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Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

ABSTRACT: Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral load was approximately 100-fold lower than that of WB-positive samples (0.01 versus 0.71 copy/100 cells). Phylogenic analysis of the complete HTLV-1 genomes of WB-indeterminate samples did not identify any specific phylogenetic groups. When we analyzed the nucleotide changes in 19 HTLV-1 isolates from WB-indeterminate samples, we identified 135 single nucleotide substitutions, composed of four types, G to A (29%), C to T (19%), T to C (19%), and A to G (16%). In the most frequent G-to-A substitution, 64% occurred at GG dinucleotides, indicating that APOBEC3G is responsible for mutagenesis in WB-indeterminate samples. Moreover, interestingly, five WB-indeterminate isolates had nonsense mutations in Pol and/or Tax, Env, p12, and p30. These findings suggest that WB-indeterminate carriers have low production of viral antigens because of a combination of a low proviral load and mutations in the provirus, which may interfere with host recognition of HTLV-1 antigens.

SUBMITTER: Kuramitsu M

PROVIDER: S-EPMC5648719 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

Kuramitsu Madoka M Sekizuka Tsuyoshi T Yamochi Tadanori T Firouzi Sanaz S Sato Tomoo T Umeki Kazumi K Sasaki Daisuke D Hasegawa Hiroo H Kubota Ryuji R Sobata Rieko R Matsumoto Chieko C Kaneko Noriaki N Momose Haruka H Araki Kumiko K Saito Masumichi M Nosaka Kisato K Utsunomiya Atae A Koh Ki-Ryang KR Ogata Masao M Uchimaru Kaoru K Iwanaga Masako M Sagara Yasuko Y Yamano Yoshihisa Y Okayama Akihiko A Miura Kiyonori K Satake Masahiro M Saito Shigeru S Itabashi Kazuo K Yamaguchi Kazunari K Kuroda Makoto M Watanabe Toshiki T Okuma Kazu K Hamaguchi Isao I

Journal of clinical microbiology 20170712 9

Western blotting (WB) for human T cell leukemia virus type 1 (HTLV-1) is performed to confirm anti-HTLV-1 antibodies detected at the initial screening of blood donors and in pregnant women. However, the frequent occurrence of indeterminate results is a problem with this test. We therefore assessed the cause of indeterminate WB results by analyzing HTLV-1 provirus genomic sequences. A quantitative PCR assay measuring HTLV-1 provirus in WB-indeterminate samples revealed that the median proviral lo ...[more]

PMID: 28701419

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Project description:BACKGROUND: In vitro studies have demonstrated that deletions and point mutations introduced into each 21 bp imperfect repeat of Tax-responsive element (TRE) of the genuine human T-cell leukemia virus type I (HTLV-1) viral promoter abolishes Tax induction. Given these data, we hypothesized that similar mutations may affect the proliferation of HTLV-1-infected cells and alter the proviral load (PvL). To test this hypothesis, we conducted a cross-sectional genetic analysis to compare the near-complete LTR nucleotide sequences that cover the TRE1 region in a sample of HTLV-1 asymptomatic carriers with different PvL burden. METHODS: A total of 94 asymptomatic HTLV-1 carriers with both sequence from the 5' long terminal repeat (LTR) and a PvL for Tax DNA measured using a sensitive SYBR Green real-time PCR were studied. The 94 subjects were divided into three groups based on PvL measurement: 31 low, 29 intermediate, and 34 high. In addition, each group was compared based on sex, age, and viral genotypes. In another analysis, the median PvLs between individuals infected with mutant and wild-type viruses were compared. RESULTS: Using a categorical analysis, a G232A substitution, located in domain A of the TRE-1 motif, was detected in 38.7% (12/31), 27.5% (8/29), and 61.8% (21/34) of subjects with low, intermediate, or high PvLs, respectively. A significant difference in the detection of this mutation was found between subjects with a high or low PvL and between those with a high or intermediate PvL (both p < 0.05), but not between subjects with a low or intermediate PvL (p > 0.05). This result was confirmed by a non-parametric analysis that showed strong evidence for higher PvLs among HTLV-1 positive individuals with the G232A mutation than those without this mutation (p < 0.03). No significant difference was found between the groups in relation to age, sex or viral subtypes (p > 0. 05). CONCLUSIONS: The data described here show that changes in domain A of the HTLV-1 TRE-1 motif resulting in the G232A mutation may increase HTLV-1 replication in a majority of infected subjects.

Project description:Western blotting has proven to be an important technique in analysis of receptor-ligand interactions (i.e., by ligand blotting) and for identifying molecules mediating cell attachment (i.e., by cell blotting). Conventional ligand blotting and cell blotting methods employ nondynamic (static) incubation conditions, whereby molecules or cells of interest are placed in suspension and overlaid on membranes. However, many cell-cell and cell-matrix adhesive interactions occur under fluid shear conditions, and shear stress itself mediates and/or facilitates the engagement of these physiologically appropriate receptors and ligands. Notably, shear forces critically influence the adhesion of circulating cells and platelets to vessel walls in physiologic cell migration and hemostasis, as well as in inflammatory and thrombotic disorders, cancer metastasis, and atherosclerosis. Use of nondynamic blotting conditions to analyze such interactions can introduce bias, overtly missing relevant effectors and/or exaggerating the relative role(s) of nonphysiologic adhesion molecules. To address this shortfall, we have developed a new technique for identifying binding interactions under fluid shear conditions, the "blot rolling assay." Using this method, molecules in a complex mixture are resolved by gel electrophoresis, transferred to a membrane that is rendered semi-transparent, and the membrane is then incorporated into a parallel-plate flow chamber apparatus. Under controlled flow conditions, cells or particles bearing adhesion proteins of interest are then introduced into the chamber and interactions with individual immobilized molecules (bands) can be visualized in real-time. The substrate molecule(s) supporting adhesion under fluid shear can then be identified by staining with specific antibodies or by excising the relevant band(s) and performing mass spectrometry or microsequencing of the isolated material. This method thus allows for the identification, within a complex mixture and without prior isolation or purification, of both known and previously uncharacterized adhesion molecules operational under dynamic conditions.

Dataset Information

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

Publications

Proviral Features of Human T Cell Leukemia Virus Type 1 in Carriers with Indeterminate Western Blot Analysis Results.

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