Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Kádas, János; Weber, Irene T.; Bagossi, Péter; Miklóssy, Gabriella; Boross, Péter; Oroszlan, Stephen; Tőzsér, József

doi:10.1074/jbc.m401868200

Cited by 45 publications

(91 citation statements)

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“…However, distinctly unique features are identified in the areas of the flaps and the putative substrate-binding sites that can be correlated with the enzymatic properties of this molecule, such as substrate specificity and the resistance to anti-HIV drugs (8,32). It will be necessary for rapid progress in future studies to overcome the propensity of the enzyme to aggregate, and the present structure will serve as a guide to surface mutations to alleviate that problem.…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Laco

Jaskólski

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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The successful development of a number of HIV-1 protease (PR) inhibitors for the treatment of AIDS has validated the utilization of retroviral PRs as drug targets and necessitated their detailed structural study. Here we report the structure of a complex of human T cell leukemia virus type 1 (HTLV-1) PR with a substratebased inhibitor bound in subsites P5 through P5 . Although HTLV-1 PR exhibits an overall fold similar to other retroviral PRs, significant structural differences are present in several loop areas, which include the functionally important flaps, previously considered to be structurally highly conserved. Potential key residues responsible for the resistance of HTLV-1 PR to anti-HIV drugs are identified. We expect that the knowledge accumulated during the development of anti-HIV drugs, particularly in overcoming drug resistance, will help in designing a novel class of antileukemia drugs targeting HTLV-1 PR and in predicting their drug-resistance profile. The structure presented here can be used as a starting point for the development of such anticancer therapies.inhibitor ͉ leukemia ͉ retroviral protease H uman T cell leukemia virus type 1 (HTLV-1) is a retrovirus that is epidemiologically associated with mature CD3 ϩ CD4 ϩ T cell-type leukemia͞lymphoma (ATL), as well as with tropical spastic paraparesis͞myelopathy (1, 2). It is estimated that up to 30 million people worldwide are infected with HTLV, with ATL being particularly prevalent in Japan (3). Only an estimated 3-5% of people infected with the virus develop ATL in their lifetime, but for those that do, the prognosis is poor (4). Although a number of treatments for ATL, such as combination chemotherapy, monoclonal antibodies directed against the ␣ chain of the interleukin 2 receptor, and antiviral therapy involving IFN-␣ and zidovudine, are used clinically, they show only very limited efficacy (3). Novel approaches under investigation use proteasome inhibitors (5) and Tax-targeted immunotherapy (4), but they have not yet been tested in practice. It is clear that new anti-ATL targets need to be found.In common with other retroviruses, HTLV-1 encodes a protease (PR) necessary for its maturation. Because inhibition of the enzyme has been shown to prevent viral proliferation, development of inhibitors targeting HTLV-1 PR is an attractive new path for chemotherapy (6). HTLV-1 PR is a homodimer, with each chain containing 125 residues. The enzymatic properties of HTLV-1 PR, including its substrate specificity, have already been studied in considerable detail (7,8). Although the design and synthesis of inhibitors specific for HTLV-1 PR have been carried out, most of the compounds are active only in micromolar concentration (9, 10). The best statine-containing inhibitor has a K i of 50 nM under high-salt conditions (7) but of only 2.3 M in a low-salt buffer (11). In comparison, a number of subpicomolar inhibitors of HIV-1 PR have been developed by using the principles of rational drug design (12).Structural investigations of HTLV-1 PR have not been su...

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Section: Resultsmentioning

confidence: 99%

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Laco

Jaskólski

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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“…Previous specificity studies of retroviral proteases established that the most important determinant of specificity is the P2-P2Ј region (nomenclature is according to Schechter and Berger (44)) of a substrate (45). Detailed specificity studies of HTLV-1 PR suggested that efficient cleavage sites require Leu, Ile, or Val at P2; Leu, Met or Phe at P1; Pro, Phe, Val, or Ala at P1Ј; and Leu, Ile, or Val at P2Ј (37,38). The LM2PV cleavage site conforms very well with these specificity determinants, whereas the inefficient LS2PV site does not contain an optimal P1 residue.…”

Section: Discussionmentioning

confidence: 99%

“…Stabilized, purified HTLV-1 PR was prepared as described previously (36). Active site titration of the PR was performed using peptide KTKVL-r-VVQPK (IB268), where r represents a reduced peptide bond, as described previously (37). The PR assays were initiated by the mixing of 5 l (34 -1700 nM) of purified HTLV-1 PR with 10 l of 2ϫ incubation buffer (0.5 M potassium phosphate buffer, pH 5.6, containing 10% glycerol, 2 mM EDTA, 10 mM dithiothreitol, 4 M NaCl) and 5 l of 0.04 -1.2 mM substrate.…”

Section: Human T-cell Lymphotropic Virus Type-1 (Htlv-1)mentioning

confidence: 99%

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

Mitchell

Tőzsér

Princler

et al. 2006

Journal of Biological Chemistry

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It is not known whether the low infectivity and low virion-associated polymerase activity of human T-cell lymphotropic virus type-1 (HTLV-1) are due to the quantity or quality of the reverse transcriptase (RT), because the protein has not yet been fully characterized. We have developed anti-RT antibodies and constructed HTLV-1 expression plasmids that express truncated or hemagglutinin-tagged Pol polyproteins to examine the maturation and composition of HTLV-1 RT. We detected virion-associated proteins corresponding to RT-integrase (IN) (pr98) and RT (p62) as well as smaller proteins containing the polymerase (p49) or RNase H domains. We have identified the amino acid sequences in the Pol polyprotein that are cleaved by HTLV-1 protease to yield RT and IN. We have also identified the cleavage sites within RT that give rise to the p49 polymerase fragment. Immunoprecipitation of an epitope-tagged p62 subunit coprecipitated p49, indicating that the HTLV-1 RT complex can exist as a p62/p49 heterodimer analogous to the RT of HIV-1 (p66/p51).Human T-cell lymphotropic virus type-1 (HTLV-1) 2 is an oncogenic retrovirus belonging to the deltaretrovirus genus, which includes HTLV-2, -3, and -4, simian T-cell lymphotropic viruses, and bovine leukemia virus. As with many other retroviruses, HTLV-1 uses ribosomal frameshifting to regulate the relative expression of Gag proteins and the viral replication enzymes. In many retroviruses, including human immunodeficiency virus (HIV) and Rous sarcoma virus (RSV), protein synthesis that initiates from the gag start codon gives rise to both Gag and Gag-Pol polyprotein precursors. A ribosomal frameshift site encoded either at the 5Ј-end of pol (HIV) or at the 3Ј-end of gag (RSV) allows the synthesis of the Gag-Pol polyprotein (1, 2). In contrast, the pro gene of some retroviruses, such as HTLV, mouse mammary tumor virus, and Mason-Pfizer monkey virus, is in a separate reading frame from both gag and pol. Thus, the expression of Gag-Pol polyprotein in these retroviruses involves two ribosomal frameshifts: one where gag and pro overlap and the second within the pro/pol overlap (3-11). In these viruses, three polyprotein precursors are synthesized. In HTLV-1, they are Gag (Pr53), Gag-Pro (Pr76), and Gag-Pro-Pol (Pr180). Based on in vitro translation of viral RNA, the molar ratio of the Gag, Gag-Pro, and Gag-Pro-Pol polyproteins was estimated to be about 100:10:1 (12). This means that the relative amounts of Pol to Gag produced by HTLV-1 would be 5-10-fold less than the amount present in singleframeshift retroviruses, such as HIV and RSV (1, 2) and 3-4-fold less than the amount found in the other two-frameshift retroviruses: mouse mammary tumor virus and Mason-Pfizer monkey virus (13-15).Retroviral pol genes encode RT and integrase (IN), which are required for the replication and integration of the viral genome. The Pol precursor is enzymatically cleaved by the viral protease to yield the subunits of the RT complex. The reverse transcriptases of HIV-1, RSV, and murine leukemia virus are t...

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“…The protease is an important target of pharmacological inhibition in the treatment of both HTLVand HIV-associated diseases. While the substrate binding region of HTLV-1 protease shares 45% sequence homology with that of HIV-1, their substrate specificity and inhibition profiles differ substantially (85,114). One currently-approved therapeutic agent, Indinavir, is an effective inhibitor of both proteases.…”

Section: Proteasementioning

confidence: 99%

“…Maturation of this aspartyl protease into a functional homodimer requires autoproteolysis (85). HTLV protease is responsible for Gag polyprotein processing into its functional products and is, therefore, crucial for virion formation and, hence, viral replication (140).…”

Section: Proteasementioning

confidence: 99%

HTLV-1 Tax Effects on Cellular Mitotic Regulation

Merling¹

2007

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The human T-cell leukemia virus 1 (HTLV-1) encodes the tax gene (transactivator encoded by the pX region) which is a potent activator of viral transcription and Nuclear , this cyclin-dependent kinase inhibitor may also play a role in tax-IRS.As part of the research presented in this dissertation, a collection of HTLV-1 tax point mutants were isolated that permit normal growth of S. cerevisiae. Similar to wild-type tax, many mutants (C23W, A108T, L159F, and L235F) transactivated both the HTLV long terminal repeat (LTR) and the NF-!B reporters. The V19M mutant preferentially activated NF-!B, but was attenuated in LTR activation. None of the mutants significantly elevated the levels of p21 CIP1/WAF1 and p27 KIP1 , indicating that dramatic Tax-induced surges in p21and p27 KIP1 occurs by mechanisms distinct from NF-!B and LTR activation. Importantly, the ability of these mutants to activate APC was attenuated or abrogated. These data show that Tax-induced rapid senescence is causally associated with APC activation and suggest iv that the mitotic abnormalities that are associated with premature APC activation might play an important role in cell transformation by Tax.In a parallel study, we demonstrate that Tax interacts with both centrosomal Nek2-associated protein 1 (C-Nap1), a large coiled-coil protein that maintains centrosome cohesion, and protein phosphatase 1 (PP1) and that these interactions localize to the centrosome. Furthermore, in cells expressing tax, C-Nap1 phosphorylation is greatly diminished. C-Nap1 phosphorylation status plays an important role in centrosome cycle progression; PP1-mediated dephosphorylation of C-Nap1 maintains centriolar cohesion during interphase, and never in mitosis A (NIMA)-related kinase 2 (Nek2A)-mediated phosphorylation of C-Nap1 during G2/M triggers C-Nap1 dissociation from duplicated centrosomes permitting normal centrosome disjunction. Our data suggest that Tax, through recruitment of PP1 to C-Nap1, may permit prolonged C-Nap1 dephosphorylation thereby disrupting normal centrosome disjunction leading to observed Tax-induced centrosome abnormalities and distinct microtubule organizing center (MTOC) loss.Our studies have revealed two distinct pathways through which Tax could contribute to oncogenesis. First, our Tax mutants that retained LTR and NF-!B activation properties but were impaired in APC activation were also defective in the ability to transform Rat1 fibroblasts. Therefore, premature APC activation by Tax appears to correlate with the ability of Tax to induce cellular transformation. Second, our demonstration of Tax-mediated alterations in C-Nap1 phosphorylation cycle offers an explanation for the centrosome abnormalities and mitotic apparatus defects observed in response to Tax expression.Chromosomal abnormalities resulting from such mitotic apparatus defects may be expected to contribute to the oncogenic potential of Tax.v

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Narrow Substrate Specificity and Sensitivity toward Ligand-binding Site Mutations of Human T-cell Leukemia Virus Type 1 Protease

Cited by 45 publications

References 56 publications

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Crystal structure of human T cell leukemia virus protease, a novel target for anticancer drug design

Synthesis, Processing, and Composition of the Virion-associated HTLV-1 Reverse Transcriptase

HTLV-1 Tax Effects on Cellular Mitotic Regulation

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