the CD4+ T lymphocyte response against nuclear human CMV IE1 protein depends in part on endogenous MHC class II presentation. To optimize presentation by HLA-DR of the nuclear IE1 protein and increase the response by CD4+ T cells, we have constructed two different adenovirus vectors containing mutant versions of IE1, containing a HLA-DR3 epitope, fused to GFP. The first construct consisted of a sequence of 46 aa encoded by exon 4, called GFP-IE1 (86-131). The second construct consisted of the whole IE1 mutated on exon 4 nuclear localization signals, identified in this study, and deleted of already known exon 2 nuclear localization signals (GFP-IE1M). Both of these IE1 vectors expressed proteins with cytoplasmic localization, as evidenced by GFP expression, as opposed to control GFP-IE1, which was nuclear. GFP-IE1 (86-131) induced IE1-specific CD4+ T cell clone response that was >30-fold more potent than that against GFP-IE1 and GFP-IE1M. The CD4+ T cell response was due to endogenous presentation followed by exogenous presentation at later time points. Presentation was dependent on both proteasome and acidic compartments. GFP-IE1 (86-131) was rapidly degraded by the APC, which may account for better presentation. Our data show potentiation of the CD4+ T cell response to a specific epitope through shortening and relocation of an otherwise nuclear protein and suggest applications in vaccination.     

A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

Protein targeting to subnuclear higher order structures: A new level of regulation and coordination of nuclear processes

Though there are no separating membranes within the nucleus, different factors are often concentrated at sites where their respective function is required, a phenomenum referred to as functional organization of the nucleus. How is then this organization achieved and how are the different metabolic processes integrated in the nucleus? One emerging principle was revealed by the identification of protein domains that, though not involved in catalysis, regulate enzyme activity at a higher order level by targeting enzymes to the right place at the right time. These targeting sequences constitute an assembly code for nuclear ‘protein factories,’ which ensure the extremely high efficiency and accuracy needed in a complex and competitive environment as the living mammalian cell. J. Cell. Biochem. 70:222– 230, 1998. © 1998 Wiley-Liss, Inc.     

Determinants of small ubiquitin-like modifier 1 (SUMO1) protein specificity, E3 ligase, and SUMO-RanGAP1 binding activities of nucleoporin RanBP2

The RanBP2 nucleoporin contains an internal repeat domain (IR1-M-IR2) that catalyzes E3 ligase activity and forms a stable complex with SUMO-modified RanGAP1 and UBC9 at the nuclear pore complex. RanBP2 exhibits specificity for SUMO1 as RanGAP1-SUMO1/UBC9 forms a more stable complex with RanBP2 compared with RanGAP1-SUMO2 that results in greater protection of RanGAP-SUMO1 from proteases. The IR1-M-IR2 SUMO E3 ligase activity also shows a similar preference for SUMO1. We utilized deletions and domain swap constructs in protease protection assays and automodification assays to define RanBP2 domains responsible for RanGAP1-SUMO1 protection and SUMO1-specific E3 ligase activity. Our data suggest that elements in both IR1 and IR2 exhibit specificity for SUMO1. IR1 protects RanGAP1-SUMO1/UBC9 and functions as the primary E3 ligase of RanBP2, whereas IR2 retains the ability to interact with SUMO1 to promote SUMO1-specific E3 ligase activity. To determine the structural basis for SUMO1 specificity, a hybrid IR1 construct and IR1 were used to determine three new structures for complexes containing UBC9 with RanGAP1-SUMO1/2. These structures show more extensive contacts among SUMO, UBC9, and RanBP2 in complexes containing SUMO1 compared with SUMO2 and suggest that differences in SUMO specificity may be achieved through these subtle conformational differences.     

SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes

SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

Binding and nuclear relocalization of protein kinase R by human cytomegalovirus TRS1

The human cytomegalovirus (HCMV) TRS1 and IRS1 genes block the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and the consequent shutoff of cellular protein synthesis that occur during infection with vaccinia virus (VV) deleted of the double-stranded RNA binding protein gene E3L (VVDeltaE3L). To further define the underlying mechanism, we first evaluated the effect of pTRS1 on protein kinase R (PKR), the double-stranded RNA (dsRNA)-dependent eIF2alpha kinase. Immunoblot analyses revealed that pTRS1 expression in the context of a VVDeltaE3L recombinant decreased levels of PKR in the cytoplasm and increased its levels in the nucleus of infected cells, an effect not seen with wild-type VV or a VVDeltaE3L recombinant virus expressing E3L. This effect of pTRS1 was confirmed by visualizing the nuclear relocalization of PKR-EGFP expressed by transient transfection. PKR present in both the nuclear and cytoplasmic fractions was nonphosphorylated, indicating that it was unactivated when TRS1 was present. PKR also accumulated in the nucleus during HCMV infection as determined by indirect immunofluorescence and immunoblot analysis. Binding assays revealed that pTRS1 interacted with PKR in mammalian cells and in vitro. This interaction required the same carboxy-terminal region of pTRS1 that is necessary to rescue VVDeltaE3L replication in HeLa cells. The carboxy terminus of pIRS1 was also required for rescue of VVDeltaE3L and for mediating an interaction of pIRS1 with PKR. These results suggest that these HCMV genes directly interact with PKR and inhibit its activation by sequestering it in the nucleus, away from both its activator, cytoplasmic dsRNA, and its substrate, eIF2alpha.