Both of the major immediate-early (IE) proteins IE1 and IE2 of human cytomegalovirus (HCMV) as well as input viral DNA and sites of viral IE transcription colocalize with or adjacent to punctate PML domains (PML oncogenic domains [PODs] or nuclear domain 10) in the nucleus within the first few hours after infection of permissive human fibroblasts. However, colocalization of IE1 and PML in PODs is only transient, with both proteins subsequently redistributing into a nuclear diffuse form. These processes are believed to promote efficient viral IE transcription and initiation of DNA synthesis especially at low multiplicities of infection. To examine the mechanism of PML displacement by IE1, we carried out indirect immunofluorescence assay experiments with plasmids expressing intact or deleted forms of PML and IE1 in DNA-transfected cells. The results demonstrated that deletion of the C-terminal acidic region of IE1 uncouples the requirements for displacement of both endogenous and coexpressed PML from those needed to target to the PODs. Mutant PML proteins containing either a Cys point mutation within the N-terminal RING finger domain or a small deletion (of positions 281 to 304) within the coiled-coil region did not localize to the PODs but instead gave a nuclear diffuse distribution, similar to that produced by intact PML in the presence of IE1. Endogenous PML also colocalized with IE1 in metaphase chromosomes in HCMV or recombinant adenovirus type 5-IE1-infected HF cells undergoing mitosis, implying that there may be a direct physical interaction between IE1 and PML. Indeed, a specific interaction between IE1 and PML was observed in a yeast two-hybrid assay, and the strength of this interaction was comparable to that of IE2 with the retinoblastoma protein. The RING finger mutant form of PML showed a threefold-lower interaction with IE1 in the yeast system, and deletion of the N-terminal RING finger domain of PML abolished the interaction. Consistent with the IFA results, a mutant IE1 protein that lacks the C-terminal acidic region was sufficient for interaction with PML in the yeast system. The two-hybrid interaction assay also showed that both the N-terminal RING finger domain and the intact coiled-coil region of PML are required cooperatively for efficient self-interactions involving dimerization or oligomerization. Furthermore, truncated or deleted GAL4/PML fusion proteins that retained the RING finger domain but lacked the intact coiled-coil region displayed an unmasked cryptic transactivator function in both yeast and mammalian cells, and the RING finger mutation abolished this transactivation property of PML. Therefore, we suggest that a direct interaction between IE1 and the N-terminal RING finger domain of PML may inhibit oligomerization and protein-protein complex formation by PML, leading to displacement of PML and IE1 from the PODs, and that this interaction may also modulate a putative conditional transactivator function of PML.Several herpesvirus nuclear regulatory proteins expressed at immediate-ea...
Six predicted Kaposi's sarcoma virus herpesvirus (KSHV) proteins have homology with other well-characterized herpesvirus core DNA replication proteins and are expected to be essential for viral DNA synthesis. Intact Flag-tagged protein products from all six were produced from genomic expression vectors, although the ORF40/41 transcript encoding a primase-helicase component proved to be spliced with a 127-bp intron. The intracellular localization of these six KSHV replication proteins and the mechanism of their nuclear translocation were investigated. SSB (single-stranded DNA binding protein, ORF6) and PPF (polymerase processivity factor, ORF59) were found to be intrinsic nuclear proteins, whereas POL (polymerase, ORF9), which localized in the cytoplasm on its own, was translocated to the nucleus when cotransfected with PPF. PAF (primaseassociated factor, ORF40/41), a component of the primase-helicase tripartite subcomplex together with PRI (primase, ORF56) and HEL (helicase, ORF44), required the presence of all five other replication proteins for efficient nuclear translocation. Surprisingly, even in the absence of a lytic cycle replication origin (ori-Lyt) and any known initiator or origin binding protein, the protein products of all six KSHV core replication genes cooperated in a transient cotransfection assay to form large globular shaped pseudo-replication compartments (pseudo-RC), which excluded cellular DNA. These pseudo-RC structures were confirmed to include POL, SSB, PRI, and PAF but did not contain any newly synthesized DNA. Similar to the human cytomegalovirus system, the peripheries of these KSHV pre-RC were also found to be surrounded by punctate PML oncogenic domains (PODs). Furthermore, by transient cotransfection, the six KSHV core replication machinery proteins successfully replicated a plasmid containing EBV ori-Lyt in the presence of the Epstein-Barr virus-encoded DNA binding initiator protein, ZTA. The KSHV-encoded K8 (ORF-K8) protein, which is a distant evolutionary homologue to ZTA, was incorporated into pseudo-RC structures formed by transient cotransfection with the six core KSHV replication genes. However, unlike ZTA, K8 displayed a punctate nuclear pattern both in transfected cells and at early stages of lytic infection and colocalized with the cellular PML proteins in PODs. Finally, K8 was also found to accumulate in functional viral RC, detected by incorporation of pulse-labeled bromodeoxyuridine into newly synthesized DNA in both tetradecanoyl phorbol acetate-induced JSC-1 primary effusion lymphoblasts and in KSHV lytically infected endothelial cells.
The major histocompatibility complex (MHC) class I molecules present peptides on the cell surface by CD8 + T cells, which is critical for killing of virally infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by endoplasmic reticulum aminopeptidase 1 (ERAP1). The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and harbors 3 microRNAs (miRNAs). We show here the HCMV miR-US4-1 specifically down-regulates ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides is inhibited, leading to reduced susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings reveal a novel viral miRNA-based CTL evasion mechanism that targets a key step in the MHC class I antigen-processing pathway.MHC class I molecule binds peptides and presents them on the cell surface for recognition by CD8 + T cells. Cytosolic peptides generated by the proteasomes and prematurely translated peptides are transported to the endoplasmic reticulum (ER) through the transporter associated with antigen processing (TAP) complex 1, 2 . Some of these peptides are further processed by ER-resident aminopeptidases, such as ERAP1, and peptide trimming by ERAP1 (also known as A-LAP, ARTS-1, and PILS-AP) in the ER is a crucial step for determining the quality and quantity of optimal antigenic peptide production and the stability of the MHC class I-β 2 m-peptide heterotrimer [3][4][5][6] . ERAP1 trims relatively long * To whom correspondence should be addressed: Department of Biological Sciences, Seoul National University, Seoul 151-747, Korea, (Phone) +822-880-9233 (Fax) +822-872-1993 S.R.R cloned the HCMV-specific CTLs. S.K and K.A designed the overall study and wrote the paper. NIH Public AccessAuthor Manuscript Nat Immunol. Author manuscript; available in PMC 2012 December 20. peptides efficiently in a sequence-specific manner, resulting in the accumulation of 8-9 amino acid-long optimal peptides 5,7,8 . ERAP1 therefore acts as a 'molecular ruler' for antigenic peptide production 9 . In addition, genome-wide association studies have associated nonsynonymous single nucleotide polymorphisms in ERAP1 with ankylosing spondylitis 4 . Additionally, ERAP1 has non-peptide processing functions via its role in shedding of cytokine receptors 4 .MiRNAs are small RNAs 19 to 23 nucleotides long that regulate gene expression by complete or partial base-pairing with the 3′-untranslated region (UTR) of their target mRNA which leads to mRNA cleavage, destabilization, or translational repression 10 . Since the first report in 2004 that viruses express miRNAs 11 , numerous viral miRNAs have been discovered and are mainly related to viral proliferation and survival-related immune evasion, although this is based on a limited number of studies 12 . The β-herpesvirus HCMV expresses at least 14 miRNAs during productive infection 13,14 . One HCMV-encoded miRNA, miR-UL112-1 targets 3 HCMV genes involved in viral r...
Inherited mutations in the ATM gene lead to a complex clinical phenotype characterized by neuronal degeneration, oculocutaneous telangiectasias, immune dysfunction, and cancer predisposition. Using the yeast two-hybrid system, we demonstrate that ataxia telangiectasia mutated (ATM) binds to -adaptin, one of the components of the AP-2 adaptor complex, which is involved in clathrin-mediated endocytosis of receptors. The interaction between ATM and -adaptin was confirmed in vitro, and coimmunoprecipitation and colocalization studies show that the proteins also associate in vivo. ATM also interacts in vitro with -NAP, a neuronal-specific -adaptin homolog that was identified as an autoantigen in a patient with cerebellar degeneration. Our data describing the association of ATM with -adaptin in vesicles indicate that ATM may play a role in intracellular vesicle and͞or protein transport mechanisms.
In human cytomegalovirus (HCMV) infection, both of the major immediate-early proteins IE1(IE68, UL123) and IE2(IE86, UL122) target to PML protein-associated nuclear bodies known as PODs or ND10 at very early times after infection. IE1 causes a redistribution of both PML and IE1 from the PODs into a nuclear diffuse form, whereas IE2 initially localizes adjacent to PODs but later associates with viral DNA replication compartments. The peripheries of PODs are also believed to be sites for initiation of both viral IE transcription and DNA replication. However, because IE1 is nonessential at high multiplicity of infection (m.o.i.) in HF cells, the exact role of these processes in viral infection has been enigmatic. Therefore, we investigated the effects of overexpression of PML in the presence or absence of IE1 on the intranuclear distribution of IE2 and formation of viral DNA replication compartments, as well as on the levels of delayed-early and late viral transcription and protein accumulation. Infection with wild-type HCMV(Towne) and the IE1-deleted derivative HCMV(CR208), which fails to disrupt PODs, was compared in a pair of related astrocytoma/glioblastoma cell lines, the U373-Neo control and a variant U373-PML that constitutively overexpresses PML(560) in much larger than normal PODs. IFA studies on the localization patterns for IE1, IE2, and PML showed that, although the numbers of IE2-positive cells were not significantly reduced in either the wild-type virus-infected U373-PML cell line or in DeltaIE1-infected control cells, POD disruption by IE1 in wild-type virus infection was delayed by up to 6 h in U373-PML cells compared to control cells. Furthermore, there was considerable enhancement of IE2 colocalization with PODs in Delta IE1-infected U373-PML cells. Formation of viral DNA replication compartments in the U373-PML cell line was also greatly delayed, measured at fivefold lower after wild-type virus infection and 12-fold lower after infection with Delta IE1 than in the control cell line at 48 h at an m.o.i. of 1.0. The levels of representative early and late viral proteins detected by Western blotting were suppressed by fivefold and 22-fold at 24 and 72 h, respectively, in the U373-PML cell line, even with high m. o.i. wild-type HCMV infection. Decreased viral protein levels also occurred when control cells were infected with the Delta IE1 virus and these two effects were additive in the U373-PML cell line. Similarly, when U373-PML cells were infected with recombinant HCMV expressing an extragenic luciferase reporter gene under the control of viral early (Pol) or late (pp28) promoters, their transcriptional activation was reduced up to fivefold at both high and low m.o.i. compared to that of the control cells. Overall, these results suggest that POD disruption by IE1 and subsequent redistribution of both PML and IE1 at very early times after infection may play an important role in the efficient utilization of cellular transcription and replication machinery by HCMV and contribute to rapid progression o...
USP DUBs cleaved both Lys48-and Lys63-linked ubiquitin dimers and oligomers, showing more activity toward Lys63 linkages. The DUB activity of the full-length UL48 protein immunoprecipitated from virus-infected cells also showed a better cleavage of Lys63-linked ubiquitinated substrates. An HCMV (Towne) mutant virus in which the UL48 DUB activity was destroyed [UL48(C24S)] produced 10-fold less progeny virus and reduced amounts of viral proteins compared to wild-type virus at a low multiplicity of infection. The mutant virus also produced perceptibly less overall deubiquitination than the wild-type virus. Our findings demonstrate that the HCMV UL48 DUB contains both a ubiquitin-specific carboxy-terminal hydrolase activity and an isopeptidase activity that favors ubiquitin Lys63 linkages and that these activities can influence virus replication in cultured 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.
Aims Posterior wall isolation (PWI) of the left atrium (LA) adjunct to pulmonary vein isolation (PVI) by radiofrequency catheter ablation has shown favourable outcomes in patients with persistent atrial fibrillation (PeAF). This study was sought to investigate the efficacy and safety of additional PWI by cryoballoon ablation (CBA) alone in patients with PeAF. Methods and results Patients who underwent de novo CBA for PeAF (n = 100) were randomly assigned (1 : 1) to the PVI only group and PVI combined with PWI (PVI+PWI) group. Procedural and clinical outcomes were prospectively compared over a 12-month follow-up. Baseline characteristics, including mean AF duration (56.2 ± 43.2 months) and LA size (48.2 ± 7.7 mm), were well-balanced between the groups. Successful PVI was achieved in all patients. In the PVI+PWI group, complete PWI by CBA was achieved in 31 (62%) patients. The LA indwelling and procedure times were significantly longer in the PVI+PWI group. The complication rates were not different between groups. During a mean follow-up of 457.9 ± 61.8 days, the recurrence rate of atrial tachyarrhythmia was significantly lower in the PVI+PWI group than in the PVI only group (24% vs. 46%; P = 0.035). The recurrence-free survival rate was significantly higher in the PVI+PWI group compared with the PVI only group, irrespective of complete PWI (log-rank P = 0.013). Multivariate analysis showed that adjunctive PWI [hazard ratio (HR) 0.255; P = 0.003] and LA size (HR 1.079; P = 0.014) were independent predictors of clinical recurrence. Conclusion Compared with PVI only, adjunctive PWI achieved exclusively by CBA resulted in better clinical outcomes without increasing complications in patients with PeAF.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.