Kinetics and intracellular compartmentalization of HTLV-1 gene expression: nuclear retention of HBZ mRNAs

Abstract

IntroductionHuman T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia-lymphoma (ATLL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). HTLV-1 uses several strategies for controlling the expression of its genome, including the production of 9 alternatively spliced transcripts ( Figure 1A). 1-6 Production of plus-strand transcripts is controlled by Tax at the level of transcription and by Rex at the level of nucleo-cytoplasmic export of unspliced and partially spliced mRNAs. 7,8 Regulation of the minus-strand HBZ transcripts, which lack elements responsive to Rex, remains to be determined.Current models suggest that plus-strand HTLV-1 mRNAs are expressed with a distinct timing during the course of the viral life cycle, with a switch from early (Rex-independent) to late (Rexdependent) transcripts. Although early studies showed a qualitative switch among classes of HTLV-1 mRNAs (multiply spliced vs unspliced), 9-12 detection of this phenomenon with quantitative transcript-specific methods has proven difficult. 13 To answer this question we used quantitative RT-PCR to quantify proviral expression during the spontaneous proviral reactivation observed in cells from infected patients. The results demonstrated a "two-phase" expression pattern. Using transfection of HTLV-1 molecular clones and subcellular RNA fractionation we demonstrated the Rex-dependency of the two-phase kinetics and determined the compartmentalization of the individual mRNAs, showing that more than 90% of the HBZ mRNAs were retained in the nucleus. Mathematical modeling 14 revealed the importance of a delay of Rex function compared with Tax, which was supported by experimental evidence of delayed accumulation and longer halflife of Rex. Methods Samples from HTLV-1-infected patientsPeripheral blood mononuclear cells (PBMCs) from ATLL and TSP/HAM patients were purified as in. 15 Patients are described in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). All samples were obtained from patients after informed consent in accordance with the Declaration of Helsinki, with approval from the Imperial College and King's College hospitals (London) Institutional Rreview Boards. Plasmids, cells, and transfectionsPlasmid pBS1-2-3 consists of the tax/rex cDNA (exons 1, 2, and 3 flanked by the 5Ј and 3ЈLTRs, from infectious molecular clone CS-HTLV-1 16 ) inserted in pBluescript (Stratagene). Plasmid ACH-Rex knockout (KO) was For personal use only. on May 10, 2018. by guest www.bloodjournal.org From derived from the HTLV-1 molecular clone ACH 17 by digestion with SphI followed by removal of 3Ј overhangs (including the Rex initiation codon) with T4 DNA polymerase and religation. Transfections were performed in the HeLa-derived cell line HLtat, 18 chosen for its high transfection efficiency. Quantitative RT-PCRRNA of PBMCs from infected patients and transfected cells was extracted and viral transcripts were quantitated as detailed in supp...

HTLV-1 bZIP Factor–Specific CD4 T Cell Responses in Adult T Cell Leukemia/Lymphoma Patients after Allogeneic Hematopoietic Stem Cell Transplantation

“…This would be consistent with the persistent expression of HBZ in HTLV-1-infected cells (2,25). In addition, insufficient HBZ-specific T cell responses may be due, in part, to the fact that the majority of the HBZ mRNA is retained in the nucleus, which may inhibit its translation (35), and probably leads to a low level of HBZ protein expression in HTLV-1-infected cells (29). In contrast, the finding that HLA-DRB1*15:01-restricted or HLA-DRB1*15:02-restricted HBZ-specific CD4 T cell responses were detected in ATL patients after allogeneic HCT requires explanation.…”

Mystery of Human T-cell Leukemia Virus Type 1: Commentary on Two Viral Genes, Tax and HBZ

“…The function of the RNA has not been characterized fully, but a report suggested that HBZ mRNA is localized mostly in the nucleus [20]. This nuclear localization may suggest other roles of HBZ mRNA in addition to its translation in the cytoplasm.…”

Cytotoxic T lymphocyte lysis of HTLV-1 infected cells is limited by weak HBZ protein expression, but non-specifically enhanced on induction of Tax expression

“…After co-incubation with either HBZ-specific or Tax-specific CTLs, there was a significant reduction in the median intensity of HLA-A*02 expression by Tax + CD4 + cells ( Figure 6C). In contrast, the level of HLA-A*02 expression was unchanged on Tax We hypothesized that Tax [11][12][13][14][15][16][17][18][19] could inhibit presentation of HBZ [26][27][28][29][30][31][32][33][34] by competing for binding to HLA-A*0201 ( Figure 7A). At saturating concentrations of HBZ 26-34 (2 uM), preincubation with Tax 11-19 had no effect on lysis of BLCL by CTLs.…”

“…However, when the concentration of HBZ [26][27][28][29][30][31][32][33][34] was limiting (0.02-0.2 uM), Tax [11][12][13][14][15][16][17][18][19] could inhibit CTL detection of HBZ, but only when present in 10,000-fold molar excess. We also quantified the frequency of CD8 + T cells specific for the Tax [11][12][13][14][15][16][17][18][19] and HBZ [26][27][28][29][30][31][32][33][34] epitopes in each of the donors in this study. IFN-γ-producing Tax [11][12][13][14][15][16][17][18][19] specific CD8 + cells were observed in all individuals, whereas no HBZ 26-34 specific CD8 + cells were detected ( Figure 7B).…”

“…Despite its significant protective potential, the binding affinity of HBZ peptides to HLA class I molecules was found to be significantly weaker than that of peptides from Tax, and the frequency of HBZ-specific CD8 + T cells in peripheral blood was extremely low [11,12], although the IL-2 secreting HBZ-specific CD8 + T cells were more frequently detected in individuals with a viral load of below 1% of PBMCs [12]. In addition, HBZ protein is present at levels barely detectable by western blot; inefficient polyadenylation and transport of mRNA from the nucleus are thought to be responsible for this low expression [4,[13][14][15]. Because of the low immunogenicity of HBZ, it has been difficult to directly test the ability of primary infected PBMCs to present HBZ to CTLs.…”