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Flow cytometric methodology for the detection of de novo human T-cell leukemia virus -1 infection in vitro: A tool to study novel infection inhibitors.

ABSTRACT: Methodology to detect and study de novo human T-cell leukemia virus (HTLV)-1 infection is required to further our knowledge of the viruses' mechanisms of infection and to study potential therapeutic interventions. Whilst methodology currently exists, utilisation of an anti-Tax antibody to detect de novo Tax expression in permissive cells labelled with cell tracker allowing for the detection by flow cytometry of new infection after co-culture with donor cell lines productively infected with HTLV-1 is an alternative strategy. Using this methodology, we have been able to detect de novo infection of the T cell line HUT78 following co-culture with the productively infected HTLV-1 donor cell line MT-2 and to confirm that infection can be effectively blocked with well characterised infection inhibitors. This methodology will benefit experimental studies examining HTLV infection in vitro and may aid identification of therapeutic agents that block this process.

SUBMITTER: Peres C

PROVIDER: S-EPMC6853161 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Flow cytometric methodology for the detection of de novo human T-cell leukemia virus -1 infection in vitro: A tool to study novel infection inhibitors.

Peres Carina C Tanaka Yuetsu Y Martin Fabiola F Fox James J

Journal of virological methods 20190908

Methodology to detect and study de novo human T-cell leukemia virus (HTLV)-1 infection is required to further our knowledge of the viruses' mechanisms of infection and to study potential therapeutic interventions. Whilst methodology currently exists, utilisation of an anti-Tax antibody to detect de novo Tax expression in permissive cells labelled with cell tracker allowing for the detection by flow cytometry of new infection after co-culture with donor cell lines productively infected with HTLV- ...[more]

PMID: 31509775

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Project description:The major advances in molecular and structural biology and automated peptide and DNA synthesis of the 1970s and 1980s generated fertile conditions in the 1990s for the exploration of designed proteins as a new approach for inorganic chemists to generate biomolecular mimics of metalloproteins. This Account follows the development of the TRI peptide family of three-stranded coiled coils (3SCC) and α3D family of three-helix bundles (3HB) as scaffolds for the preparation of metal binding sites within de novo designed constructs. The 3SCC were developed using the concept of a heptad repeat (abcdefg) putting hydrophobes in the a and d positions. The TRI peptides contain four heptads with capping glycines. Via substitution of leucine hydrophobes, metal ligands can be introduced into the a or d sites in order to bind metals. First, the ability to use cysteine-substituted 3SCC aggregates to impose higher or lower coordination numbers on Hg(II) and Cd(II) or matching the coordination preferences of As(III) and Pb(II) is discussed. Then, methods to develop dual site peptides capable of discriminating metals based on their type (e.g., Cd(II) vs Pb(II)), their preference for a vs d sites, and then their coordination number is described. Once these principles of metal site differentiation are described, we shift to building dual site peptides using both cysteine and histidine metal binding sites. This approach provides a construct with both a Hg(II) structural and a Zn(II) hydrolytic center, the latter of which is capable of hydrating CO2. With these Zn(II) proteins, we consider the relative importance of the location of the catalytic center along the primary sequence of the peptide and show that only minor perturbations in catalytic efficiencies are observed based on metal location. We then assess the feasibility of preparing enzymes competent to reduce nitrite with copper centers in a histidine-rich environment. As part of this discussion, we examine the influence of surface residues on catalyst reduction potentials and catalytic efficiencies. We end describing approaches to prepare asymmetric proteins that can incorporate acid-base catalysts or water channels. In this respect, we highlight modifications of a helix-turn-helix-turn-helix motif called α3D and show how this 3HB can be modified to bind heavy metals or to make Zn(II) centers, which are active hydrolytic catalysts. A comparison is made to the comparable parallel 3SCC.

Dataset Information

Flow cytometric methodology for the detection of de novo human T-cell leukemia virus -1 infection in vitro: A tool to study novel infection inhibitors.

Publications

Flow cytometric methodology for the detection of de novo human T-cell leukemia virus -1 infection in vitro: A tool to study novel infection inhibitors.

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