Inhibitors of Strand Transfer That Prevent Integration and Inhibit Human T-Cell Leukemia Virus Type 1 Early Replication

Abstract

The replication of the retrovirus human T-cell leukemia virus type 1 (HTLV-1) is linked to the development of lymphoid malignancies and inflammatory diseases. Data from in vitro, ex vivo, and in vivo studies have revealed that no specific treatment can prevent or block HTLV-1 replication and therefore that there is no therapy for the prevention and/or treatment of HTLV-1-associated diseases in infected individuals. HTLV-1 and human immunodeficiency virus type 1 (HIV-1) integrases, the enzymes that specifically catalyze the integration of these retroviruses in host cell DNA, share important structural properties, suggesting that compounds that inhibit HIV-1 integration could also inhibit HTLV-1 integration. We developed quantitative assays to test, in vitro and ex vivo, the efficiencies of styrylquinolines and diketo acids, the two main classes of HIV-1 integrase inhibitors. The compounds were tested in vitro in an HTLV-1 strand-transfer reaction and ex vivo by infection of fresh peripheral blood lymphocytes with lethally irradiated HTLV-1-positive cells. In vitro, four styrylquinoline compounds and two diketo acid compounds significantly inhibited HTLV-1 integration in a dose-dependent manner. All compounds active in vitro decreased cell proliferation ex vivo, although at low concentrations; they also dramatically decreased both normalized proviral loads and the number of integration events during experimental ex vivo primary infection. Accordingly, diketo acids and styrylquinolines are the first drugs that produce a specific negative effect on HTLV-1 replication in vitro and ex vivo, suggesting their potential efficiency for the prevention and treatment of HTLV-1-associated diseases.Human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus type 1 (HIV-1) are exogenous retroviruses pathogenic for humans. Although both viruses are lymphotropic, their pathogenicities depend on strongly distinct mechanisms. Schematically, in vivo, HIV infection triggers the progressive elimination of CD4 ϩ lymphocytes, leading to immunosuppression, whereas HTLV-1 infection is associated with the clonal expansion of infected cells, possibly leading to malignant CD4 ϩ proliferation or to spinal cord infiltration, infection, and inflammation. Clinically, HIV-induced cellular defects are regularly linked to the development of AIDS, whereas in a minority of carriers, HTLV-1 infection causes adult T-cell leukemia/lymphoma (ATLL) and/or tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). The median length of survival for patients with AIDS receiving modern treatment, i.e., triple therapy, is currently over 8 years; in contrast, the prognosis for HTLV-1-associated diseases remains extremely poor. To date, there is no effective treatment for TSP/HAM (32), while the median overall length of survival for patients with ATLL does not exceed a few months (3).Integration of a DNA copy of the viral RNA genome into host cellular DNA is essential and unique to the retroviral life cycle. After completion of reverse...

HTLV-1-infected CD4+ T-cells display alternative exon usages that culminate in adult T-cell leukemia

“…This study was conducted according to the principles outlined in the Declaration of Helsinki, and approved by the Institutional Review Board of the Hospices Civils de Lyon (France). As previously described [ 29 , 30 ], CD4 + clones were submitted to ex vivo culture for one month prior to HTLV-1 screening and RNA extraction. At this time, infected and uninfected cells remained non-immortalized and required IL-2 for continued growth.…”

“…For T-cell limiting dilution cloning, PBMCs were seeded at 0.1 cell/well in Terasaki plates after removal of adherent cells. T-lymphocytes were cultured in RPMI 1640 containing penicillin and streptomycin, sodium pyruvate, non-essential amino acids, 2-mercaptoethanol, 10% filtered human AB serum, 100 U/mL recombinant IL-2 (Chiron Corporation), and 75 μM HTLV-1 integrase inhibitor L-731,988 [ 30 ]. T-lymphocytes were re-stimulated every 14 days with PHA (1 μg/mL) and fresh feeder cells (5.10 5 cells/mL) that were composed of lethally irradiated PBMCs from three distinct allogenic HTLV-1-negative donors.…”

“…Background correction and probe selection were performed as described previously [ 42 ]. Statistical analyses were performed using a Student’s t -test on the splicing index (SI) that corresponds to comparison of gene-normalized exon intensity values between two experimental conditions [ 30 , 42 ]. According to exon-specific RT-PCR (Figures 1 C and D), microarray data were considered statistically significant for p < 0.05 and SI ≥ 1.2.…”

Tax gene expression and cell cycling but not cell death are selected during HTLV-1 infection in vivo

“…HTLV-1 transmission was performed by co-culturing the PBMCs with lethally irradiated (60 Gy) HTLV-1-positive MT2 cells at a ratio of 5:1, as described elsewhere [24]. The MT2 cell line is known to be chronically infected with HTLV-1 [25].…”

Structural and Functional Insights into Foamy Viral Integrase

“…Infectivity of PFV in cell culture and in vitro enzymatic activities of PFV IN showed a significant sensitivity to raltgravir and elvitegravir [24]. The high degree of amino acid sequence similarity within the active sites of HIV-1 and PFV INs and superposition of active side residues in crystals [24] implies the possibility of HIV-1 IN strand transfer inhibitors to be equally active against PFV IN [24,105,106]. Soaking PFV intasome crystals in raltegravir or other INSTIs has revealed that these compounds bind the active site and are interacted by their oxygen atoms with bound metal ions at the IN active site [25].…”