Truncation and non-natural amino acid substitution studies on HTLV-I protease hexapeptidic inhibitors

The culprit behind adult T-cell leukemia, myelopathy/tropical paraparesis, and a plethora of inflammatory diseases is the human T-cell leukemia virus type 1 (HTLV-I). We recently unveiled a potent hexapeptidic HTLV-I protease inhibitor, KNI-10166, composed mostly of natural amino acid residues. Herein, we report the derivation of potent tetrapeptidic inhibitor KNI-10516, possessing only non-natural amino acid residues.     

Synthesis and activity of tetrapeptidic HTLV-I protease inhibitors possessing different P3-cap moieties

The causative agent behind adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy is the human T-cell leukemia virus type 1 (HTLV-I). Tetrapeptidic HTLV-I protease inhibitors were designed on a previously reported potent inhibitor KNI-10516, with modifications at the P(3)-cap moieties. All the inhibitors showed high HIV-1 protease inhibitory activity (over 98% inhibition at 50nM) and most exhibited highly potent inhibition against HTLV-I protease (IC(50) values were less than 100nM).     

Identification of peptidomimetic HTLV-I protease inhibitors containing hydroxymethylcarbonyl (HMC) isostere as the transition-state mimic

Towards the development of chemotherapy for the infection by human T-cell leukemia virus type I (HTLV-I), we have established evaluation systems for HTLV-I protease (PR) inhibitors using both recombinant and chemically synthesized HTLV-I PRs. Newly synthesized substrate-based inhibitors containing hydroxymethylcarbonyl (HMC) isostere showed potent anti-HTLV-I PR activity.     

Maintaining potent HTLV-I protease inhibition without the P3-cap moiety in small tetrapeptidic inhibitors

The human T cell lymphotropic/leukemia virus type 1 (HTLV-I) causes adult T cell lymphoma/leukemia. The virus is also responsible for chronic progressive myelopathy and several inflammatory diseases. To stop the manufacturing of new viral components, in our previous reports, we derived small tetrapeptidic HTLV-I protease inhibitors with an important amide-capping moiety at the P₃ residue. In the current study, we removed the P₃-cap moiety and, with great difficulty, optimized the P₃ residue for HTLV-I protease inhibition potency. We discovered a very potent and small tetrapeptidic HTLV-I protease inhibitor (KNI-10774a, IC₅₀ = 13 nM).     

Locking the two ends of tetrapeptidic HTLV-I protease inhibitors inside the enzyme

Adult T-cell leukemia and tropical spastic paraparesis/HTLV-I-associated myelopathy are only some of the more common end results of an infection with a human T-cell leukemia virus type 1 (HTLV-I). Expanding from our previous reports, we synthesized all different permutations of tetrapeptidic HTLV-I protease inhibitors using at least eight P(3)-cap and five P(1)(')-cap moieties. The inhibitors exhibited over 97% inhibition against HIV-1 protease and a wide range of inhibitory activity against HTLV-I protease.     

HTLV-I,infective dermatitis,and tropical spastic paraparesis

Since human T-cell lymphotropic virus (HTLV-I) was identified in 1980 as causing human disease, it has been etiologically associated with adult T-cell lymphoma/leukemia (ATL) and tropical spastic paraparesis (TSP). More recently, several new diseases have been reported in association with this virus, including infective dermatitis of Jamaican children, which we reported in 1990. Studies on infective dermatitis have shown that these children have abnormalities of immune function, and some develop other HTLV-I associated disorders, including TSP. This paper reviews the work done on infective dermatitis to date, and explores the association with TSP.     

The transactivator gene of human T-cell leukemia virus type I is more variable within and between healthy carriers than patients with tropical spastic paraparesis.

Human T-cell leukemia virus type I (HTLV-1) causes T-cell leukemia and tropical spastic paraparesis (TSP) in a minority of infected people, whereas the majority remain healthy. No association between a particular HTLV-I sequence and disease manifestation has been found in previous studies. We studied here the sequence variability of the gene for the HTLV-I Tax protein, which is the dominant target antigen of the very strong cytotoxic T-lymphocyte response to the virus. In HTLV-I infection, the intraisolate nucleotide variability is much greater than the variability between isolates. The predicted protein sequence of Tax was significantly more variable in the healthy seropositive individuals' provirus than in those of the patients with TSP. Thus, tax sequence heterogeneity, rather than the presence of particular sequences, distinguishes healthy HTLV-I-seropositive individuals from patients with TSP.     

Prevalent loss of mitotic spindle checkpoint in adult T-cell leukemia confers resistance to microtubule inhibitors.

Human T-cell leukemia virus type I (HTLV-I) is the causative agent for adult T-cell leukemia (ATL). Molecularly, ATL cells have extensive aneugenic abnormalities that occur, at least in part, from cell cycle dysregulation by the HTLV-I-encoded Tax oncoprotein. Here, we compared six HTLV-I-transformed cells to Jurkat and primary peripheral blood mononuclear cells (PBMC) in their responses to treatment with microtubule inhibitors. We found that both Jurkat and PBMCs arrested efficiently in mitosis when treated with nocodazole. By contrast, all six HTLV-I cells failed to arrest comparably in mitosis, suggesting that ATL cells have a defect in the mitotic spindle assembly checkpoint. Mechanistically, we observed that in HTLV-I Tax-expressing cells human spindle assembly checkpoint factors hsMAD1 and hsMAD2 were mislocated from the nucleus to the cytoplasm. This altered localization of hsMAD1 and hsMAD2 correlated with loss of mitotic checkpoint function and chemoresistance to microtubule inhibitors.     

Tightly bound zinc in human immunodeficiency virus type 1, human T-cell leukemia virus type I, and other retroviruses.

Human immunodeficiency virus type 1 (HIV-1) and human T-cell leukemia virus type I (HTLV-I) were purified by sucrose density gradient centrifugation in the presence of 1 mM EDTA. Pelleted gradient fractions were analyzed for total protein, total Gag capsid protein, and total zinc. Zinc was found to copurify and concentrate with the virus particles. Through successive cycles of resuspending in buffer containing EDTA and repelleting, the zinc content remained constant at about 1.7 mol of zinc per mol of Gag protein. Proteins from purified virus (HIV-1 and HTLV-I) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to polyvinylidene fluoride paper, and probed with 65ZnCl2. Viral nucleocapsid (NC) proteins (HIV-1 p7NC and HTLV-I p15NC) bound 65Zn2+. Other retroviruses, including simian immunodeficiency virus, equine infectious anemia virus, bovine leukemia virus, Moloney murine leukemia virus, mouse mammary tumor virus, and Mason-Pfizer monkey virus, were found to contain amounts of zinc per milligram of total protein similar to those found in HIV-1 and HTLV-I. Collectively, these data support the hypothesis that retroviral NC proteins function as zinc finger proteins in mature viruses.     

Purification and characterization of human T-cell leukemia virus type I protease produced in Escherichia coli

Human T-cell leukemia virus type I (HTLV-I) protease has been purified to homogeneity from a strain of recombinant Escherichia coli. The protease was expressed as a larger precursor, which was autoprocessed to form a mature protease. Protein chemical analyses revealed the coding sequence of mature protease, which agreed with the putative sequence predicted from the sequence of bovine leukemia virus protease. The purified protease processed the natural substrate gag precursor (p53) to form gag p19 and gag p24. The protease activity was inhibited by pepstatin A. These results provide direct evidence that this protease belongs to the aspartic protease family and has an activity consistent with the protease in HTLV-I virion.     

Immunological risks of adult T-cell leukemia at primary HTLV-I infection

A small percentage of human T-cell leukemia virus type-I (HTLV-I)-infected individuals develop adult T-cell leukemia (ATL). In animal experiments, inoculation of HTLV-I via the oral route, which is the main route of mother-to-child viral transmission in humans as a result of breastfeeding, induced host HTLV-I-specific T-cell unresponsiveness and resulted in increased viral load. This strongly suggested that the known epidemiological risk factors for ATL (i.e. vertical HTLV-I infection and elevated viral load) are linked by an insufficient HTLV-I-specific T-cell response. Recent findings on the anti-tumor effects of Tax-targeted vaccination in rats and the reactivation of Tax-specific T cells in ATL patients as a result of hematopoietic stem cell transplantation imply promising immunological approaches for the prophylaxis and therapy of ATL.     

Effect of the recombinant vaccinia viruses that express HTLV-I envelope gene on HTLV-I infection.

The human T-lymphotropic virus type I (HTLV-I) is etiologically linked to adult T-cell leukemia (ATL). To develop a vaccine against ATL, we constructed recombinant vaccinia viruses containing the envelope gene of HTLV-I in the vaccinia virus hemagglutinin (HA) gene, a new site where foreign genes can be inserted. A single inoculation of the recombinant virus induced antibodies to the env proteins of HTLV-I in rabbits and had a protective effect against HTLV-I infection.     

Polymerase chain reaction analysis of defective human T-cell leukemia virus type I proviral genomes in leukemic cells of patients with adult T-cell leukemia.

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia, and the clonally derived leukemic cells all contain proviral genomes. Polymerase chain reaction with a variety of primers which span the HTLV-I genome was used to determine that a significant fraction of patients (at least 32%) carry deleted viral genomes in their leukemic cells. The pX region of the HTLV-I genome encoding the regulatory genes tax and rex was preferentially retained. The fact that the tax coding region was retained provides supporting evidence that the tax protein contributes to leukemogenesis in vivo. The reasonably high fraction of patients with adult T-cell leukemia carrying deleted genomes in their tumor cells suggests that the deletions have a role in leukemogenesis.     

Low degree of human T-cell leukemia/lymphoma virus type I genetic drift in vivo as a means of monitoring viral transmission and movement of ancient human populations.

We have studied the genetic variation of human T-cell leukemia/lymphoma virus type I (HTLV-I) isolates in the same individuals over time, as well as of HTLV-I isolates from various parts of the world. The viral DNA fragment studied encodes the carboxy terminus of gp46 and almost all of gp21, both of which are envelope glycoproteins. Samples were obtained from native inhabitants of five African countries, two South American countries, China, the French West Indies, and Haiti and included 14 patients with tropical spastic paraparesis/HTLV-I-associated myelopathy, 10 patients with adult T-cell leukemia, 1 patient with T-cell non-Hodgkin's lymphoma, and 3 healthy HTLV-I-seropositive individuals. DNA analyses of HTLV-I sequences demonstrated that (i) little or no genetic variation occurred in vivo in the same individual or in different hosts from the same region carrying the same virus, regardless of their clinical statuses; (ii) changes in nucleotide sequences in some regions of the HTLV-I genome were diagnostic of the geographical origin of the viruses; (iii) HTLV-I sequences from West African countries (Mauritania and Guinea Bissau) and some from the Ivory Coast and Central African Republic were virtually identical to those from the French West Indies, Haiti, French Guyana, and Peru, strongly suggesting that at least some HTLV-I strains were introduced into the New World through infected individuals during the slave trade events; and (iv) the Zairian HTLV-I isolates represent a separate HTLV-I cluster, in which intrastrain variability was also observed, and are more divergent from the other HTLV-I isolates. Because of the low genetic variability of HTLV-I in vivo, the study of the proviral DNA sequence in selected populations of infected individuals will increase our knowledge of the origin and evolution of HTLV-I and might be useful in anthropological studies.