Reporter Systems to Study HTLV-1 Transmission

Abstract

The retrovirus Human T-lymphotropic virus type 1 (HTLV-1) preferentially infects CD4 T-cells via cell-to-cell transmission, while cell-free infection of T-cells is inefficient. Substantial insights into the different routes of transmission have largely been obtained by imaging techniques or by flow cytometry. Recently, strategies to quantify infection events with HTLV-1 improved. In this chapter, we present two different methods to quantitate virus transmission. Both methods are based on measuring gene activity of luciferase with a cost-saving in-house luciferase assay. First, we established a reporter Jurkat T-cell line carrying a luciferase gene under the control of the HTLV-1 core promoter U3R. Upon co-culture with chronically HTLV-1-infected T-cell lines, reporter cells are infected, and upon expression of the viral transactivator Tax, the viral promoter is activated resulting in enhanced luciferase activity. However, this assay as presented here does not exclude cell fusion as the mechanism allowing intracellular Tax-dependent activation of luciferase gene expression. Therefore, we describe a second method, the single-cycle replication-dependent reporter system developed by Mazurov et al. (PLoS Pathog 6:e1000788, 2010) that allows quantitation of HTLV-1 infection in co-cultured cells. Taken together, both methods facilitate quantitation of HTLV-1 transmission and will help to unravel pathways required for cell-to-cell transmission on a quantitative basis.

Nucleic acid recognition and antiviral activity of 1,4-substituted terphenyl compounds mimicking all faces of the HIV-1 Rev protein positively-charged α-helix

“…Assuming similar structures for the RNA-terphenyl complexes, this suggested that 1a and 1d had greater affinity for loop IIB relative to 3c, in line with the results obtained with fluorescence experiments. We also detected intermolecular NOEs between H1' of the extrahelical A19 loop nucleotide and the aminoethyl protons of compound 1a, implying that the ligand associated to the loop from the major groove side, as observed for Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] (Fig. 1C) 11,12 .…”

“…The terminal synthons were aryl bromides bearing nitrile groups on the two side chains as masked amines, ready to be revealed in later steps, whereas the central synthon presented two alkyl side chains and two boronic esters. A simple double palladium-catalyzed Suzuki-Miyaura cross-coupling between two terminal synthons to every one central synthon constructed the desired terphenyl scaffold, and a subsequent borane-mediated reduction of the nitrile groups resulted in the amines required to mimic the arginine residues in Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] (Fig. S1).…”

“…The Rev protein adopts a helix-turn-helix conformation [8][9][10] , and the Nt-segment of the second helix contains a positively-charged arginine-rich motif, T 34 RQARRNRRRRWRERQR 50 (hereafter identified as Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] ) that drives RNA-binding and also functions as a nuclear localization signal (NLS). The Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] helix forms a high-affinity interaction with an internal loop located within subdomain IIB of the Rev Recognition Element (RRE) of the viral RNA (Fig. 1C) 11,12 .…”

“…1C) 11,12 . This interaction is essential for virus viability, as it triggers the cooperative incorporation of additional Rev molecules into the complex through interactions between Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] helices and further sites on the RRE as well as protein-protein contacts 13 , allowing nuclear export of unspliced or singly-spliced viral RNA molecules in the late phase of the virus cycle 14 . Nevertheless, despite its importance in the viral replication cycle, the RRE-Rev ribonucleoprotein complex remains an unexploited target for HIV-1 chemotherapy.…”

“…The image was generated with MOE 2019.0102 (www.chemcomp.com). (D) Secondary structure of RNA hairpins IIB h , containing the high-affinity Rev [34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] binding site, and TAR h , used as a specificity control. For fluorescence intensity assays a fluorescein probe was linked to the extra-helical loop residues U23 of IIB h and U8 of TAR h (indicated with asterisks).…”