Monoclonal gammopathy of undetermined significance (MGUS) is associated with an increased frequency of Epstein-Barr Virus (EBV) latently infected B lymphocytes in long-term renal transplant patients.

Monoclonal gammopathy of undetermined significance (MGUS) is a common phenomenon in kidney transplant patients that might be a prestage of posttransplant lymphoproliferative disease (PTLD). Because the role of Epstein-Barr virus (EBV) for PTLD development is well established, we wondered about the association between EBV and MGUS. Thus, B-cells from kidney transplant patients (25 with and 100 without MGUS) and from 100 healthy controls were analyzed for EBV latent (EBER1) and lytic (EA, VCA) gene expression by RT-nested PCR. The EBV load was measured by real-time PCR. A significantly higher EBV load and expression of the nonimmunogenic latency associated EBER 1 gene was observed in patients with MGUS compared to both control groups (P < .001). In addition, a rare detection of the highly immunogenic lytic transcripts demonstrated a linkage between latency-associated EBV infection and MGUS in transplant patients. This pattern was similar to EBV-associated B-cell lymphomas in nonimmunosuppressed patients. It contrasted with PTLD patients who express higher EBV loads and both lytic and latent EBV transcripts. These data suggest that transplant patients with MGUS demonstrate a more sufficient control of EBV-infected B-cells. Nevertheless, EBV monitoring should be performed in patients with EBV-associated MGUS for early detection of later PTLD.

A 30 kb region of the Epstein-Barr virus genome is colinear with the rearranged human immunoglobulin gene loci: implications for a "ping-pong evolution" model for persisting viruses and their hosts. A review.

The left part of the Epstein-Barr virus (EBV) genome exhibits a strong colinearity of structural and functional elements with the immunoglobulin (Ig) gene loci which is only partially reflected in nucleotide sequence homologies. We propose that this colinearity may be the result of an inter-dependent co-evolution of the immunoglobulin loci together with EBV. Our observation could help elucidating the mechanisms of somatic hypermutation, explaining the ability of EBV to accidentally cause tumors, and shedding more light on the general mechanisms of viral and organismal evolution. We suggest that persisting viruses served as a complement for the organismal germline like in a ping-pong game and outline The Ping-Pong Evolution Hypothesis.

Association between Epstein-Barr virus infection and late acute transplant rejection in long-term transplant patients.

Recently we reported about a possible involvement of extrarenal systemic cytomegalovirus (CMV) infection in graft deteriorating immune processes. We now examined whether Epstein-Barr virus (EBV) may also be associated with late renal graft injury. We analyzed the expression of early antigen-, viral capsid antigen-, and a latency-associated EBV-RNA-transcript, which is not translated into protein in peripheral blood mononuclear cells of kidney transplant patients with histologically proven late acute rejection and no signs of CMV or any other infection (A), patients with stable graft function (B), and healthy probands (C). A total of 40% in group A vs. 5 and 0% in groups B and C, respectively, expressed early antigen-mRNA (P<0.05) suggesting an activation of lytic EBV infection. Response to steroid bolus therapy in group A was comparably poor with that observed in CMV-related graft injury. Our data suggest that extrarenal lytic EBV infection may also be involved in the pathogenesis of late graft injury. A controlled ganciclovir trial may prove the significance of our observation.

Latency of Epstein-Barr virus is stabilized by antisense-mediated control of the viral immediate-early gene BZLF-1.

The ability of the Epstein-Barr virus (EBV) to avoid lytic replication and to establish a latent infection in B-lymphocytes is fundamental for its lifelong persistence and the pathogenesis of various EBV-associated diseases. The viral immediate-early gene BZLF-1 plays a key role for the induction of lytic replication and its activity is strictly regulated on different levels of gene expression. Recently, it was demonstrated that BZLF-1 is also controlled by a posttranscriptional mechanism. Transient synthesis of a mutated competitor RNA saturated this mechanism and caused both expression of the BZLF-1 protein and the induction of lytic viral replication. Using short overlapping fragments of the competitor, it is shown that this control acts on the unspliced primary transcript. RT-PCR demonstrated unspliced BZLF-1 RNA in latently infected B-lymphocytes in the absence of BZLF-1 protein. Due to the complementarity of the gene BZLF-1 and the latency-associated gene EBNA-1 on the opposite strand of the genome, we propose an antisense-mediated mechanism. RNase protection assays demonstrated transcripts in antisense orientation to the BZLF-1 transcript during latency, which comprise a comparable constellation to other herpesviruses. A combined RNAse protection/RT-PCR assay detected the double-stranded hybrid RNA, consisting of the unspliced BZLF-1 transcript and a noncoding intron of the EBNA-1 gene. Binding of BZLF-1 transcripts is suggested to be an important backup control mechanism in addition to transcriptional regulation, stabilizing latency and preventing inappropriate lytic viral replication in vivo.

A case of severe chronic active infection with Epstein-Barr virus: immunologic deficiencies associated with a lytic virus strain.

Infectious mononucleosis (IM) is a self-limiting, lymphoproliferative disease induced by primary infection with the Epstein-Barr virus (EBV). Infection with EBV leads in general to lifelong asymptomatic persistence of the virus. We report the case of a woman who acquired IM at the age of 15 years and then suffered from recurrent high fever, fatigue, and signs of immunologic disorder for more than 12 years until she died of liver failure. In an attempt to describe and to define the course of chronic active infection with EBV, we performed immunologic and molecular assays that demonstrated lytic replication of EBV in the B and T cells of the peripheral blood. In addition to signs of humoral and cellular immune deficiency, we detected an EBV strain with an impaired capability to immortalize B cells and a tendency to lytic replication, thus contributing to the pathogenesis of this chronic active infection.

Epstein-Barr virus infection of human astrocyte cell lines.

Epstein-Barr virus (EBV) is implicated in different central nervous system syndromes. The major cellular receptor for EBV, complement receptor type 2 (CR2) (CD21), is expressed by different astrocyte cell lines and human fetal astrocytes, suggesting their susceptibility to EBV infection. We demonstrated the infection of two astrocyte cell lines, T98 and CB193, at low levels. As infection was mediated by CR2, we used two stable CR2 transfectant astrocyte cell lines (T98CR2 and CB193CR2) to achieve a more efficient infection. We have monitored EBV gene expression for 2 months and observed the transient infection of T98 and T98CR2 cells and persistent infection of CB193 and CB193CR2 cells. The detection of BZLF1, BALF2, and BcLF1 mRNA expression suggests that the lytic cycle is initiated at early time points postinfection. At later time points the pattern of mRNA expressed (EBER1, EBNA1, EBNA2, and LMP1) differs from latency type III in the absence of LMP2A transcription and in the expression of BALF2 and BcLF1 but not BZLF1. A reactivation of the lytic cycle was achieved in CB193CR2 cells by the addition of phorbol esters. These studies identify astrocyte cell lines as targets for EBV infection and suggest that this infection might play a role in the pathology of EBV in the brain.

The control of lytic replication of Epstein-Barr virus in B lymphocytes (Review).

Uncontrolled replication of a virus, which is harmful to the host is also disadvantageous to the virus. Most viruses cannot compete with the various immune mechanisms and become eliminated in the course of infection. Therefore, only the time between infection and eradication remains for these viruses to proliferate. A few viruses, like the Herpesviruses or the papillomaviruses, however, have developed a sophisticated strategy for persisting lifelong, usually asymptomatically in the host, hiding from the immune system and producing infectious progeny at the same time. This strategy depends on a separation of latency and the lytic replication, either by time due to differentiation-dependent mechanisms or by spatial separation as the result of different host cell types. Both are true for the Epstein-Barr virus (EBV). B cells and epithelial cells have a pivotal role in the life cycle of the virus. The former can become latently infected and are thought to be the virus reservoir in vivo, whereas the latter were shown to be permissive for lytic replication. However, replication of EBV in vivo is controlled primarily by host immune mechanisms selecting for cells that are not permissive for viral replication as the result of a particular set of transcription factors. These factors control the activity of the regulatory immediate-early genes and, in addition, lytic and latent cycle regulatory genes negatively interfere with each other and thus link cellular and viral gene regulatory mechanisms. Disturbance of both the immune surveillance as well as viral gene regulation may result in EBV-associated disease.

Epstein-Barr virus small RNA (EBER) genes: differential regulation during lytic viral replication.

In every latently Epstein-Barr virus-infected cell the viral genes EBER-1 and EBER-2 are transcribed by polymerase III. In lytically infected cells in vivo the EBER genes could not be detected. However, in cell culture downregulation could not be confirmed, and hence the relevance of this shutdown to the replication of the virus was not clear. We assayed the transcriptional activity of the EBER genes by nuclear run-on assays with enriched lytically infected cells and demonstrated that EBER-1 and EBER-2 are differentially downregulated on the transcriptional level during the switch to lytic viral replication. This downregulation was an early event during the lytic replication of the virus.

Epstein-Barr viral gene expression in B-lymphocytes.

The strategy of the Epstein-Barr virus to persist lifelong in the host depends on establishing a reservoir, which cannot be detected by the immune system but allows reactivation of the virus for shedding and transmission to a new host. Epithelial cells and B-cells play a major role in this viral strategy of EBV, since differentiating epithelial tissues were shown to be permissive for lytic replication in vivo, whereas the B-lymphocytes become predominantly latently infected. However, which cells are the reservoir and which the sites of lytic replication are not quite clear. With the technique of reverse transcription, PCR and immunohistochemistry, we demonstrated that the B-cells of the peripheral blood are a major site of virus production during the primary infection during infectious mononucleosis. These permissive B-cells were also detected after convalescence, however, the absence of any lytic transcripts suggested an efficient immunological control very early in the viral lytic cycle. Serological data on reactivation of EBV correlated with the detection of lytic cycle transcripts in the blood and thus demonstrated that the site of virus production during infectious mononucleosis must be different from that of the persistent state. In those cases, where the infection takes a chronic active course, control of lytic replication is insufficient, either on the level of immune surveillance or of viral gene regulation. We have demonstrated a virus strain with a lytic phenotype in an individual suffering chronic active infection. The impaired capability of this virus to immortalise B-cells correlated with an enhanced expression of the lytic switch gene BZLF-1 and down-regulation of latent regulatory genes in the early phase of infection.

Epstein-Barr virus-infected B cells of males with the X-linked lymphoproliferative syndrome stimulate and are susceptible to T-cell-mediated lysis.

Primary infection with the Epstein-Barr virus (EBV) results in fatal infectious mononucleosis in up to 70% of males affected by the X-linked lymphoproliferative syndrome (XLP). This rare disease is often associated with diverse natural killer (NK)-, B- and T-cell deficiencies. We describe experiments testing whether the B lymphocytes of affected males play a role in the pathogenesis of XLP due to a low susceptibility to T-cell-mediated immunity. Using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry we detected in these B cells the expression of viral proteins EBNA-1, EBNA-2, EBNA-3A, EBNA-3C, LMP-1 and LMP-2A, which provide targets for cytotoxic T cells. Major histocompatibility complex (MHC) class I, MHC class II and the B7 costimulatory molecule were present on the cell surface. Accordingly, the EBV-infected B cells were lysed in 51Cr-release assays by T lymphocytes sharing MHC determinants with the targets. This MHC-restricted and specific lysis was confirmed in competition experiments using MHC-specific monoclonal antibodies (MAbs) and synthetic peptides. XLP-derived LCLs could also induce MHC class I-restricted memory and cytotoxic T lymphocytes. Thus, these XLP-derived B cells resembled normal LCIs in vitro with respect to induction of EBV-specific cytotoxic T cells (CTL), the ability to present EB viral antigens and the susceptibility to EBV-specific and MHC-restricted CTL-mediated killing. The failure of the immune system to eliminate these virus-infected B cells in XLP is clearly not caused by a B-cell-specific defect.

Post-transplantation lymphoproliferative disorders in Mexico: an aggressive clonal disease associated with Epstein-Barr virus type A.

Post-transplantation lymphoproliferative disorders (PT-LPDs) are a complication of immunosuppression with variable clinical behavior and frequent Epstein-Barr virus (EBV) association. There is geographic variation in the association of EBV with certain tumors and a lack of studies of PT-LPDs from developing countries, so we decided to study in detail a series of PT-LPDs from Mexico to identify similarities and differences between populations in Mexico and those in Europe and the United States. We used paraffin-embedded tissue from eight PT-LPDs (six from men, two from women) that arose after renal transplantation. Clinical data, morphologic features, and clonality on the basis of immunoglobulin (Ig) light chain restriction, as well as polymerase chain reaction (PCR) for Ig heavy chain genes, were studied. The presence of EBV was investigated with PCR, immunohistochemical analysis for latent membrane protein (LMP)-1, and in situ hybridization for EBV early RNA transcripts. In addition, the subtype of EBV based on the EBNA 2A and 2B genes and the presence of a 30-bp deletion in the LMP-1 gene were investigated by PCR. Seven (87.5%) of eight cases presented with gastrointestinal involvement; five patients died. Three cases were polymorphic PT-LPDs, four were monomorphic large cell lymphomas (one diffuse large cell, three immunoblastic), and one was unclassifiable. All showed a B-cell phenotype, with a clonal population demonstrated in seven of the eight cases. Tumor cells expressed EBERs in all of the cases and LMP-1 in six of seven evaluable cases. Seven of seven cases showed EBV subtype A. Two (25%) of eight cases had the 30-bp LMP-1 deletion. This study shows that PT-LPDs in Mexico are clonal disorders associated with EBV subtype A. In contrast to series from Europe and the United States, our cases showed a significantly higher incidence of gastrointestinal tract involvement (P < .001), and a lower incidence of the 30-bp LMP-1 deletion, although this was not statistically significant (P < .28).

Lytic replication of Epstein-Barr virus in the peripheral blood: analysis of viral gene expression in B lymphocytes during infectious mononucleosis and in the normal carrier state.

Epstein-Barr virus (EBV) has been shown to establish latency in resting B lymphocytes of the peripheral blood. This creates a virus reservoir in contrast to lytic virus replication, which is thought to be restricted to differentiated epithelial cells in vivo. So far, the route of transmission between B cells and the production of progeny virus in the epithelial tissue has remained unclear. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry analysis of 16 patients with acute infectious mononucleosis (IM) and 25 healthy seropositive donors was performed to detect lytic replication gene products in B lymphocytes of the peripheral blood. Transcriptional activity was found in peripheral blood B lymphocytes (PBLs) for BZLF1 in 88%, BALF2 in 50%, and BcLF1 in 25% of the tested IM patients. All positive results were further confirmed in enriched B-cell populations by antigen determination using immunostaining with the APAAP technique. Furthermore, we detected transcripts for BZLF1 in 72% and for BALF2 in 16% of peripheral B lymphocytes of healthy seropositive donors. In contrast to patients with IM, no signals for BcLF1 were ever found in healthy seropositive donors. In these individuals, lytic replication of EBV is probably restricted by immunologic and gene regulatory mechanisms, whereas in the absence of immunologic control, reflected here by IM patients, the production of infectious virus becomes visible in PBLs.

Pathogenesis of chronic Epstein-Barr virus infection: detection of a virus strain with a high rate of lytic replication.

In rare cases Epstein-Barr virus (EBV) leads to chronic active infection (CAEBV) which is characterized by persistant symptoms of infectious mononucleosis. Previously we described a case of persisting polyclonal B-cell lymphocytosis (PPBL) that was associated with CAEBV. Using reverse transcription and polymerase chain reaction we showed that in late passages of a spontaneous cell line, SM, latent EB viral genes such as EBNA1, EBNA2, EBNA3A/3B/ 3C, LMP1 and LMP2A were active. The master gene of the lytic cycle, BZLF1, was silent. This indicated that there was no general defect in immortalization and establishing latency by this CAEBV isolate SM. We obtained virus from the standard immortalizing strain B95-8 and the CAEBV strain SM from latently infected LCL quantified the number of virus particles by competitive PCR and demonstrated that the impaired capacity to immortalize umbilical cord blood lymphocytes was a virus strain-specific property, and was not due to an incapability to infect purified CD19+ B lymphocytes. Transcription of latency- and immortalization-associated genes such as EBNA1, EBNA2 and LMP2A was reduced, in contrast to a strongly enhanced activity of the master gene of the lytic cycle, BZLF1. A scenario for an antagonistic regulation of lytic and latent cycle genes is presented and a role for the pathogenesis of CAEBV is discussed.

Chronic active Epstein-Barr virus disease in a case of persistent polyclonal B-cell lymphocytosis.

Persistent polyclonal B-cell lymphocytosis (PPBL) is a rare haematological disorder. It is characterized by activated and morphologically atypical B lymphocytes and polyclonal IgM production and has been associated with female sex, cigarette smoking, and HLA-DR7 expression. We report a case of PPBL with intermitting symptoms compatible with a chronic fatigue syndrome, recurrent erythema nodosum and multiforme. Serological findings suggested a chronic active Epstein-Barr virus (EBV) infection. Messenger RNA of EBV immediate early gene transactivation BZLF1 was detected in peripheral blood lymphocytes by reverse transcriptase PCR indicating a persistent replication of the virus. Over 2 years of observation we detected varying numbers of atypical lymphocytes. These cells hybridized with a probe specific for the EBV internal repeat region (BamHI W) which indicates a productive infection. Of interest, no reaction was observed with a probe specific for the latency-associated small RNAs (EBERs). The immunological phenotype of the polyclonal B cells was similar to B-cell lines immortalized by EBV in vitro, expressing a number of activation molecules (CD23, CD25, CD54) and the bcl-2 protein. In summary, our findings suggest that persistent EBV replication might be crucial in the development of lymphoproliferative disorders such as PPBL.

Role of Epstein-Barr virus and soluble CD21 in persistent polyclonal B-cell lymphocytosis.

The expression of EBV proteins and immunological properties were studied in the first stable cell line (SM) established from a patient presenting with persistent polyclonal B-cell lymphocytosis (PPBL). SM cells which represent a small population of EBV-positive atypical cells found in the peripheral blood of the patient express the KI-1 antigen (CD30) as well as the proto-oncogene bcl-2 product and cell surface markers of mature activated B lymphocytes. The cells harbour an EBV subtype A genome and contain EBNA2 protein. This argues against a transformation-incompetent virus as the main cause of the chronic active EBV infection observed in our patient. Latent membrane protein (LMP1) was weakly expressed and found predominantly in a perinuclear localization, a location which could lead to decreased immunogenicity in vivo. Similar to the EBV-transformed marmoset cell line B95-8, SM cells were in part productively infected as transcription of the immediate early gene BZLF1 could be shown and in some cells high levels of EBV-genome were detected by in situ hybridization with a BamH1 W-probe. Comparable to the atypical cells in the peripheral blood of the patient. EBV small RNAs were not detected with EBER-specific probes. Of interest, we noticed a markedly increased production of soluble CD21 (sCD21) antigen by SM cells as compared to LCL-type Burkitt's lymphoma cell lines. This could explain the elevated sCD21 levels observed in the serum of our PPBL patient and confirms our previous findings in patients with acute EBV infection. It also suggests a possible role of sCD21 in EBV-mediated regulation of the immune response and provides a possible explanation for the dysregulation of the humoral immune system observed in PPBl patients.

Epstein-Barr virus lytic replication is controlled by posttranscriptional negative regulation of BZLF1.

Regulation of the immediate-early gene BZLF1 is assumed to play a key role in triggering the lytic replication of Epstein-Barr virus (EBV). The expression of BZLF1 is regulated on multiple levels, including control of transcription by several positive and negative cis-acting elements as well as posttranslational modifications and protein-protein interactions. Localization of BZLF1 on one strand of the genome and the latent EBNA1 transcription unit on the complementary strand suggests a regulatory mechanism via hybridization of antisense RNA. With a plasmid encoding a defective BZLF1 RNA, which could not be translated, we were able to induce expression of endogenous BZLF1 gene product Zta and other proteins of the lytic cycle. Our data show for the first time that latent replication is stabilized by negative regulation of an immediate-early gene of the lytic cycle by a posttranscriptional mechanism. This might be a common theme of herpes simplex virus and EBV latency.

Negatively cis-acting elements in the distal part of the promoter of Epstein-Barr virus trans-activator gene BZLF1.

Epstein-Barr virus (EBV) replicates in a latent or a lytic way in the infected organism, depending on the type and level of differentiation of the host cell. The switch between latency and lytic replication was previously shown, for Burkitt's lymphoma cell lines, to depend on the viral BZLF1 gene product. Protein-DNA assays were used to identify the cis-acting elements that represent the link between regulating signal transduction pathways and the viral cascade of gene expression. Specific binding of proteins to several sites of the BZLF1 promoter during latency was shown. Induction of the lytic cycle by stimulation with 12-O-tetradecanoyl-phorbol 13-acetate abolished the binding of these proteins to the distal promoter (positions -227 to -551), suggesting a functional role for the down-regulation of promoter activity during latency. Computer analysis identified a multiply repeated sequence motif, HI, in this region and exonuclease III footprints confirmed that these sites act as specific protein recognition sites. Using a set of reporter plasmids we were able to demonstrate a negative regulatory effect of the HI motif in some B lymphoid cell lines, in contrast to epithelial HeLa cells. The HI silencer elements are different from other silencer elements described so far in respect of their sequence and protein-binding pattern during the activation of BZLF1.

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.

[Current perspectives in molecular virology]. / Aktuelle Perspektiven in der molekularen Virologie.

For reproduction viruses depend on the metabolism of higher organisms that use a variety of different antiviral mechanisms for defense. Insights into virus-host cell interactions and in the strategies viruses employ to produce progeny virions were possible by sophisticated methods of modern virology. Knowing about the molecular mechanisms during infection and replication we have been able to counteract viral attacks on the molecular level by rationally designed vaccines. On the other hand these viruses can be used as molecular vector tools for treatment of e.g. genetically determined diseases. On the basis of new molecular-biological methods the sensitivity of detection of infectious agents and diagnosis of diseases could be drastically improved.

Epstein-Barr virus and its interaction with the host.

Epstein-Barr virus (EBV) as a member of the herpesvirus family persists lifelong in the human body and causes diseases associated with virus replication (infectious mononucleosis, oral hairy leukoplakia) as well as neoplastic conditions such as nasopharyngeal carcinoma, B-cell lymphoma, Hodgkin's disease associated with viral latency. This complex biology relates to a highly regulated control of the persisting virus. Still, EBV is lytically produced in certain compartments of the human body. Epithelial cells were found to be of key importance for this. Various routes (cell fusion, IgA receptor-mediated uptake) were described for EBV to enter epithelial cells in the absence of CR2 receptor. Viral entry into cells, however, via CR2 receptor fusion or IgA mediated was not found to be sufficient for viral production. The molecular mechanisms for the lack of viral production in most target cells are primarily the presence of silencer activities and the early elimination of cells entering the lytic cycle. Only terminally differentiated epithelial cells are capable of supporting an efficient lytic cycle of EBV replication. EBV-mediated suppression of apoptosis as well as down-regulation of cellular and viral gene products, such as HLA molecules, which mediate recognition by the immune system, are important contributing factors to the development of these neoplasias where viral genes, possibly via interaction with anti-oncogenes, such as p53, in context with genetic and environmental factors play a key role. Novel diagnostic tools and a vaccine have been developed which could help to control EBV-related diseases.