Non-canonical translation initiation of the spliced mRNA encoding the human T-cell leukemia virus type 1 basic leucine zipper protein

Abstract

Human T-cell leukemia virus type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL). The HTLV-1 basic leucine zipper protein (HBZ) is expressed in all cases of ATL and is directly associated with virus pathogenicity. The two isoforms of the HBZ protein are synthesized from antisense messenger RNAs (mRNAs) that are either spliced (sHBZ) or unspliced (usHBZ) versions of the HBZ transcript. The sHBZ and usHBZ mRNAs have entirely different 5′untranslated regions (5′UTR) and are differentially expressed in cells, with the sHBZ protein being more abundant. Here, we show that differential expression of the HBZ isoforms is regulated at the translational level. Translation initiation of the usHBZ mRNA relies on a cap-dependent mechanism, while the sHBZ mRNA uses internal initiation. Based on the structural data for the sHBZ 5′UTR generated by SHAPE in combination with 5′ and 3′ deletion mutants, the minimal region harboring IRES activity was mapped to the 5′end of the sHBZ mRNA. In addition, the sHBZ IRES recruited the 40S ribosomal subunit upstream of the initiation codon, and IRES activity was found to be dependent on the ribosomal protein eS25 and eIF5A.

Post-translational modifications of hnRNP A1 differentially modulate retroviral IRES-mediated translation initiation

“…As reported ( 10 , 23 , 36 ), the overexpression of hnRNP A1 increased FLuc activity without having a significant ( P <0.05) impact over the expression RLuc (Figure 2C ). Analysis of the FLuc/RLuc ratio (relative translational activity, RTA), index of IRES activity ( 7 , 9 , 12 , 13 , 29 , 32 ), better illustrates the significantly higher activity of the HIV-1 IRES when the wt-hnRNP A1-HA protein is overexpressed (50–500 ng DNA; Figure 2D ). Also, a partial knockdown of the endogenous hnRNP A1 mRNA using the siA1-RNA, that reflected in a reduction ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\sim$\end{document} 30–40%) of protein levels (Figure 2E ), was sufficient to significantly ( P <0.05) reduce HIV-1 IRES activity (FLuc) without impacting on cap-dependent translation (RLuc) (Figure 2F ).…”

“…For hnRNP A1 silencing, a silencer RNA (siRNA) duplex (5′-AATGGGGAACGCTCACGGACT-3′; Integrated DNA Technologies, IDT, Coralville, IA, USA) previously described ( 23 ), targeting the open reading frame (ORF) of the hnRNP A1 mRNA was used at 150 nM for 48h, together with 200 ng of dl HIV-1 IRES plasmid, or at 100, 150 or 250 nM for 48 hr in combination with 200 ng of pNL4.3-RLuc plasmids. For RLuc silencing, 100 nM of a duplex siRNA targeting the RLuc ORF (Renilla siRNA 2, 5′-UAUAAGAACCAUUACCAGAUUUGCCUG-3′, Integrated DNA Technologies) ( 12 , 29 , 32 ), was co-transfected with 150 ng of dual-luc plasmids and pSP64 Poly(A) or hnRNP A1-HA plasmids for 48h. The Silencer Select Negative Control #1 siRNA (#4390844, Ambion, Thermo Fisher Scientific Inc.), was used as a non-related scrambled RNA (scRNA) negative control.…”

“…The activity of Firefly luciferase (FLuc) and Renilla luciferase (RLuc) were measured using the DLR ® Assay System (#E1960, Promega Corporation) according to the manufacturer's instructions, on 20 μl of cell lysates using a Sirius Single Tube Luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). Data are expressed as a percentage of the Relative Luciferase Activity (RLA) or as Relative Translation Activity (RTA); the latter corresponding to the FLuc/RLuc ratio, an index of the IRES activity ( 7 , 9 , 12 , 13 , 29 , 32 ).…”

“…Furthermore, how the HIV-1 IRES, or any other retroviral IRES, recruits the translational machinery also continues unclear ( 5 , 6 , 16 ). However, compelling results suggest that the activity of the HIV-1 IRES, and other retroviral IRESs, rely on host factors such as the eukaryotic initiation factors (eIF) 4A and eIF5A ( 29–31 ), ribosomal protein S25 ( 12 , 29 , 32 ), and the availability of IRES trans-acting factors (ITAFs) that activate or repress its activity ( 9 , 23 , 29 , 30 , 32–35 ). For the HIV-1 IRES, one such ITAF is the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) ( 10 , 23 , 36 ), a host protein that binds the viral genomic mRNA in the nucleus and is exported to the cytoplasm as part of the full-length HIV-1 mRNA ribonucleoprotein (RNP) complex ( 23 ).…”

Strategies of Human T-Cell Leukemia Virus Type 1 for Persistent Infection: Implications for Leukemogenesis of Adult T-Cell Leukemia-Lymphoma

“…and its promoter activity is stronger than that of usHBZ (Yoshida et al, 2008). It has been also shown that the sHBZ protein is more abundant than in cells than that of usHBZ (Cavanagh et al, 2006;Murata et al, 2006;Caceres et al, 2018), suggesting that the effect of sHBZ is dominant in infected cells.…”