BET-Inhibitors Disrupt Rad21-Dependent Conformational Control of KSHV Latency.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) establishes stable latent infection in B-lymphocytes and pleural effusion lymphomas (PELs). During latency, the viral genome persists as an epigenetically constrained episome with restricted gene expression programs. To identify epigenetic regulators of KSHV latency, we screened a focused small molecule library containing known inhibitors of epigenetic factors. We identified JQ1, a Bromodomain and Extended Terminal (BET) protein inhibitor, as a potent activator of KSHV lytic reactivation from B-cells carrying episomal KSHV. We validated that JQ1 and other BET inhibitors efficiently stimulated reactivation of KSHV from latently infected PEL cells. We found that BET proteins BRD2 and BRD4 localize to several regions of the viral genome, including the LANA binding sites within the terminal repeats (TR), as well as at CTCF-cohesin sites in the latent and lytic control regions. JQ1 did not disrupt the interaction of BRD4 or BRD2 with LANA, but did reduce the binding of LANA with KSHV TR. We have previously demonstrated a cohesin-dependent DNA-loop interaction between the latent and lytic control regions that restrict expression of ORF50/RTA and ORF45 immediate early gene transcripts. JQ1 reduced binding of cohesin subunit Rad21 with the CTCF binding sites in the latency and lytic control regions. JQ1 also reduced DNA-loop interaction between latent and lytic control regions. These findings implicate BET proteins BRD2 and BRD4 in the maintenance of KSHV chromatin architecture during latency and reveal BET inhibitors as potent activators of KSHV reactivation from latency.

Primary effusion lymphoma presenting as a cutaneous intravascular lymphoma.

Primary effusion lymphoma (PEL) is a rare and aggressive lymphoma that arises in the context of immunosuppression and is characterized by co-infection with Epstein-Barr virus (EBV) and human herpesvirus-8/Kaposi sarcoma-associated herpesvirus (HHV-8/KSHV). It was originally described as arising in body cavity effusions, but presentation as a mass lesion (extracavitary PEL) is now recognized. Here, we describe a case of PEL with an initial presentation as an intravascular lymphoma with associated skin lesions. The patient was a 53-year-old man with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS) who presented with fevers, weight loss and skin lesions concerning for Kaposi sarcoma (KS). A skin biopsy revealed no evidence of KS; however, dermal vessels contained large atypical cells that expressed CD31 and plasma cell markers but lacked most B- and T-cell antigens. The atypical cells expressed EBV and HHV-8. The patient subsequently developed a malignant pleural effusion containing the same neoplastic cell population. The findings in this case highlight the potential for unusual intravascular presentations of PEL in the skin as well as the importance of pursuing microscopic diagnosis of skin lesions in immunosuppressed patients.

Self-limited effusion large B-cell lymphoma: two cases of effusion lymphoma maintaining remission after drainage alone.

We report two cases of human herpesvirus-8 (HHV-8)-negative large B-cell lymphoma involving pericardial and/or pleural effusion that regressed after drainage alone. Case 1 is a 70-year-old man showing massive pericardial effusion. Cytology of the drained effusion showed monotonous infiltration of CD3-, CD20+, CD79a+, and CD138- large B-cells. Monoclonality was shown by Southern blot analysis. Case 2 is a 70-year-old man with massive pericardial and bilateral pleural effusion. Cytology of pericardial effusion showed infiltration of CD20+, CD45RO-, CD138-, immunoglobulin lambda chain+, and kappa chain- large B cells. In both cases, effusion resolved after drainage and no relapse has been observed. HHV-8 was not demonstrated in either case. Clinical presentation of our two cases resembled primary effusion lymphoma (PEL), but cytomorphology, immunophenotype, and prognosis were clearly distinct from those of PEL. HHV-8-negative effusion lymphomas might include prognostically favorable self-limited tumors that could regress without any cytotoxic therapy.

[Primary effusion lymphoma in two kidney transplant recipients]. / Lymphome des séreuses chez le transplanté rénal: deux cas.

BACKGROUND: Primary effusion lymphoma (PEL) is a highly malignant non-Hodgkin lymphoma associated with Kaposi's sarcoma-associated herpesvirus/human herpesvirus-8 infection (KSHV/HHV-8). It is chiefly seen in HIV patients and is rare in transplant recipients, possibly going unrecognized. OBSERVATION: We describe two male kidney transplant recipients, aged 47 and 51 years, followed for Kaposi's sarcoma in skin, lymph nodes, gastrointestinal (GI) tract and lung whose disease was poorly controlled by sirolimus and chemotherapy. Recurrent pleural effusion contrasted with reduction of cutaneous Kaposi lesions. KHSV viral loads were negative or very low in plasma, were negative or very low, whereas those in pleural effusion were high. Lymphoma cells were discovered only seven to nine months after the initial effusion despite repeated needle biopsies. In one patient, tumour cells were co-infected with Epstein-Barr virus. CONCLUSION: The contrast between a very low KHSV viral load in plasma and a very high viral load pleural effusion should alert physicians and prompt suspicion of PEL in Kaposi's sarcoma patients with recurrent serous effusion. The potential inhibitory role of sirolimus on PEL progression is discussed.

Role of viral induced vascular endothelial growth factor (VEGF) production in pleural effusion and malignant mesothelioma.

Vascular endothelial growth factor (VEGF) plays a key role in formation of pleural effusions and in tumorigenesis and progression of malignant mesothelioma. Mesothelial cells (MC) express the viral receptors Toll-like receptor 3 (TLR3), RIG-I and MDA5. Activation of these receptors by viral RNA exemplified by poly(I:C) RNA leads to a time- and dose-dependent increase of mesothelial VEGF synthesis. To show the specific effect of viral receptors knockdown experiments with siRNA for TLR3, RIG-I and MDA5 were performed. This finding of viral induced mesothelial VEGF synthesis may indicate a novel link between viral infections and formation of pleural effusions and progression of malignant mesothelioma.

Extracavitary tumor after primary effusion lymphoma: relapse or second distinct lymphoma?

HHV-8-associated solid lymphomas which develop in extracavitary sites during the course of primary effusion lymphoma (PEL) could represent the relapse of original PEL tumors in different anatomical sites, or newly occurring distinct HHV-8-associated lymphomas, such as multicentric Castleman disease-related microlymphomas. HHV-8 episome clonality might help identify which event takes place.

EBV-associated but HHV8-unrelated double-hit effusion-based lymphoma.

Effusion-based lymphoma is a rare and unique type of large B-cell lymphoma presenting in effusion without a mass lesion. It shares many clinicopathological features with primary effusion lymphoma (PEL), but is distinct from PEL by the absence of HHV8 association. Double hit lymphoma (DHL) is an aggressive B-cell lymphoma, defined by concurrent rearrangement of MYC and BCL2 or BCL6. DHL often presents as lymphadenopathy or an extranodal mass, but rarely occurs in effusion. Here we report a 61-year-old male with alcoholic cirrhosis presenting as massive ascites and left pleural effusion. He has no HIV, HBV or HCV infection and no mass lesion by CT scans. Cytology of both pleural effusion and ascites show large lymphoma cells with plasmablastic morphology characterized by pleomorphic and eccentric nuclei, prominent nucleoli and frequent mitoses. Immunohistochemical study with cell block shows that the lymphoma cells express plasma cell-related markers (CD138, MUM-1 and EMA), but not CD3, CD30, CD45, B-cell markers (CD19, CD20, CD79a, and PAX5), HHV8, ALK or cytokeratin. EBER is positive in most lymphoma cells. Fluorescence in situ hybridization reveals rearrangement at the IGH, BCL2, and MYC loci, but not at BCL6. It is diagnosed as an EBV-associated but HHV8-unrelated double hit effusion-based lymphoma with plasmablastic features. The patient passed away soon after diagnosis without chemotherapy. This is the first reported case of double-hit effusion-based lymphoma with MYC and BCL2 rearrangement. This case illustrates the importance of integrating clinical, cytological, immunophenotypical, and molecular findings to reach a correct diagnosis. Diagn. Cytopathol. 2017;45:257-261. © 2016 Wiley Periodicals, Inc.

Susceptibility of KSHV-Infected PEL Cell Lines to the Human Complement System.

Pleural effusion lymphoma (PEL) is a rare B-cell lymphoma that has a very poor prognosis with a median survival time of around 6 months. PEL is caused by Kaposi's sarcoma-associated herpesvirus, and is often co-infected with the Epstein Barr virus. The complement system is fundamental in the innate immune system against pathogen invasion and tumor development. In the present study, we investigated the activation of the complement system in PEL cells using human serum complements. Interestingly, two widely used PEL cell lines, BCP-1 and BCBL-1, showed different susceptibility to the complement system, which may be due to CD46 expression on their cell membranes. Complement activation did not induce apoptosis but supported cell survival considerably. Our results demonstrated the susceptibility of PEL to the complement system and its underlying mechanisms, which would provide insight into understanding the pathogenesis of PEL.

A requirement for NF-kappaB induction in the production of replication-competent HHV-8 virions.

The gammaherpesvirus human herpesvirus 8 (HHV-8) infects endothelial and B-lymphoid cells and is responsible for the development of Kaposi's sarcoma and primary effusion lymphoma (PEL). In the present study, we demonstrate that the activation of the NF-kappaB pathway during HHV-8 lytic replication is required for the generation of replication-competent virions capable of initiating a de novo infection of endothelial cells. In the HHV-8-positive PEL cell line BCBL-1, tetradecanoyl phorbol acetate (TPA) induction of the lytic cycle activates the NF-kappaB pathway, and this activation requires the induction of the IKKbeta component of the classical IkappaB kinase (IKK) complex. To further investigate the role of NF-kappaB activation in HHV-8 lytic replication, the NF-kappaB super-repressor IkappaBalpha-2NDelta4 was introduced into BCBL-1 cells by retroviral transduction. Expression of IkappaBalpha-2NDelta4 completely abolished NF-kappaB activity, as demonstrated by the loss of NF-kappaB DNA-binding activity and the absence of expression of the endogenous, NF-kappaB-regulated IkappaBalpha gene. NF-kappaB blockade dramatically impaired the ability of HHV-8 to produce infectious particles capable of initiating an effective de novo infection of endothelial EA.hy926 cells, as demonstrated by the lack of viral protein production in the target cells. Diminished infectivity did not appear to be caused by a reduction in virus titer, as demonstrated by equivalent viral DNA content in the supernatant of TPA-stimulated BCBL-1 and BCBL-1/2N4 cells. Although the viral and/or cellular products affected by NF-kappaB inactivation remain to be fully characterized, these data demonstrate an unexpected role for NF-kappaB induction during lytic reactivation in the production of replication-competent HHV-8 virions.

HHV-8 infection is specific for cell lines derived from primary effusion (body cavity-based) lymphomas.

Human herpes virus 8 (HHV-8; or KSHV, Kaposi's sarcoma-associated herpes virus) is a gamma herpes virus with sequence homology to Epstein-Barr virus (EBV). It was first isolated from Kaposi's sarcoma tumor cells and subsequently from tumor cells and peripheral blood mononuclear cells from patients with primary effusion lymphomas (PEL; or body cavity-based lymphomas). PEL has been recognized as an individual nosologic entity based on its distinctive biological-pathological features and its consistent infection with HHV-8 (commonly, but not always co-infected with EBV), occurring predominantly in human immunodeficiency virus (HIV)-infected patients but occasionally also in HIV-negative cases. Whether HHV-8 sequences can be found also in non-hematopoietic tumor cells other than Kaposi's sarcoma and in malignant hematopoietic malignancies other than PEL, has been the focus of the present studies. We examined the presence of HHV-8 sequences by polymerase chain reaction (PCR) using (1) a panel of 133 human cell lines established from a large variety of solid tumors; (2) a spectrum of 114 hematopoietic cell lines derived from the different cell lineages including 50 B cell leukemia/lymphoma-derived cell lines and seven cell lines established from patients with PEL. Besides the seven PEL cell lines, 46 cell lines that were derived from malignant pleural effusion or ascitic fluid material (25 non-hematopoietic and 21 hematopoietic cell lines) were examined. Except for the seven PEL cell lines that were strongly HHV-8+ in the PCR, all solid tumor cell lines and all hematopoietic cell lines scored consistently negative for the presence of HHV-8 sequences. These results confirm the absolute specificity of HHV-8 infection (within the hematopoietic malignancies) for PEL. PEL cell lines represent useful tools for the analysis of the biology of this neoplasm and of the pathogenetic role of the virus in the disease development.

Lymphoma cell lines: in vitro models for the study of HHV-8+ primary effusion lymphomas (body cavity-based lymphomas).

Primary effusion lymphoma (PEL; also known as body cavity-based lymphoma) is recognized as a new and unique lymphoma entity occurring predominantly, but not exclusively in human immunodeficiency virus (HIV)-seropositive patients with acquired immunodeficiency syndrome (AIDS). PEL grows exclusively in body cavities as serous lymphomatous effusion without evidence of mass disease or dissemination. Their most unique feature is infection with the newly discovered human herpesvirus-8 (HHV-8; also known as Kaposi's sarcoma-associated herpesvirus), often accompanied by co-infection with Epstein-Barr virus (EBV). A number of continuous lymphoma cell lines have been established from the malignant pleural effusion, ascitic fluid and peripheral blood of patients with AIDS- and non-AIDS-associated PEL. While all cell lines are HHV-8+, about half of them also contain EBV sequences. Stimulation of the cell lines causes switch from latent to lytic HHV-8 infection. The cells are generally negative for T and B cell immunomarkers (except for CD138 suggesting a pre- or terminal plasma cell stage) and positive for some activation and adhesion markers; they are genotypically B cells with their immunoglobulin genes rearranged. Complex, hyperdiploid karyotypes with multiple structural abnormalities are seen in the cell lines examined. No alterations of known proto-oncogenes are detected in PEL, with the exception of BCL-6 mutations occurring in a large percentage of cases. Heterotransplantation of the cell lines into immunodeficient mice leads to the development of lymphomatous effusion and marked angiogenesis. As HHV-8 contains DNA sequences of several protein homologues, the cell lines express various cytokines, cytokine receptors, chemokines, cell cycle and anti-apoptosis modulators which are upregulated upon stimulation. Indeed, some cell lines produce high levels of (human) interleukin-6 and interleukin-10. Taken together, these cell lines represent very important model systems for the elucidation of the pathobiology of PEL; furthermore, the cell lines are extremely useful scientific tools providing a resource to pursue studies of HHV-8-mediated pathogenic mechanisms.

Characterization of a novel HHV-8-positive cell line reveals implications for the pathogenesis and cell cycle control of primary effusion lymphoma.

Primary effusion lymphoma (PEL) represents a peculiar type of B cell lymphoma which associates with HHV-8 infection and preferentially grows in liquid phase in the serous body cavities. In this report, we provide the detailed characterization of a newly established PEL cell line, termed CRO-AP/6. The cell line was obtained from the pleural effusion of a HIV-positive patient with PEL. Its derivation from the tumor clone was established by immunogenotypic analysis. Detailed phenotypic investigations defined that CRO-AP/6 reflects pre-terminally differentiated B cells expressing the CD138/syndecan-1 antigen. Karyotypic studies of CRO-AP/6 identified several chromosomal abnormalities, whereas genotypic studies ruled out the involvement of molecular lesions associated with other types of B cell lymphoma. Both CRO-AP/6 and the parental tumor sample harbored infection by HHV-8. Conversely, EBV infection was present in the parental tumor sample although not in CROAP/6, indicating that CRO-AP/6 originated from the selection of an EBV-negative tumor subclone. The pattern of viral (HHV-8 v-cyclin) and cellular (p27Kip1) regulators of cell cycle expressed by CRO-AP/6, together with the results of growth fraction analysis, point to abrogation of the physiological inverse relationship between proliferation and p27Kip1 expression. Also, both CRO-AP/6 and the parental tumor sample display biallelic inactivation of the DNA repair enzyme gene O6-methylguanine-DNA methyltransferase (MGMT) by promoter methylation. Overall, the CRO-AP/6 cell line may help understand cell cycle control of PEL cells, may clarify the relative contribution of HHV-8 and EBV to the disease growth and development and may facilitate the identification of recurrent cytogenetic abnormalities highlighting putative novel cancer related loci relevant to PEL.

Primary body cavity-based AIDS-related lymphomas.

Five patients with advanced AIDS developed a unique type of high grade primary body cavity-based non-Hodgkin's lymphoma (NHL). The lymphomas were exclusively in serous effusions with no detectable mass disease in the body cavities and no lymphadenopathy or organomegaly. All of the lymphomas exhibited virtually identical morphology, which could not be precisely classified, but appeared to bridge features of large cell immunoblastic and anaplastic large cell lymphomas. Immunophenotypically the lymphoma cells lacked expression of any B- or T-lymphocyte antigens, but expressed CD45 and the activation antigens CD30, CD38, CD71, and HLA-DR. Clonally rearranged immunoglobulin heavy chain and kappa light chain genes were identified by Southern blot analysis. Molecular studies also revealed Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) genomes and germline configuration of the c-myc protooncogene. In two cases studied cytogenetically, the lymphoma cells manifested complex chromosome abnormalities. These lymphomas are clinically and biologically unique and found predominantly in patients with advanced AIDS, in many cases with pre-existing Kaposi's sarcoma.

Virokines in the pathogenesis of cancer: focus on human herpesvirus 8.

During evolution, DNA viruses have captured a broad array of cellular genes involved in immune recognition and growth control that are nonessential for viral replication. The encoded virokines and viroceptors may act as mimetics or antagonists of their cellular homologues, altering signal transduction and cell communication towards survival of virus-infected cells. Human herpesvirus type 8 (HHV8) is the most recently identified human oncogenic herpesvirus. It is associated with Kaposi's sarcoma and lymphoproliferative diseases, such as pleural effusion lymphomas and multicentric Castleman's disease. HHV8 has captured a unique number of cellular regulatory genes, which redirect gene expression and cell growth, prevent apoptosis and immune recognition, and interfere with tumor suppressor gene function. HHV8 encodes a unique virokine, viral interleukin-6, which is particularly relevant for the pathogenesis of HHV8-associated tumors, since it participates in transformation and mediates autocrine and paracrine mitogenic and proinflammatory effects. Viral IL-6 differs fundamentally from human IL-6 in receptor engagement for signal transduction and thus constitutes a singular model to understand the facets of human and viral cytokine biology. We provide an overview of the role of virokines in cancer, with a particular focus on the differences of human and viral IL-6 in the pathophysiology of HHV8-associated tumors.

Establishment of a primary effusion lymphoma cell line (RM-P1) and in vivo growth system using SCID mice.

Primary effusion lymphoma (PEL) is recognized as a unique lymphoma entity, which occurs exclusively in body cavities as a serous lymphomatous effusion without tumor formation or organ infiltration. We established a cell line of B-cell origin from a pericardial effusion of a 63-year-old Japanese PEL patient who did not have human immunodeficiency virus infection. This PEL cell line had human herpesvirus-8 (HHV-8) and Epstein-Barr virus (EBV) infection. We named this cell line RM-P1. This cell line showed complex chromosomal abnormalities that could not be identified by G-banding. However, spectral karyotyping analysis determined the origin and organization of all unidentified chromosomal abnormalities. When inoculated into the peritoneal cavity of 8 severe combined immunodeficiency (SCID) mice depleted of natural killer cells, RM-P1 cells induced solid tumor with ascites in all animals tested. These tumor and ascitic cells had the same immunogenotypic features as those of the original RM-P1. These 2 types of cells were positive for both HHV-8 and EBV as demonstrated using polymerase chain reaction. Fluorescence-activated cell sorting analyses showed that neither tumors nor ascitic cells grown in SCID mice expressed leukocyte function-associated antigen (LFA)-1alpha (CD11a), LFA-1lbeta (CD18), LFA-2 (CD2), LFA-3 (CD58), intercellular adhesion molecule (ICAM)-1 (CD54), ICAM-2 (CD102), ICAM-3 (CD50), or leukocyte endothelial adhesion molecule (LECAM)-1 (CD62L), suggesting that these cytoadhesion molecules are not involved in tumor formation of RM-P1 cells in vivo. The establishment of the RM-P1 cell line and the animal model of PEL may provide insights for understanding the relationship between these viruses and PEL and for understand the mechanism for PEL.

Human herpes virus 8-negative primary effusion lymphoma in a patient with a ventriculoperitoneal shunt tube.

A 60-year-old woman was referred to our hospital in 1996 due to an abdominal distension in the right lower quadrant. She had undergone a partial resection of a cholesteatoma at the right temporal lobe of the cerebrum 30 years previously, and a ventriculoperitoneal shunt (VPS) tube had been placed with drainage into the right lower peritoneal cavity. The patient developed paralytic ileus in December 1966, and ultrasound and computed tomography of the abdomen revealed a cystic mass in the right lower quadrant without lymphadenopathies or masses. Cytologic examinations of the fluid in the cystic mass revealed signs of malignant lymphoma. After the resection of the cystic mass, lymphoma cells were detected in the fluid, but the wall of the cyst consisted of only fibrous tissues. Results of immunophenotypic analysis of the lymphoma cells by immunocytochemistry or flow cytometry were positive for CD19, CD20, CD22, CD45, and HLA-DR but negative for CD45RO, CD3, CD4, and CD8. The genome of human herpes virus (HHV)-8 was not detected in the lymphoma cells, but Epstein-Barr (EB) nuclear antigen 1 and EB virus (EBV)-encoded small nuclear RNAs were detected. Chromosome analysis by the G-banding method showed complicated abnormalities including der(8)t(2;8)(q31;q24), but Southern blotting analysis suggested that the c-myc oncogene did not participate in the lymphomagenesis. The patient's disease was diagnosed as HHV-8-negative primary effusion lymphoma (PEL). The long-standing inflammatory stimulation by a VPS tube might have contributed to the clonal evolution of EBV-infected lymphocytes. resulting in the development of PEL.

Body-cavity-based lymphoma in an elderly AIDS-unrelated male.

Body-cavity-based lymphoma (BCBL) is a recently described subtype of human non-Hodgkin's lymphoma characterized by the localization of neoplastic cells exclusively in the body cavities. BCBL is found most commonly in AIDS patients, and is known to be highly associated with Kaposi's sarcoma-associated herpes virus (KSHV). We describe here a case of BCBL that occurred in a 101-year-old man. He was successively treated with etoposide, but died 8 months after the diagnosis of BCBL. No lymphoma masses were noted at pre- and postmortem examinations. KSHV was demonstrated in large numbers in the neoplastic cells using semiquantitative PCR analysis. Although there have been four brief reports of HIV negative BCBL, the present case is the first for which the detailed clinical course and response to chemotherapy have been recorded.

Induction of human herpesvirus 8 gene expression in a posttransplantation primary effusion lymphoma cell line.

Human herpesvirus 8 (HHV-8 or Kaposi's sarcoma herpesvirus) is a gamma herpesvirus that is most likely the etiologic agent of both Kaposi's sarcoma and primary effusion lymphoma (PEL), a rare HIV-associated lymphoma. The role of HHV-8 in post-transplant lymphoma is less well characterized. We demonstrate that HHV-8 is constitutively present in LH5-21 cells, an atypical patient derived posttransplant PEL cell line. LH5-21 cells lack detectable Epstein-Barr virus, express T cell-associated surface markers and have undergone immunoglobulin heavy chain gene rearrangement. Incubation with 12-O-tetradecanoyl-phorbol- 13-acetate or butyrate induces high levels of several HHV-8 encoded genes that are associated with lytic replication. The patient from whom this cell line was derived demonstrated a dramatic clinical response to withdrawal of immunosuppressive therapy. While HHV-8 associated PELs in the post-transplant setting are rare, this study suggests that improvement in the host immunologic function might help in the management of some PELs.

Alterations of mRNA splicing in primary effusion lymphomas.

Primary effusion lymphoma (PEL) is a unique form of malignant lymphoma associated with infection by the Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8). The majority of PELs also contain the EBV genome. Although viral infection is believed to play a critical role in the pathogenesis of PEL, it has been suggested that additional molecular lesions are required for the development of PEL. Alternative splicing of pre-mRNA is an important mechanism in the regulation of cellular and viral gene expression. Deregulation of pre-mRNA splicing may shift the gene expression balance and lead to the development of cancer. In order to investigate mRNA splicing in PELs, we examined mRNA splicing of three genes, DNA polymerase beta (pol beta), Bcl-x and CD45, in eight PEL cell lines. We found that the average variant percentage of pol beta in PEL cell lines is two times higher than in peripheral blood mononuclear cells (PBMC) and that the variant pattern of genes bcl-x and CD45 is quite different in PEL cell lines than in PBMC. In addition, we also found that the percentage of variant pol beta increased two-fold in PBMC following Epstein-Barr virus (EBV) infection. Therefore, viral infection may contribute to mRNA alternative splicing in PEL. In order to explore the mechanism by which viral infection affects mRNA splicing, we also examined the roles of genes KS-SM, SM and EBERs and viral copies in mRNA splicing. Our findings indicate that various factors acting as positive or negative regulators may be involved in mRNA alternative splicing caused by viral infection. In conclusion, mRNA splicing in PEL can be altered by viral infection and this alteration may contribute to the pathogenesis of PEL.