Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubi... more Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.
Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety o... more Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.
Malignant cells achieve replicative immortality by two alternative mechanisms, a common one depen... more Malignant cells achieve replicative immortality by two alternative mechanisms, a common one dependent on de novo synthesis of telomeric DNA by telomerase, and a rare one based on telomere recombination known as alternative lengthening of telomeres (ALT). Epstein-Barr virus (EBV) transforms human B-lymphocytes into lymphoblastoid cell lines with unlimited growth potential in vitro and in vivo. Here we show that newly EBV-infected cells exhibit multiple signs of telomere dysfunction, including the occurrence of extra-chromosomal telomeres, telomere fusion and telomere length heterogeneity, and undergo progressive increase in telomere length without a parallel increase in telomerase activity. This phenotype is accompanied by the accumulation of telomere-associated promyelocytic leukemia nuclear bodies and telomeric-sister chromatid exchange, suggesting that EBV infection promotes the activation of ALT. Newly infected cells also display a significant reduction of telomere-associated TRF2 and express low levels of TRF1, TRF2, POT1 and ATRX, pointing to telomere de-protection as an important correlate of ALT activation. Collectively, these findings highlight the involvement of recombination-dependent mechanisms for maintenance of telomere homeostasis in EBV-induced B-cell immortalization.
Interleukin 10 (IL-10) is a cytokine with a variety of reported effects including inhibition of m... more Interleukin 10 (IL-10) is a cytokine with a variety of reported effects including inhibition of monocyte major histocompatibility complex (MHC) class II-dependent antigen presentation, type 1 helper T cell cytokine production, and inhibition of T cell proliferation. Herein we report the effect of IL-10 pretreatment on antigen presentation to tumor-and allo-specific CD8 + cytotoxic T lymphocytes (CTL). Prior incubation of human melanoma cells with recombinant IL-10 (rlL-10) for 48-72 h resulted in a dose-dependent, up to 100% inhibition, of autologous CTL-mediated, HLA-A2.1-restricted, tumor-specific lysis. Allo-specific CTL cytotoxicity against Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) was also inhibited, demonstrating a protective effect also on lymphoid calls. In contrast, IL-10 pretreatment of allogeneic LCL or K562 targets had either no effect or slightly enhanced cytotoxic activity mediated by freshly isolated or IL-2-activated natural killer cells. Flow cytometric analysis with monoclonal antibodies against HLA-A2, or nonpolymorphic determinants of MHC class I proteins, revealed a 20-50% reduction in call-surface expression, whereas intercellular adhesion molecules 1, and 2, and lymphocyte function-associated antigen 3 levels were not affected. In addition, relative to untreated target cells, IL-10 pretreated tumor ceils were unaltered in their capacity to affect CTL-mediated lysis by cold target inhibition, demonstrating that the effect of IL-10 is unrelated to the initial binding of CTL to their targets. These results are compatible with an effect of IL-10 on the MHC class I antigen presentation pathway, and suggest a novel mechanism of immune tolerance, based on escape from CTL-mediated tumor and allo-transplant rejection.
We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) All-r... more We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) All-restricted cytotoxic T lymphocyte (CTL) epitopes derived fi:om amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation oflymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences ofCTL eflaciency since clones specific for the two epitopes lysed equally well All-positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT-specific effectors, suggesting that a higher density of All-IVT complexes is presented at the cell surface. In accordance, 10--50-fold higher amounts oflVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350--12,800 IVT and 8-760 AVF molecules per cell. Peptide-mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of All expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal eflSciency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface tumover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of,'-'3.5 as compared to ~2 h for molecules induced at 26~ in the absence of exogenous petpides and >12 h for IVT-containing complexes. This difference in persistence is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules, and may be an important factor contributing to their immunogenicity.
Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotox... more Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M.
SUMMARYThe strategies adopted by viruses to reprogram the protein translation and quality control... more SUMMARYThe strategies adopted by viruses to reprogram the protein translation and quality control machineries to promote infection are poorly understood. Here, we discovered that the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of Epstein- Barr virus (EBV) regulates ribosomal stress responses. The vDUB participates in protein complexes that include the ubiquitin ligases ZNF598 and LTN1 and the UFM1 ligase UFL1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of 40S ribosome subunits, inhibits the degradation of translation-stalled polypeptides by the proteasome, and prevents UFMylation of the 60S particle, which impairs the ER-phagy- dependent clearance of stalled products. Inhibition of the ribosome quality control activates a GCN2-dependent integrated stress response that decreases global protein translation while promoting the readthrough of stall-inducing mRNAs. The vDUB enhances viral mRNAs translation and virus release during produc...
Proceedings of the National Academy of Sciences of the United States of America, Nov 11, 1997
The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B... more The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATPdependent ubiquitination and degradation by the proteasome. We have investigated the inf luence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP͞ubiquitin͞ proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin͞proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.
A proportion of patients with carcinomas and sarcomas possess blood lymphocytes that can lyse the... more A proportion of patients with carcinomas and sarcomas possess blood lymphocytes that can lyse their freshly harvested tumor cells in vitro (I-4). To enhance such cytotoxicity, we activated the lymphocytes either in mixed cultures with autologous tumor biopsy cells (ATS) 1 or in conventional mixed lymphocyte cultures (MLC). In accordance with the results of Zarling et al. (5), with leukemia cells, MLC-activated lymphocytes were often cytotoxic for autologous and third-party allogeneic solid tumor cells (6). Cocultivation with the patients' tumors generated autotumor cytotoxicity in the majority of experiments (7-9). In contrast to the MLC, these lymphocytes did not lyse allogeneic tumor biopsy cells. The effects against autologous and third-party tumors by the patients' MLClymphocytes might be a consequence of activation of lymphocyte sets that recognize (a) antigens that cross-react with the stimulator lymphocytes (b) tumor-related antigens shared by tumor cells of the lymphocyte donor and the allogeneic tumor, and (c) distinct allo-and/or tumor-related antigens on the different targets. Cold target competition experiments presented in this paper suggest that the third alternative is valid. Establishment of growing T cell lines with maintained specific functions (cytotoxicity or proliferative capacity) is an important step in the progress of tumor immunology (10-17). Such T cell populations reacting with autologous tumor cells might help in the study of the cell surface antigens of tumor cells, and they may be exploited in therapeutical measures. Because T cell growth factor (TCGF) (interleukin 2; IL-2) promotes the growth of activated T cells (18), its use imposes a selection for propagation of specific clones when applied to populations preexposed to the relevant antigen. To raise specific autotumor killer T cell cultures from patients with solid tumors, we cultured the blood lymphocytes first with the autologous tumor cells and thereafter added TCGF to
ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector c... more ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector cells in a population. LDA are dose-response assays that allow detection of an all-or-none (positive or negative) immunoresponse in each individual culture within replicates that vary in the number of responder cells tested (reviewed in ref. 1). The frequency of positive cultures is not informative because it is never clear whether one or more precursors in the culture well are giving the positive response. The negative response instead demonstrates that there are no precursors of a given specificity. Therefore, the evaluation of the cell frequency in the original population is possible by determining the number of cultures that are negative in the experiment. Multiple cultures are set up at different cell concentrations and the larger the number of replicates used for each cell concentration, the more precise the estimate will be. If the percentage of negative cultures is converted to its negative logarithm, the results can be plotted graphically. The fraction of negative cultures is plotted on the ordinate while the cell concentration is plotted on the abscissa to give a straight line. Suitable statistic tests are used to fit this line, including regression analysis and the least square method (2,3).
An OKT3+T4+T8-DR+ lymphocyte line was developed from an Epstein-Barr virus (EBV) seropositive ind... more An OKT3+T4+T8-DR+ lymphocyte line was developed from an Epstein-Barr virus (EBV) seropositive individual by repeated stimulation in vitro with autologous EBV-infected B cells. The T cell population designated E-44 was carried for eight months in the presence of Interleukin-2 and was repeatedly tested for cytotoxicity, proliferation and lymphokine production in response to the autologous and a panel of allogenic B cells. The E-44 cells lysed the autologous lymphoblastoid cell lines (LCL) and allogenic B cell lines sharing the DR6.1 major histocompatibility complex antigen with the lymphocyte donors. The EBV genome-negative lymphoma line BJAB and its two, infected in vitro, EBV-positive sublines were lysed with similar efficiencies. Autologous Staphylococcus aureus protein A (Prot-A) induced B, but not Phytohaemagglutinin (PHA)-induced T blasts were also lysed. It is likely that E-44 recognized an antigenic component derived from the fetal calf serum in association with class II determinants expressed on the B cells. Preincubation of E-44 cells with saturating amounts of OKT3 and Leu3a monoclonal antibodies abrogated the lytic effect on the autologous LCL. Cold target competition experiments demonstrated that, within, the population, the same cells reacted with the autologous Prot-A-induced blasts, the EBV-transformed LCL, and also with Daudi (an EBV genome-positive BL line). Although Daudi was the target which was lysed with the greatest efficiency, the avidity of interaction was highest with the autologous LCL because these cells competed best. Among the cells that were sensitive for the lytic effect, only the autologous LCL and Prot-A-induced B blasts triggered release of detectable amounts of Interleukin-2 and induced proliferation of the culture. The results suggest that the affinity of interaction with the target may be decisive for the triggering of the various T cell functions.
Blood lymphocyte subsets separated on the basis of densities from 14 healthy donors and 25 patien... more Blood lymphocyte subsets separated on the basis of densities from 14 healthy donors and 25 patients with solid tumors were tested for lytic effect against allogeneic or autologous tumor cells derived from surgical specimens. The allogeneic combinations comprised 36 tests on 28 tumors. In these the total lymphocyte population was cytotoxic in one experiment. On the other hand, 16 experiments with density separated subsets were positive. The lytic effect resided in the light subset. Auto-tumor lysis excreted by total lymphocyte population occurred in 7 of 25 cases. Additional 11 tests showed cytotoxicity with the separated subsets. The autologous tumor cells were damaged by the light lymphocytes of 15 patients. Among these 6 had cytotoxic cells also in the high density subset. In seven cases auto-tumor lysis was exerted only by the dense lymphocytes. Thus in 13 cases the profile of auto tumor lysis differed from that observed with the allogeneic combinations and against K562. The autologous system may represent an immune situation and therefore it may not be unexpected that a proportion of the active cells have different phenotypic characteristics compared to the operationally natural killer system.
Several lines of evidence indicate that an impairment of EBV-specific immune responses may contri... more Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's disease (HD). At present, however, it is not clear whether a defective immunity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in standard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the A11 and B44 HLA alleles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a Th0 phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.
After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist ind... more After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.
ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV... more ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV can be monitored in vitro as the capacity of T-cell lymphocytes to inhibit virusinduced proliferation of autologous B cells. In vitro infection of peripheral blood mononuclear cells (PBMCs) from EBV-seronegative and EBV-seropositive donors is accompanied by the appearance of foci of proliferating cells expressing the EBV nuclear antigens (EBNAs) within 7–14 d postinfection. These cells continue to expand giving rise to EBV-transformed lymphoblastoid cell lines (LCLs) in cultures from EBVseronegative individuals. In corresponding cultures from EBV-seropositive donors, the initial proliferation is followed by a regression of growth, seen preferentially at high cell concentration, that culminates within 1 mo in the complete degeneration of the culture (1). This phenomenon has been termed regression or outgrowth inhibition and can be monitored by the regression assay. The strength of the regression can be quantitatively expressed as the minimal number of cells required for 50% growth inhibition.
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that ... more Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, β-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.
Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr vir... more Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr virus-induced B-cell transformation was tested. Macrophages were as efficient as B cells and B blasts in presenting the virus to T lymphocytes. This function required antigen processing, because it was inhibited by chloroquine treatment and by fixation of the antigen-presenting cells immediately after viral exposure but not 18 h later. T cells exposed to the purified Epstein-Barr virus envelope antigen gp350 coupled to immunostimulating complexes also showed inhibitory function. These results suggest that recognition of processed virion antigens elicits the generation of T-cell-mediated inhibition of Epstein-Barr virus-induced B-cell transformation.
Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubi... more Posttranslational modifications of cellular proteins by covalent conjugation of ubiquitin and ubiquitin-like polypeptides regulate numerous cellular processes that are captured by viruses to promote infection, replication, and spreading. The importance of these protein modifications for the viral life cycle is underscored by the discovery that many viruses encode deconjugases that reverse their functions. The structural and functional characterization of these viral enzymes and the identification of their viral and cellular substrates is providing valuable insights into the biology of viral infections and the host's antiviral defense. Given the growing body of evidence demonstrating their key contribution to pathogenesis, the viral deconjugases are now recognized as attractive targets for the design of novel antiviral therapeutics.
Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety o... more Post-translational modification by the Small Ubiquitin-like Modifier (SUMO) regulates a variety of cellular functions, and is hijacked by viruses to remodel the host cell during latent and productive infection. Here we have monitored the activity of the SUMO conjugation machinery in cells productively infected with Epstein-Barr virus (EBV). We found that SUMO2/3 conjugates accumulate during the late phase of the productive virus cycle, and identified several viral proteins as bone fide SUMOylation substrates. Analysis of the mechanism involved in the accumulation of SUMOylated proteins revealed upregulation of several components of the SUMO-conjugation machinery and post-transcriptional downregulation of the SUMO-targeted ubiquitin ligase RNF4. The latter effect was mediated by selective inhibition of RNF4 protein expression by the viral miR-BHRF1-1. Reconstitution of RNF4 in cells expressing an inducible miR-BHRF1-1 sponge or a miR-BHRF1-1 resistant RNF4 was associated with reduced levels of early and late viral proteins and impaired virus release. These findings illustrate a novel strategy for viral interference with the SUMO pathway, and identify the EBV miR-BHRF1-1 and the cellular RNF4 as regulators of the productive virus cycle.
Malignant cells achieve replicative immortality by two alternative mechanisms, a common one depen... more Malignant cells achieve replicative immortality by two alternative mechanisms, a common one dependent on de novo synthesis of telomeric DNA by telomerase, and a rare one based on telomere recombination known as alternative lengthening of telomeres (ALT). Epstein-Barr virus (EBV) transforms human B-lymphocytes into lymphoblastoid cell lines with unlimited growth potential in vitro and in vivo. Here we show that newly EBV-infected cells exhibit multiple signs of telomere dysfunction, including the occurrence of extra-chromosomal telomeres, telomere fusion and telomere length heterogeneity, and undergo progressive increase in telomere length without a parallel increase in telomerase activity. This phenotype is accompanied by the accumulation of telomere-associated promyelocytic leukemia nuclear bodies and telomeric-sister chromatid exchange, suggesting that EBV infection promotes the activation of ALT. Newly infected cells also display a significant reduction of telomere-associated TRF2 and express low levels of TRF1, TRF2, POT1 and ATRX, pointing to telomere de-protection as an important correlate of ALT activation. Collectively, these findings highlight the involvement of recombination-dependent mechanisms for maintenance of telomere homeostasis in EBV-induced B-cell immortalization.
Interleukin 10 (IL-10) is a cytokine with a variety of reported effects including inhibition of m... more Interleukin 10 (IL-10) is a cytokine with a variety of reported effects including inhibition of monocyte major histocompatibility complex (MHC) class II-dependent antigen presentation, type 1 helper T cell cytokine production, and inhibition of T cell proliferation. Herein we report the effect of IL-10 pretreatment on antigen presentation to tumor-and allo-specific CD8 + cytotoxic T lymphocytes (CTL). Prior incubation of human melanoma cells with recombinant IL-10 (rlL-10) for 48-72 h resulted in a dose-dependent, up to 100% inhibition, of autologous CTL-mediated, HLA-A2.1-restricted, tumor-specific lysis. Allo-specific CTL cytotoxicity against Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) was also inhibited, demonstrating a protective effect also on lymphoid calls. In contrast, IL-10 pretreatment of allogeneic LCL or K562 targets had either no effect or slightly enhanced cytotoxic activity mediated by freshly isolated or IL-2-activated natural killer cells. Flow cytometric analysis with monoclonal antibodies against HLA-A2, or nonpolymorphic determinants of MHC class I proteins, revealed a 20-50% reduction in call-surface expression, whereas intercellular adhesion molecules 1, and 2, and lymphocyte function-associated antigen 3 levels were not affected. In addition, relative to untreated target cells, IL-10 pretreated tumor ceils were unaltered in their capacity to affect CTL-mediated lysis by cold target inhibition, demonstrating that the effect of IL-10 is unrelated to the initial binding of CTL to their targets. These results are compatible with an effect of IL-10 on the MHC class I antigen presentation pathway, and suggest a novel mechanism of immune tolerance, based on escape from CTL-mediated tumor and allo-transplant rejection.
We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) All-r... more We have investigated the reactivity to two human histocompatibility leukocyte antigen (HLA) All-restricted cytotoxic T lymphocyte (CTL) epitopes derived fi:om amino acids 416-424 (IVTDFSVIK, designated IVT) and 399-408 (AVFDRKSVAK, designated AVF) of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) 4. A strong predominance of CTL clones specific for the IVT epitope was demonstrated in polyclonal cultures generated by stimulation oflymphocytes from the EBV-seropositive donor BK with the autologous B95.8 virus-transformed lymphoblastoid cell line (LCL). This was not due to intrinsic differences ofCTL eflaciency since clones specific for the two epitopes lysed equally well All-positive phytohemagglutinin blasts and LCLs pulsed with the relevant synthetic peptide. Irrespective of the endogenous levels of EBNA4 expression, untreated LCLs were lysed more efficiently by the IVT-specific effectors, suggesting that a higher density of All-IVT complexes is presented at the cell surface. In accordance, 10--50-fold higher amounts oflVT peptides were found in high-performance liquid chromatography fractions of acid extracts corresponding to an abundance of about 350--12,800 IVT and 8-760 AVF molecules per cell. Peptide-mediated competition of CTL sensitization, transport assays in streptolysin-O permeabilized cells, and induction of All expression in the transporter associated with antigen presentation-deficient T2/A11 transfectant demonstrated that the IVT and AVF peptides bind with similar affinities to A11, are translocated with equal eflSciency to the endoplasmic reticulum, and form complexes of comparable stability over a wide range of temperature and pH conditions. A rapid surface tumover of A11 molecules containing the AVF peptide was demonstrated in metabolically active T2/A11 cells corresponding to a half-life of,'-'3.5 as compared to ~2 h for molecules induced at 26~ in the absence of exogenous petpides and >12 h for IVT-containing complexes. This difference in persistence is likely to determine the representation of individual class I-restricted CTL epitopes within the cell surface pool of molecules, and may be an important factor contributing to their immunogenicity.
Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotox... more Epstein-Barr virus (EBV), a ubiquitous herpesvirus, induces potent HLA class I-restricted cytotoxic T-lymphocyte (CTL) responses. Analyses of target antigen choice have shown that the very strong CTL responses which are often observed through the HLA All allele map are due almost entirely to a single transformation-associated EBV protein, the nuclear antigen EBNA4. Here, we sought to determine the number and relative immunogenicities of HLA All-restricted epitopes within this 938-amino-acid protein. An initial screening with a series of recombinant vaccinia virus vectors encoding progressively truncated forms of EBNA4 was followed by peptide sensitization experiments using overlapping 14-or 15-mers from the entire sequence. These two approaches allowed the identification of five epitope regions located between residues 101 and 115, 416 and 429, 396 and 410, 481 and 495, and 551 and 564 of the EBNA4 molecule. CTL preparations from all seven HLA All-positive donors tested had demonstrable reactivities against the 416-to-429 peptide, whereas reactivities against the other epitopes either tended to be lost on serial passage or, for some of the donors, were never detected. The immunodominance of the 416-to-429 epitope was further supported by peptide dilution assays using polyclonal effectors and by CTL cloning experiments. Analysis of the 416-to-429 region identified the nanomer 416-424 (IVTDFSVIK) as the cognate peptide. This peptide was able to sensitize targets to lysis by All-restricted CTL clones at concentrations as low as 5 x 10-14 M.
SUMMARYThe strategies adopted by viruses to reprogram the protein translation and quality control... more SUMMARYThe strategies adopted by viruses to reprogram the protein translation and quality control machineries to promote infection are poorly understood. Here, we discovered that the viral ubiquitin deconjugase (vDUB) encoded in the large tegument protein of Epstein- Barr virus (EBV) regulates ribosomal stress responses. The vDUB participates in protein complexes that include the ubiquitin ligases ZNF598 and LTN1 and the UFM1 ligase UFL1. Upon ribosomal stalling, the vDUB counteracts the ubiquitination of 40S ribosome subunits, inhibits the degradation of translation-stalled polypeptides by the proteasome, and prevents UFMylation of the 60S particle, which impairs the ER-phagy- dependent clearance of stalled products. Inhibition of the ribosome quality control activates a GCN2-dependent integrated stress response that decreases global protein translation while promoting the readthrough of stall-inducing mRNAs. The vDUB enhances viral mRNAs translation and virus release during produc...
Proceedings of the National Academy of Sciences of the United States of America, Nov 11, 1997
The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B... more The Epstein-Barr virus (EBV) encoded nuclear antigen (EBNA) 1 is expressed in latently infected B lymphocytes that persist for life in healthy virus carriers and is the only viral protein regularly detected in all EBV associated malignancies. The Gly-Ala repeat domain of EBNA1 was shown to inhibit in cis the presentation of major histocompatibility complex (MHC) class I restricted cytotoxic T cell epitopes from EBNA4. It appears that the majority of antigens presented via the MHC I pathway are subject to ATPdependent ubiquitination and degradation by the proteasome. We have investigated the inf luence of the repeat on this process by comparing the degradation of EBNA1, EBNA4, and Gly-Ala containing EBNA4 chimeras in a cell-free system. EBNA4 was efficiently degraded in an ATP͞ubiquitin͞ proteasome-dependent fashion whereas EBNA1 was resistant to degradation. Processing of EBNA1 was restored by deletion of the Gly-Ala domain whereas insertion of Gly-Ala repeats of various lengths and in different positions prevented the degradation of EBNA4 without appreciable effect on ubiquitination. Inhibition was also achieved by insertion of a Pro-Ala coding sequence. The results suggest that the repeat may affect MHC I restricted responses by inhibiting antigen processing via the ubiquitin͞proteasome pathway. The presence of regularly interspersed Ala residues appears to be important for the effect.
A proportion of patients with carcinomas and sarcomas possess blood lymphocytes that can lyse the... more A proportion of patients with carcinomas and sarcomas possess blood lymphocytes that can lyse their freshly harvested tumor cells in vitro (I-4). To enhance such cytotoxicity, we activated the lymphocytes either in mixed cultures with autologous tumor biopsy cells (ATS) 1 or in conventional mixed lymphocyte cultures (MLC). In accordance with the results of Zarling et al. (5), with leukemia cells, MLC-activated lymphocytes were often cytotoxic for autologous and third-party allogeneic solid tumor cells (6). Cocultivation with the patients' tumors generated autotumor cytotoxicity in the majority of experiments (7-9). In contrast to the MLC, these lymphocytes did not lyse allogeneic tumor biopsy cells. The effects against autologous and third-party tumors by the patients' MLClymphocytes might be a consequence of activation of lymphocyte sets that recognize (a) antigens that cross-react with the stimulator lymphocytes (b) tumor-related antigens shared by tumor cells of the lymphocyte donor and the allogeneic tumor, and (c) distinct allo-and/or tumor-related antigens on the different targets. Cold target competition experiments presented in this paper suggest that the third alternative is valid. Establishment of growing T cell lines with maintained specific functions (cytotoxicity or proliferative capacity) is an important step in the progress of tumor immunology (10-17). Such T cell populations reacting with autologous tumor cells might help in the study of the cell surface antigens of tumor cells, and they may be exploited in therapeutical measures. Because T cell growth factor (TCGF) (interleukin 2; IL-2) promotes the growth of activated T cells (18), its use imposes a selection for propagation of specific clones when applied to populations preexposed to the relevant antigen. To raise specific autotumor killer T cell cultures from patients with solid tumors, we cultured the blood lymphocytes first with the autologous tumor cells and thereafter added TCGF to
ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector c... more ABSTRACT Limiting dilution assays (LDA) are designed to define an unknown frequency of effector cells in a population. LDA are dose-response assays that allow detection of an all-or-none (positive or negative) immunoresponse in each individual culture within replicates that vary in the number of responder cells tested (reviewed in ref. 1). The frequency of positive cultures is not informative because it is never clear whether one or more precursors in the culture well are giving the positive response. The negative response instead demonstrates that there are no precursors of a given specificity. Therefore, the evaluation of the cell frequency in the original population is possible by determining the number of cultures that are negative in the experiment. Multiple cultures are set up at different cell concentrations and the larger the number of replicates used for each cell concentration, the more precise the estimate will be. If the percentage of negative cultures is converted to its negative logarithm, the results can be plotted graphically. The fraction of negative cultures is plotted on the ordinate while the cell concentration is plotted on the abscissa to give a straight line. Suitable statistic tests are used to fit this line, including regression analysis and the least square method (2,3).
An OKT3+T4+T8-DR+ lymphocyte line was developed from an Epstein-Barr virus (EBV) seropositive ind... more An OKT3+T4+T8-DR+ lymphocyte line was developed from an Epstein-Barr virus (EBV) seropositive individual by repeated stimulation in vitro with autologous EBV-infected B cells. The T cell population designated E-44 was carried for eight months in the presence of Interleukin-2 and was repeatedly tested for cytotoxicity, proliferation and lymphokine production in response to the autologous and a panel of allogenic B cells. The E-44 cells lysed the autologous lymphoblastoid cell lines (LCL) and allogenic B cell lines sharing the DR6.1 major histocompatibility complex antigen with the lymphocyte donors. The EBV genome-negative lymphoma line BJAB and its two, infected in vitro, EBV-positive sublines were lysed with similar efficiencies. Autologous Staphylococcus aureus protein A (Prot-A) induced B, but not Phytohaemagglutinin (PHA)-induced T blasts were also lysed. It is likely that E-44 recognized an antigenic component derived from the fetal calf serum in association with class II determinants expressed on the B cells. Preincubation of E-44 cells with saturating amounts of OKT3 and Leu3a monoclonal antibodies abrogated the lytic effect on the autologous LCL. Cold target competition experiments demonstrated that, within, the population, the same cells reacted with the autologous Prot-A-induced blasts, the EBV-transformed LCL, and also with Daudi (an EBV genome-positive BL line). Although Daudi was the target which was lysed with the greatest efficiency, the avidity of interaction was highest with the autologous LCL because these cells competed best. Among the cells that were sensitive for the lytic effect, only the autologous LCL and Prot-A-induced B blasts triggered release of detectable amounts of Interleukin-2 and induced proliferation of the culture. The results suggest that the affinity of interaction with the target may be decisive for the triggering of the various T cell functions.
Blood lymphocyte subsets separated on the basis of densities from 14 healthy donors and 25 patien... more Blood lymphocyte subsets separated on the basis of densities from 14 healthy donors and 25 patients with solid tumors were tested for lytic effect against allogeneic or autologous tumor cells derived from surgical specimens. The allogeneic combinations comprised 36 tests on 28 tumors. In these the total lymphocyte population was cytotoxic in one experiment. On the other hand, 16 experiments with density separated subsets were positive. The lytic effect resided in the light subset. Auto-tumor lysis excreted by total lymphocyte population occurred in 7 of 25 cases. Additional 11 tests showed cytotoxicity with the separated subsets. The autologous tumor cells were damaged by the light lymphocytes of 15 patients. Among these 6 had cytotoxic cells also in the high density subset. In seven cases auto-tumor lysis was exerted only by the dense lymphocytes. Thus in 13 cases the profile of auto tumor lysis differed from that observed with the allogeneic combinations and against K562. The autologous system may represent an immune situation and therefore it may not be unexpected that a proportion of the active cells have different phenotypic characteristics compared to the operationally natural killer system.
Several lines of evidence indicate that an impairment of EBV-specific immune responses may contri... more Several lines of evidence indicate that an impairment of EBV-specific immune responses may contribute to the pathogenesis of Hodgkin's disease (HD). At present, however, it is not clear whether a defective immunity to EBV is a characteristic restricted to EBV-associated HD cases or a more generalized phenomenon, part of the inherent immune deficiency of HD patients. In this study, we have addressed this issue by analyzing EBV-specific responses in infiltrating T lymphocytes (TILs) from one HD biopsy, where the virus was confined to a small proportion of apparently normal lymphocytes. TIL cultures were established using low amounts of recombinant interleukin 2 and in the absence of specific stimulation, conditions that preferentially induce the proliferation of in vivo activated T cells. An EBV-specific cytotoxic component was revealed by the capacity of these TILs to lyse autologous EBV-positive lymphoblastoid cell lines (LCLs) obtained by spontaneous transformation from the lesion but not HLA-mismatched LCLs and autologous phytohemagglutinin blasts. This cytotoxic activity closely resembled that of EBV-specific memory T cells, which may be reactivated from the blood lymphocytes of healthy donors by in vitro stimulation with autologous LCLs. The use of a panel of appropriately HLA-matched B95.8-transformed LCLs as targets in standard 51Cr release assays revealed EBV-specific cytotoxic responses to be restricted mainly through the A11 and B44 HLA alleles with a minor HLA-A26-restricted component. Using autologous fibroblasts infected with recombinant vaccinia viruses expressing the EBV latent antigens, the TIL culture was shown to recognize latent membrane protein 2 and, to a lesser extent, EBV-encoded nuclear antigen 6. In addition, a strong proliferative response was induced by coculture of TILs with autologous but not with allogeneic LCLs or autologous phytohemagglutinin blasts. Six CD4-positive, EBV-specific T-cell clones were isolated by limiting dilution. The study of cytokine mRNA expression, carried out by reverse transcriptase-assisted PCR, revealed that three of these T-cell clones expressed a Th0 phenotype, whereas 1 had a Th2 phenotype. These findings are consistent with the presence in this HD lesion of an ongoing immune response against EBV-carrying cells and suggest that the complex immune deficiency that characterizes HD patients probably does not include a generalized, constitutional defect of EBV-specific T-cell responses.
After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist ind... more After Epstein-Barr virus (EBV) infection in vivo, B-cells with latent virus infection persist indefinitely through life. These cells grow in vitro on explanation and can be established as immortal B-cell lines. To reconcile the unlimited growth potential in vitro with the maintenance of a low proportion of B-cells infected by EBV in vivo, a strict in vivo control mechanism has to be postulated. Certain aspects of this control are apparent when the primary infection is followed by infectious mononucleosis. This is characterized by lymphocytosis and the presence of activated T-cells. The T-cell proliferation is probably the manifestation of the immune response against EBV antigens. However, the reaction of T-cells upon encounter of B-blasts is also likely to contribute to the events. At present, it is difficult to detect an EBV-specific component in the action of the T-cells in the acute phase of mononucleosis exerted on B-cells. However, for the clinical course of the disease the activation of T-cells is important. The activated T-cells may control and also eliminate the B-cells infected by EBV. In addition to the immunity which develops during the disease, th immunoregulatory mechanism is likely to have a role in the inhibition of B-cell proliferation.
ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV... more ABSTRACT In Epstein-Barr virus (EBV)-seropositive individuals, cell-mediated immunity against EBV can be monitored in vitro as the capacity of T-cell lymphocytes to inhibit virusinduced proliferation of autologous B cells. In vitro infection of peripheral blood mononuclear cells (PBMCs) from EBV-seronegative and EBV-seropositive donors is accompanied by the appearance of foci of proliferating cells expressing the EBV nuclear antigens (EBNAs) within 7–14 d postinfection. These cells continue to expand giving rise to EBV-transformed lymphoblastoid cell lines (LCLs) in cultures from EBVseronegative individuals. In corresponding cultures from EBV-seropositive donors, the initial proliferation is followed by a regression of growth, seen preferentially at high cell concentration, that culminates within 1 mo in the complete degeneration of the culture (1). This phenomenon has been termed regression or outgrowth inhibition and can be monitored by the regression assay. The strength of the regression can be quantitatively expressed as the minimal number of cells required for 50% growth inhibition.
Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that ... more Ubiquitin C-terminal hydrolase-L1 (UCH-L1) is a deubiquitinating enzyme of unknown function that is highly expressed in neurons and overexpressed in several human cancers. UCH-L1 has been implicated in the regulation of phenotypic properties associated with malignant cell growth but the underlying mechanisms have not been elucidated. By comparing cells expressing catalytically active or inactive versions of UCH-L1, we found that the active enzyme enhances cell adhesion, spreading, and migration; inhibits anoikis; and promotes anchorage independent growth. UCH-L1 accumulates at the motile edge of the cell membrane during the initial phases of adhesion, colocalizes with focal adhesion kinase (FAK), p120-catenin, and vinculin, and enhances the formation of focal adhesions, which correlates with enhanced FAK activation. The involvement of UCH-L1 in the regulation of focal adhesions and adherens junctions is supported by coimmunoprecipitation with key components of these complexes, including FAK, paxillin, p120-catenin, β-catenin, and vinculin. UCH-L1 stabilizes focal adhesion signaling in the absence of adhesion, as assessed by reduced caspase-dependent cleavage of FAK following cell detachment and sustained activity of the AKT signaling pathway. These findings offer new insights on the molecular interactions through which the deubiquitinating enzyme regulates the survival, proliferation, and metastatic potential of malignant cells.
Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr vir... more Barr virion antigens to autologous T cells and trigger their capacity to inhibit Epstein-Barr virus-induced B-cell transformation was tested. Macrophages were as efficient as B cells and B blasts in presenting the virus to T lymphocytes. This function required antigen processing, because it was inhibited by chloroquine treatment and by fixation of the antigen-presenting cells immediately after viral exposure but not 18 h later. T cells exposed to the purified Epstein-Barr virus envelope antigen gp350 coupled to immunostimulating complexes also showed inhibitory function. These results suggest that recognition of processed virion antigens elicits the generation of T-cell-mediated inhibition of Epstein-Barr virus-induced B-cell transformation.
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Papers by Maria Masucci