Examination of a Reporter Vector for HTLV-1 Infectivity Using MT2, a HTLV-1 Producer Cell Line

Abstract

Background: HTLV-1 (Human T_cell lymphotropic virus type 1), with about 20 million individuals infected worldwide, is a global health problem that is endemic in certain areas such as Japan and Khorasan province of Iran.HTLV-1 is the causative agent of progressive diseases, Adult T cell Leukemia (ATL), and HTLV-I Associated Myelopathy/Tropical Spastic Paraparesis (HAM/TSP), which do not yet have approved effective treatment. Due to the non-cytolytic characteristic of this virus and cell associated properties, there is no routine Procedure for drug study in vitro and it's confined to some HTLV infected cell lines. Therefore construction of a reporter vector is necessary to evaluate HTLV-1 infectivity in cell culture for drug studies, since designed reporters for retroviruses are usually based on long terminal repeat (LTR) transactivation. LTR region of HTLV-1 contains a virus promoter that plays the most important role in replication and transcription by the Tax transactivation effect. Objectives: In this project we attempted to construct a reporter vector containing the luciferase gene coupled to the HTLV-1 promoter for evaluation of HTLV-1 infectivity. Material and Methods: LTR region was digested from pUCLTR-Lac by HindIII and sub-cloned into the Multiple cloning site (MCS) region of a promoter-less reporter vector, pGL4.17, upstream to the luciferase gene. Colonies were screened by Colony PCR, then selected colonies were confirmed by restriction enzyme digestion and sequencing. Human embryonic kidney (HEK) 293T cell line was transfected by a recombinant vector and inducible expression of luciferase was evaluated. Results: Recombinant vector, pGL4LTR-Luc, has expression levels of more than 50 folds compared to the control when co-transfected with the Tax expressing vector into HEK 293T cells. Moreover, cells display more than 30 folds of luciferase expression over 2 days when cocultured with MT2 cell, a HTLV-1 producer cell line. Conclusions: According to the high functionality of the produced recombinant vector, it seems a good applicable tool for making an indicator cell line in subsequent basic and drug studies.