NXF1 and CRM1 nuclear export pathways orchestrate nuclear export, translation and packaging of murine leukaemia retrovirus unspliced RNA.

Cellular mRNAs are exported from the nucleus as fully spliced RNAs. Proofreading mechanisms eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. Retroviruses need to export partially spliced and unspliced full-length RNAs to the cytoplasm where they serve as templates for protein synthesis and/or as encapsidated RNA in progeny viruses. Genetically complex retroviruses such as HIV-1 use Rev-equivalent proteins to export intron-retaining RNA from the nucleus using the cellular CRM1-driven nuclear export machinery. By contrast, genetically simpler retroviruses such as murine leukaemia virus (MLV) recruit the NXF1 RNA export machinery. In this study, we reveal for the first time that MLV hijacks both NXF1 and CRM1-dependent pathways to achieve optimal replication capacity. The CRM1-pathway marks the MLV full-length RNA (FL RNA) for packaging, while NXF1-driven nuclear export is coupled to translation. Thus, the cytoplasmic function of the viral RNA is determined early in the nucleus. Depending on the nature of ribonucleoprotein complex formed on FL RNA cargo in the nucleus, the FL RNA will be addressed to the translation machinery sites or to the virus-assembly sites at the plasma membrane.

IgM peak independently predicts treatment-free survival in chronic lymphocytic leukemia and correlates with accumulation of adverse oncogenetic events.

We examined the significance of IgM peaks in chronic lymphocytic leukemia (CLL), including its association with newly reported MYD88, BIRC3, NOTCH1 and SF3B1 mutations. A total of 27, 25, 41 and 57 patients with monoclonal IgM or IgG peaks (IgM and IgG groups), hypogammaglobulinemia (Hypo-γ group) and normal immunoglobulin serum levels (normal-γ group) were, respectively, included. IgM peaks were mainly associated with Binet stage C and the del(17p). Biased usage of IGHV3-48 was shared by both IgM and IgG groups. IGHV3-74 and IGHV4-39 gene rearrangements were specific for IgM and IgG peaks, respectively. SF3B1, NOTCH1, MYD88 and BIRC3 mutation frequencies were 12%, 4%, 2% and 2%, respectively, being over-represented in IgM, IgG and Hypo-γ groups for SF3B1, and being equal between normal-γ and IgM groups for MYD88. Overall, 76%, 87%, 49% and 42% of cases from IgM, IgG, Hypo-γ and normal-γ groups had at least one intermediate or poor prognosis genetic marker, respectively. By multivariate analysis, IgM peaks were associated with shorter treatment-free survival independently from any other univariate poor prognosis biological parameters, including IgG peaks, Hypo-γ, IGHV status, SF3B1 mutations, cytogenetics and lymphocytosis. Therefore, as with IgG peaks, IgM peaks aggravated the natural course of CLL, with increased accumulation of adverse genetic events.

Reversion of apoptotic resistance of TP53-mutated Burkitt lymphoma B-cells to spindle poisons by exogenous activation of JNK and p38 MAP kinases.

Defects in apoptosis are frequently the cause of cancer emergence, as well as cellular resistance to chemotherapy. These phenotypes may be due to mutations of the tumor suppressor TP53 gene. In this study, we examined the effect of various mitotic spindle poisons, including the new isocombretastatin derivative isoNH2CA-4 (a tubulin-destabilizing molecule, considered to bind to the colchicine site by analogy with combretastatin A-4), on BL (Burkitt lymphoma) cells. We found that resistance to spindle poison-induced apoptosis could be reverted in tumor protein p53 (TP53)-mutated cells by EBV (Epstein Barr virus) infection. This reversion was due to restoration of the intrinsic apoptotic pathway, as assessed by relocation of the pro-apoptotic molecule Bax to mitochondria, loss of mitochondrial integrity and activation of the caspase cascade with PARP (poly ADP ribose polymerase) cleavage. EBV sensitized TP53-mutated BL cells to all spindle poisons tested, including vincristine and taxol, an effect that was systematically downmodulated by pretreatment of cells with inhibitors of p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases. Exogenous activation of p38 and JNK pathways by dihydrosphingosine reverted resistance of TP53-mutated BL cells to spindle poisons. Dihydrosphingosine treatment of TP53-deficient Jurkat and K562 cell lines was also able to induce cell death. We conclude that activation of p38 and JNK pathways may revert resistance of TP53-mutated cells to spindle poisons. This opens new perspectives for developing alternative therapeutic strategies when the TP53 gene is inactivated.

RelA and RelB cross-talk and function in Epstein-Barr virus transformed B cells.

In this study, we determined the respective roles of RelA and RelB NF-κB subunits in Epstein-Barr virus (EBV)-transformed B cells. Using different EBV-immortalized B-cell models, we showed that only RelA activation increased both survival and cell growth. RelB activity was induced secondarily to RelA activation and repressed RelA DNA binding by trapping the p50 subunit. Reciprocally, RelA activation repressed RelB activity by increasing expression of its inhibitor p100. To search for such reciprocal inhibition at the transcriptional level, we studied gene expression profiles of our RelA and RelB regulatable cellular models. Ten RelA-induced genes and one RelB-regulated gene, ARNTL2, were repressed by RelB and RelA, respectively. Apart from this gene, RelB signature was included in that of RelA Functional groups of RelA-regulated genes were for control of energy metabolism, genetic instability, protection against apoptosis, cell cycle and immune response. Additional functions coregulated by RelA and/or RelB were autophagy and plasma cell differentiation. Altogether, these results demonstrate a cross-inhibition between RelA and RelB and suggest that, in fine, RelB was subordinated to RelA. In the view of future drug development, RelA appeared to be pivotal in both classical and alternative activation pathways, at least in EBV-transformed B cells.

IGHV gene features and MYD88 L265P mutation separate the three marginal zone lymphoma entities and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas.

To clarify the relationships between marginal zone lymphomas (MZLs) and Waldenström macroglobulinemia/lymphoplasmacytic lymphomas (WM/LPLs), immunoglobulin heavy chain variable gene (IGHV) features were analyzed and the occurrence of MYD88 L265P mutations was identified in a series of 123 patients: 53 MZLs from the spleen (SMZLs), 11 from lymph nodes (NMZLs), 28 mucosa-associated lymphatic tissue (MALT) lymphomas and 31 WM/LPLs. SMZLs were characterized by overrepresentation of IGHV1-2 gene rearrangements with a canonical motif, without selection pressure and with long CDR3 segments. NMZLs had increased frequencies of IGHV3 genes. The IGHV gene was unmutated in most cases, often with long CDR3 segments. MALT lymphomas were usually associated with a mutated IGHV gene, but with the absence of selection pressure. WM/LPLs were associated with an IGHV3-23 overrepresentation and high IGHV mutation rate, with features of selection pressure and short CDR3 segments. MYD88 L265P mutations were almost restricted exclusively to WM/LPL patients. Taken all diagnoses together, all patients with MYD88 L265P mutations had an immunoglobulin M peak and almost all patients except one had bone marrow infiltration. These results demonstrate that the history of antigen exposure of the four entities studied was different and MYD88 L265P was specifically associated with WM/LPLs. WM/LPL may thus be functionally associated with constitutive nuclear factor-κB activation.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group.

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as 'normal', 'suggestive of', or 'diagnostic of' MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Identification of a novel p53-dependent activation pathway of STAT1 by antitumour genotoxic agents.

Chemotherapeutic drugs such as fludarabine*, doxorubicin or cisplatin are very potent activators of the anti-oncogene p53. Convergent studies suggest that p53 and STAT1 (signal transducer and activator of transcription 1) cooperate in the induction of cell death. We show that these drugs are also activators of STAT1 in p53-expressing cells, but not in p53-null cells. STAT1 activation was obtained in the presence of both the secretion inhibitor brefeldine A and the inhibitor of RNA synthesis, actinomycin D. p53-dependent STAT1 activation was reversed by overexpression of MDM2 and siRNAs against p53. Genetic analysis of p53 showed that expression of transcriptionally inactive p53 punctual mutants markedly increased Y701-STAT1 phosphorylation, and suggests that the p53 DNA-binding domain was alternatively involved in STAT1 activation or p53 multimerization. Immunoprecipitation experiments showed that ataxia telangiectasia mutated, p53, STAT1 and c-Abl1 (Abelson murine leukaemia viral oncogene homologue 1) were associated together. Treatment of cells with the c-Abl1 tyrosine kinase inhibitor STI571 decreased STAT1 activation by genotoxic drugs. Finally, genotoxic agents sensitized cells in response to very low doses of both interferon alpha and gamma (IFNalpha and gamma). These results show that genotoxic drugs induce STAT1 activation, an effect that depends on p53 protein but not on p53 transcriptional activity, and point to a novel pathway of STAT1 activation by genotoxic drugs, with involvement of c-Abl1 tyrosine kinase in sensitizing cells to IFN response.

Heterogeneous Epstein-Barr virus latent gene expression in AIDS-associated lymphomas and in type I Burkitt's lymphoma cell lines.

Epstein-Barr virus (EBV) is associated with lymphoma in immunocompromised patients. This study provides evidence that the expression of EBV nuclear antigen-3 genes can be directed from the F promoter in different type I Burkitt's lymphoma cell lines and in some lymphomas from human immunodeficiency virus-infected patients. This expression occurs predominantly after induction of the EBV lytic cycle.

Identification of a leukemic counterpart of the plasmacytoid dendritic cells.

This work aims to demonstrate that CD4(+)CD56(+) malignancies arise from transformed cells of the lymphoid-related plasmacytoid dendritic cell (pDC) subset. The analysis of malignant cells from 7 patients shows that in all cases, like pDCs, leukemic cells are negative for lineage markers CD3, CD19, CD13, CD33, and CD11c but express high levels of interleukin-3 receptor alpha chain (IL-3Ralpha), HLA-DR, and CD45RA. Tumor cells produce interferon-alpha in response to influenza virus, while upon maturation with IL-3 they become a powerful inducer of naive CD4(+) T-cell proliferation and promote their T-helper 2 polarization. As pDCs, leukemic cells also express pre-Talpha and lambda-like 14.1 transcripts, arguing in favor of a lymphoid origin. In addition, malignant cells express significant levels of CD56 and granzyme B. Overall, those observations suggest that CD4(+)CD56(+) leukemic cells could represent the malignant counterpart of pDCs, both of which are closely related to B, T, and NK cells.

Investigation of human spleen dendritic cell phenotype and distribution reveals evidence of in vivo activation in a subset of organ donors.

Although the mouse spleen dendritic cell (DC) is perhaps the most intensively studied DC type, little has been published concerning its human equivalent. In this report, rare event flow cytometry and in situ immunofluorescence were used to study the surface phenotype and distribution of HLA-DR(+) CD3(-)14(-)16(-)19(-) human spleen DC. Spleens from organ donors with different clinical histories were used. Most (81% +/- 9%; n = 14) spleen DCs expressed high levels of the integrin CD11c. CD11c(+) DCs were distributed in 3 distinct regions-the peri-arteriolar T-cell zones, the B-cell zones, and the marginal zone, where they formed a ring of cells surrounding the white pulp, just inside a ring of CD14(+) red pulp macrophages, apparently more regularly organized than the previously described marginating DC population in the mouse spleen. The T-cell zones contained CD86(+) DCs, among which a subpopulation expressed CD83. These mature/activated CD86(+) DCs represented a minority (12% +/- 8%) of total spleen DCs in most organ donors: most spleen DCs are immature. In 3 of 18 (17%) donors, however, most (54%-81%) of spleen DCs were CD86(+), suggesting that in vivo DC activation had occurred. In one donor, a radical shift in DC distribution from the marginal zone to the T-cell zones was also observed. This activation of spleen DCs in vivo was reminiscent of the effects of experimental microbial product injection in mice, and it seemed to correlate with bacterial infection or multiple trauma. (Blood. 2001;97:3470-3477)

Protein analysis by mass spectrometry and sequence database searching: a proteomic approach to identify human lymphoblastoid cell line proteins.

Lymphoblastoid cell lines correspond to in vitro EBV-immortalized lymphocyte B-cells. These cells display a suitable model for experiments dealing with changes in protein expression occurring upon B-cell differentiation, after drug treatment, or after inhibition of some transcription factors. For all these reasons we have undertaken an effort aimed at developing a hematopoietic cell line protein two-dimensional electrophoresis (2-DE) database, containing B-lymphoblastoid 2-DE maps. In this work, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) peptide mass fingerprinting analysis was adopted for protein identification. The peptide mass fingerprinting identification and the sequence coverage obtained on colloidal Coomassie blue (CBB) stained gel was close to that obtained using zinc-imidazole staining. Everything considered, CBB being more comfortable for subsequent spot manipulations, CBB staining was chosen for identification of a larger number of polypeptides. The results suggest that reticulation of the gel can interfere preventing the uptake of the enzyme during the in-gel digestion step. Consequently, low molecular mass proteins appear more difficult to identify by mass fingerprinting. Finally, the information provided in this study allows the construction of a new annoted reference map of human lymphoblastoid cell proteins. Among the identified proteins 60% were not yet positioned on 2-DE maps in three of the most important well-documented databases. The annoted map will be accessible via Internet on the LBPP server at URL:http:// www-smbh.univ-paris13.fr/lbtp/index.htm.

In vivo identification of lymphocyte subsets exhibiting transcriptionally active NF-kappaB/Rel complexes.

To analyze the NF-kappaB/Rel activity pattern in a living organism, we previously generated transgenic mice carrying a kappaB-dependent lacZ gene. In situ analysis of both primary and secondary lymphoid organs revealed a strong NF-kappaB transcriptional activity in antigen-presenting cells, some endothelial cells and sinus lining cells of the lymph node capsula with very little activity in lymphocytes and thymocytes. Using fluorescein-di-beta-D-galactopyranoside (FDG) as a vital substrate for the beta-galactosidase, we re-examined by flow cytometry the NF-kappaB/Rel transcriptional activity in our mouse model. We report here that such constitutive NF-kappaB/Rel activity was significantly detected in thymocytes at the CD44+CD25(-) stage. This constitutive activity extended with CD25 expression to the majority of the CD44(-)CD25(+) thymocytes and was then restricted to a few mature T cells. In the spleen, constitutive NF-kappaB/Rel activity was found in most B cells, unlike T cells which were largely negative. Virgin IgD(+) B cells expressed higher levels of NF-kappaB transcriptional activity than other B cell types. Altogether, these results suggest that NF-kappaB/Rel complexes are key players in the in vivo differentiation of IgD(+) B lymphocytes and possibly CD25(+) thymocytes.

Inducible loss of NF-kappaB activity is associated with apoptosis and Bcl-2 down-regulation in Epstein-Barr virus-transformed B lymphocytes.

The Epstein-Barr virus (EBV)-encoded latent membrane protein-1 induces NF-kappaB activity by targeting IkappaBalpha. To understand the role of NF-kappaB activation in EBV-related oncogenesis, we have subcloned mutated IkappaBalpha(32/36A) cDNA into a pHEBo vector containing doxycycline regulatory sequences and stably transfected this construct into a lymphoblastoid cell line. Two tightly regulated clones were obtained in which IkappaBalpha(32/36A) was inducible in a doxycycline dose-dependent manner. Levels of inducible IkappaBalpha(32/36A) peaked at day 2. Inhibition of NF-kappaB activity was closely correlated with levels of inducible IkappaBalpha(32/36A). Levels of 3 well-known NF-kappaB-dependent genes, CD54, p105, and endogenous IkappaBalpha, were decreased when IkappaBalpha(32/36A) was induced, and the growth of IkappaBalpha(32/36A)-induced EBV-infected cells was slightly reduced. Loss of NF-kappaB activity was associated with decreased Bcl-2 protein levels. Finally, the induction of apoptosis was strongly increased in IkappaBalpha(32/36A)-overexpressing cells. Together these results show that it is possible to control IkappaBalpha(32/36A) levels, ie, NF-kappaB activity, in EBV-infected B-lymphocytes using a doxycycline-inducible vector. Moreover, our results indicate that NF-kappaB can protect EBV-infected cells from apoptosis by Bcl-2. Finally, our results suggest that a cellular model with doxycycline-inducible IkappaBalpha(32/36A) may be useful in the identification of genuine NF-kappaB target genes in EBV-infected B cells. (Blood. 2000;95:2068-2075)

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)

A computer-assisted two-dimensional gel electrophoresis approach for studying the variations in protein expression related to an induced functional repression of NFkappaB in lymphoblastoid cell lines.

Strategies are needed for conclusive interpretation of two-dimensional gel electrophoresis (2-D PAGE) maps in order to identify pertinent differences in protein expression during regulation of the transcription of discrete sets of genes. The model used in this study was a human lymphoblastoid cell line in which a functional repression of the transcription factors NFkappaB was obtained by induction of overexpression of IkappaBalpha, a physiological inhibitor of NFkappaB. The analytical methodology used relies on the comparison of two sets of 2-D PAGE maps for detecting differences in protein expression between samples overexpressing or not overexpressing IkappaBalpha. The analysis was based on a combination of an automatic computerized analysis, constituting an actual aid for deciding, and of an interactive visual validation, corresponding to the interpretation of computer propositions. This strategy is proposed as a rapid way to detect potential variations in protein expression applicable to any biological model. In this study, correspondence analysis data made it possible to discrimate between the samples overexpressing or not overexpressing IkappaBalpha, and pointed out some of the potential meaningful spots characterizing the samples in which NFkappaB was active. Then, after visual validation of the computer data, 53 polypeptides were considered to be different in the two classes of gels. Five polypeptides were specifically found in both samples overexpressing IkappaBalpha. The overexpression of IkappaB also induced a lower expression of 11 polypeptides. Finally, 15 polypeptides were only expressed in samples in which IkappaBalpha was not overexpressed and, consequently, in which NFkappaB factors were active. Thus, these polypeptides are candidates for further analysis as putative target gene products of NFkappaB.

Relationship between IkappaBalpha constitutive expression, TNFalpha synthesis, and apoptosis in EBV-infected lymphoblastoid cells.

In order to understand the role of NF-kappaB in EBV transformation we have established stably transfected IkappaBalpha into lymphoblastoid cells. Two clones were obtained in which the loss of NF-kappaB binding activity correlated with the constitutive expression of the transgenic IkappaBalpha. Protein latency expression was determined by immunocytochemistry. Expression of surface markers, intracytoplasmic content of cytokines cell cycle analysis after BrdU incorporation and DNA staining with propidium iodide were studied by flow cytometry. Percentage of apoptotic cells was determined by in-situ labelling of DNA strand breaks. No significative changes in EBV latency nor in cell surface marker expression was found. In contrast, intracytoplasmic TNFalpha levels were strongly reduced in transfected clones. Furthermore, 30% of IkappaBalpha transfected cells were apoptotic after 8 h of TNFalpha treatment. This correlated with a strong reduction of BrdU incorporation after 24 h of TNFalpha treatment. No effect was seen with non transfected cells or with cells transfected with a control plasmid. Our results suggest that the TNFalpha gene could be one of the targets of NF-kappaB in EBV infected cells and that NF-kappaB protects EBV-infected cells from apoptosis induced by TNFalpha, which may favour the proliferative effect of this cytokine.

CD80 expression is decreased in hyperplastic lymph nodes of HIV+ patients.

A centrofollicular hyperplasia is present within secondary lymphoid organs during all the asymptomatic phase of the HIV disease. Although this hyperplasia has been well characterized by histological studies, the nature of the phenotypic alterations in B cell populations occurring within HIV+ lymphoid organs remains to be established. By immunohistochemistry, we thus investigated whether a particular germinal center (GC) B cell population was increased during HIV-induced hyperplasia and whether any phenotypic change was specific to HIV-1 infection. As compared to normal tonsils (three cases) and HIV- hyperplastic lymph nodes (eight patients), we observed a loss of GC polarization in all HIV+ sections (11 patients), with no more delineation between dark and light zones, as shown by Ki67, CD10, CD77, CD95 and CD86 staining. In contrast to CD86 expression which remained as intensive in HIV+ as in HIV- lymph nodes, CD80 staining was strongly decreased in GC of HIV+ lymph nodes but not in their extrafollicular zones. The loss of CD80 expression from CD19+ B cells was also observed by cytometric analysis of cell suspensions of three HIV+ patients. Although we found no evidence of an increase in a particular GC B cell subset in HIV-1-induced hyperplasia, the strong GC disorganization observed may induce impaired cell-cell interactions and thus participate in the loss of CD80 antigen. In contrast to HIV- situations where CD80 and CD86 was similarly expressed on B cells, the lower level of CD80 expression in HIV+ GC may favor Th2 T cell responses through CD86-CD28 interactions.

Detection rate and intratumoral virus load of human herpesvirus-8 in immunodeficiency-related B-cell lymphoid malignancies.

Human herpesvirus-8 (HHV-8), associated with Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, has been found in circulating B-cells and might have a causative role in B-cell malignancies associated with immunodeficiency syndromes. We determined the rate of detection and intratumoral virus load of HHV-8 by means of a semiquantitative approach in post-transplant lymphoproliferative diseases (PTLDs), AIDS-related non-Hodgkin's lymphomas (NHLs), including both Burkitt's lymphomas (BLs) and large cell lymphomas (LCLs), as well as in control groups consisting of follicular hyperplasias (FHs) and HIV-negative LCLs. HHV-8 sequences were detected at a similar rate in HIV-negative PTLDs (24%), HIV-negative LCLs (22%) and HIV-negative FHs (17%). The detection rate was significantly higher in HIV-positive BLs (73%), HIV-positive LCLs (67%), and HIV-positive FHs (65%) supporting the view of an epidemiological link between HHV-8 and HIV infections. The viral load was 10(2) genome copies per cell in the single case of primary effusion lymphoma included in the LCL group while it was 10(-3) copy per cell (median value; range: 10(-4)-10(-1)) in all the other HHV-8-positive samples. No significant difference of viral load was found according to HIV status. The virus loads of PTLDs and HIV-positive LCLs were significantly higher than those observed in HIV-positive BLs and FHs, suggesting, to some extent, that the degree of immunodeficiency may influence HHV-8 replication. However, with the exception of the single case of primary effusion lymphoma studied, the low intratumoral load of HHV-8 strongly argues against a direct causative agent of the virus in the occurrence of PTLDs and AIDS-related NHLs.