Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

Activation of NF-kappaB by the human herpesvirus 8 chemokine receptor ORF74: evidence for a paracrine model of Kaposi's sarcoma pathogenesis.

Infection with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma (KS)-associated herpesvirus, is necessary for the development of KS. The HHV-8 lytic-phase gene ORF74 is related to G protein-coupled receptors, particularly interleukin-8 (IL-8) receptors. ORF74 activates the inositol phosphate/phospholipase C pathway and the downstream mitogen-activated protein kinases, JNK/SAPK and p38. We show here that ORF74 also activates NF-kappaB independent of ligand when expressed in KS-derived HHV-8-negative endothelial cells or primary vascular endothelial cells. NF-kappaB activation was enhanced by the chemokine GROalpha, but not by IL-8. Mutation of Val to Asp in the ORF74 second cytoplasmic loop did not affect ligand-independent signaling activity, but it greatly increased the response to GROalpha. ORF74 upregulated the expression of NF-kappaB-dependent inflammatory cytokines (RANTES, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor) and adhesion molecules (VCAM-1, ICAM-1, and E-selectin). Supernatants from transfected KS cells activated NF-kappaB signaling in untransfected cells and elicited the chemotaxis of monocytoid and T-lymphoid cells. Expression of ORF74 conferred on primary endothelial cells a morphology that was strikingly similar to that of spindle cells present in KS lesions. Taken together, these data, demonstrating that ORF74 activates NF-kappaB and induces the expression of proangiogenic and proinflammatory factors, suggest that expression of ORF74 in a minority of cells in KS lesions could influence uninfected cells or latently infected cells via autocrine and paracrine mechanisms, thereby contributing to KS pathogenesis.

Common human T cell leukemia virus type 1 (HTLV-1) integration sites in cerebrospinal fluid and blood lymphocytes of patients with HTLV-1-associated myelopathy/tropical spastic paraparesis indicate that HTLV-1 crosses the blood-brain barrier via clonal HTLV-1-infected cells.

In the spinal cord of patients with human T cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP), infiltrating CD4(+) lymphocytes seem to be the major reservoir for the virus. Little, however, is known about the mechanisms by which HTLV-1 crosses the blood-brain barrier. An oligoclonal proliferation of HTLV-1-infected CD4 lymphoid T cells is present in the peripheral blood of all HTLV-1-infected individuals. Here, such oligoclonal distribution of HTLV-1-infected cells is evidenced in the cerebrospinal fluid (CSF) derived from 5 patients with HAM/TSP. Furthermore, clonal populations of HTLV-1-infected lymphocytes sharing the same HTLV-1 proviral flanking sequences (i.e. , integration sites in the cellular DNA), and thus derived from a single HTLV-1-infected progenitor, were found, for a given patient, in both the CSF and the peripheral blood. These data demonstrate that HTLV-1 crosses the blood-brain barrier by migration of HTLV-1-infected lymphocytes in vivo.

Host sequences flanking the human T-cell leukemia virus type 1 provirus in vivo.

Human pathogenic retroviruses do not have common loci of integration. However, many factors, such as chromatin structure, transcriptional activity, DNA-protein interaction, CpG methylation, and nucleotide composition of the target sequence, may influence integration site selection. These features have been investigated by in vitro integration reactions or by infection of cell lines with recombinant retroviruses. Less is known about target choice for integration in vivo. The present study was conducted in order to assess the characteristics of cellular sequences targeted for human T-cell leukemia virus type 1 (HTLV-1) integration in vivo. Sequencing integration sites from >/=200 proviruses (19 kb of sequence) isolated from 29 infected individuals revealed that HTLV-1 integration is not random at the level of the nucleotide sequence. The virus was found to integrate in A/T-rich regions with a weak consensus sequence at positions within and without of the hexameric repeat generated during integration. These features were not associated with a preference for integration near active regions or repeat elements of the host chromosomes. Most or all of the regions of the genome appear to be accessible to HTLV-1 integration. As with integration in vitro, integration specificity in vivo seems to be determined by local features rather than by the accessibility of specific regions.

Semiquantitative analysis of residual disease in patients treated for adult T-cell leukaemia/lymphoma (ATLL).

Many adult T-cell leukaemia/lymphoma (ATLL) patients who respond to induction treatment, then relapse. Knowing the clonality pattern of residual tumourous clones during treatment could help understand disease evolution and aid therapeutic decisions. We developed a sensitive and semi-quantitative molecular analysis of these clones in ATLL patients. DNA samples from PBMCs derived from eight ATLL patients were studied over time by quadruplicate linker mediated PCR (LMPCR) amplification of HTLV-1 integration sites. Patients were treated with combination chemotherapy, zidovudine-interferon-alpha and/or by peripheral stem cell transplantation or allogeneic bone marrow transplantation. Persistence of tumourous clones at a high frequency (>1/300 PBMCs) was frequently observed, even in complete responders, and was invariably correlated with relapse and/or poor outcome. Fluctuation in the frequency of some tumourous clones was observed with evidence for clonal change under treatment in one patient, indicating that treatment of ATLL can result in the selection of resistant clones. Finally, allogeneic bone marrow transplantation (BMT) using an HTLV-1 infected sibling as donor was found to be associated with long-lasting disappearance of tumourous clones and a possible cure of the disease. Long-term persistent clonal expansion of circulating HTLV-1 bearing T cells which derived from the donor bone marrow was evidenced in this patient. In conclusion, variable success in treatment of ATLL is probably due to the clonal heterogeneity which results in the selection of resistant clones. Semi-quantitative assessment of residual disease (RD) through LMPCR may predict treatment failure. Accordingly, additional therapy may be tailored to the clonality pattern observed after first-line therapy.

Implication of HTLV-I infection, strongyloidiasis, and P53 overexpression in the development, response to treatment, and evolution of non-Hodgkin's lymphomas in an endemic area (Martinique, French West Indies).

A clinicopathologic study was conducted to assess the implication of HTLV-I infection, Strongyloides stercoralis (Ss) infection, and P53 overexpression in the development, response to treatment, and evolution of non-Hodgkin's lymphoma (NHL) in Martinique, French West Indies. Two groups of patients, with 22 and 41 participants with B-cell and T-cell lymphoma, respectively, were analyzed. HTLV-I antibodies were detected in 24 (59%) patients with T-cell lymphoma of whom 19 (46%) fulfilled diagnostic criteria of adult T-cell leukemia/lymphoma (ATLL). By comparison with other T-cell lymphomas, patients with ATLL were significantly younger (52 versus 63 years; p = .03), had a significantly higher incidence of hypercalcemia (60% versus 0%; p = .0001), a trend for higher incidence of digestive tract localization (21% versus 4%; p = .1) and significantly shorter median survival (6 versus 17 months; p = .03). Similar results were observed when all 24 HTLV-I-infected patients with T-cell lymphoma were compared with the 17 seronegative patients. Strongyloidiasis was diagnosed in 11 of 34 patients tested for Ss infection. All 4 Ss-infected (Ss-positive) ATLL patients treated with combination chemotherapy achieved complete remission (CR) versus only 2 of 7 Ss-negative ATLL patients (p = .04). In addition, survival of Ss-positive patients with ATLL was better than that of the uninfected patients: 27 versus 5 months, p = .04, respectively). P53 expression was assessed by immunohistochemistry on lymph node biopsies from 37 patients including 18 B-cell lymphomas, 14 ATLL, and 5 other T-cell lymphomas. P53 overexpression (P53-positive) was observed in 6 samples that corresponded in all 6 patients with ATLL. All P53-positive ATLL patients had stage IV disease with elevated lactate dehydrogenase (LDH) levels. By comparison with other ATLL patients studied for p53 expression, P53-positive ATLL were characterized by a lower response rate to combination chemotherapy (CR: 0 of 6 versus 4 of 6; p = .04) and a shorter survival (2 versus 9 months, p = .04). Our results suggest that ATLL represents almost 50% of T-cell lymphomas in Martinique; Ss infection during ATLL seems to be linked with a high response rate to chemotherapy and prolonged survival; and P53 overexpression is observed in almost 50% of aggressive ATLL from Martinique and, even in advanced clinical subtypes, is associated with resistance to chemotherapy and short-term survival.

Persistent oligoclonal expansion of human T-cell leukemia virus type 1-infected circulating cells in patients with Tropical spastic paraparesis/HTLV-1 associated myelopathy.

The pattern of HTLV-1 replication was assessed through PCR amplification of the 3' proviral integration sites in patients with TSP/HAM at different times. Integration sites were sequenced and oligonucleotides specific for the flanking sequences were synthesized. Together with HTLV-1 LTR specific primers, clonotypic nested PCR was performed on peripheral blood from two patients. The frequencies of five clones studied ranged from 1/300 to 1/1500 PBMCs while four clones persisted for more than 1-5 years. It would seem that Tax driven expansion of T cells may persist for considerable periods of time in TSP/HAM despite strong cellular immunity. This may provide a background for the accumulation of subsequent mutations leading to malignancy.

Oligoclonal proliferation of human T-cell leukaemia virus type 1 bearing T cells in adult T-cell leukaemia/lymphoma without deletion of the 3' provirus integration sites.

We report a new case of an asymptomatic carrier with a deletion of a 3' HTLV-1 integration site. We further investigated whether these 3' deletions of flanking sequences may explain the oligoclonal pattern of HTLV-1 replication, evidenced by inverse PCR (IPCR) analysis of tumourous samples from patients with adult T-cell leukaemia (ATLL). 48 HTLV-1 3' integration sites, derived from tumourous DNA of five ATLL patients were sequenced. One dominant flanking sequence was obtained in the four samples harbouring a unique band after Southern-blotting. In one sample, which harboured two signals after Southern-blotting, IPCR amplification of diluted tumourous DNA revealed that these two sequences corresponded to one clone harbouring two integrated proviruses rather than to two distinct cellular clones, a result consistent with superinfection of the tumourous sample. In addition to integration sites corresponding to malignant clones, two to six oligoclonal forms were sequenced in four samples. No flanking sequence homology was found between clones derived from each patient, indicating that integration sites deletion in the vicinity of the provirus is a rare event in ATLL. The oligoclonal pattern of HTLV-1 replication in ATLL may result from clonal expansion of non-malignant HTLV-1-bearing clones within the sample and partly from HTLV-1 superinfection of monoclonal tumour cells.

Adult T-cell leukemia/lymphoma on a background of clonally expanding human T-cell leukemia virus type-1-positive cells.

Tumorous and nontumorous samples from patients with various forms of adult T-cell leukemia/lymphoma (ATLL) were analyzed using the sensitive inverse polymerase chain reaction (PCR) technique. In all samples, oligoclonal expansion of human T-cell leukemia virus (HTLV)-1 bearing T cells were detected, even for the tumorous samples that were mainly monoclonal by Southern blotting. For one case of smouldering ATLL, chemotherapy apparently reduced the number of detectable clones. Taken together with similar data on asymptomatic and symptomatic HTLV-1 carriers without malignancy, it would appear that ATLL appears on a prior background of HTLV-1-initiated oligoclonal expansion.

Proliferation of HTLV-1 infected circulating cells in vivo in all asymptomatic carriers and patients with TSP/HAM.

Assuming that the clonal expansion of T cells harbouring the human T-cell leukemia virus type 1 (HTLV-1) provirus is a central feature of HTLV-1 infection, the identification of such cells was sought among a series of 19 asymptomatic carriers and 19 cases of tropical spastic paraparesis/HTLV-1 associated myelopathy (TSP/HAM) devoid of malignancy. Two PCR based protocols designed to amplify the host cell-HTLV-1 proviral integration sites were used. In all cases large numbers of proliferating clones could be identified. The proportion of some clones was > 1/1500 peripheral blood mononuclear cells (PBMCs) with the suggestion that their number increased as a function of age among asymptomatic carriers.

Clonal expansion of infected cells: a way of life for HTLV-I.

Human T-cell lymphotropic virus type I (HTLV-I) is characterized by a remarkable genetic stability and high proviral loads in the absence of malignant disease. This results from the effect of tax on cell cycling. The virus replicates essentially in concert with the cell that is, via mitosis, which can be shown by polymerase chain reaction amplification of the HTLV-I integration sites. This is true of all stages of HTLV-I infection and accompanies adult T-cell leukemia/lymphoma. The very low viremia results from its genetic organization.

Stochastic events in the amplification of HTLV-I integration sites by linker-mediated PCR.

Human T-cell leukaemia virus type I (HTLV-I) proviral integration sites from an asymptomatic carrier and from the MT4 cell line were analysed by linker-mediated PCR (LMPCR) and inverse PCR (IPCR). LMPCR was more sensitive, allowing detection of a greater number of integrated proviruses. Reconstruction experiments using a cloned integrated HTLV-1 provirus indicated that > 100 copies were necessary to be detected frequently by LMPCR. To circumvent this problem, the LMPCR analysis was performed approximately 20 times per sample. Thus, for the MT4 cell line, the seven major integration sites were accompanied by approximately 20 clones of lesser frequency. For an asymptomatic HTLV-I carrier, nine integration sites were identified in a single amplfication, while a further 9 followed from 14 additional reactions. These findings show that there is a stochastic element to sampling HTLV-I integration sites by LMPCR, which tends to underestimate the actual number of HTLV-I bearing clones. Accordingly, those detected in at least two reactions represent the most abundant clones.