Increased production of viral proteins by a 3'-LTR-deleted infectious clone of human T-cell leukemia virus type 1
© Ohsugi; licensee BioMed Central Ltd. 2009
Received: 23 October 2009
Accepted: 24 December 2009
Published: 24 December 2009
We previously reported that a full-length provirus of HTLV-1 was directly constructed from the HTLV-1-transformed cell line MT-2 using overlapping polymerase chain reaction (PCR) and cloned into a plasmid vector (pFL-MT2). 293T cells transfected with pFL-MT2 alone did not produce virus particles because there was no expression of the viral transactivator protein Tax, whereas cells transfected with pFL-MT2 plus a Tax expression vector produced virus-like particles. In the process of constructing the HTLV-1 provirus by overlapping PCR, we also constructed an incomplete molecular clone, in which the 3' long terminal repeat (LTR) was replaced with the endogenous human gene, which resulted in the expression of a tax gene shorter by 43 bp. This incomplete molecular clone alone expressed Tax and produced the viral protein in transfected cells. Various clones were then constructed with different lengths of the 3' LTR and lacking the reverse-direction TATA box. The clones contained over 113 bp of the 3' LTR, with no reverse-direction TATA box, which might express the full-length tax gene, and did not produce the viral antigen. These results suggest that Tax in which the C-terminal portion is deleted is more strongly expressed than the wild-type protein and has transcriptional activity.
Human T-cell leukemia virus type 1 (HTLV-1) was the first human retrovirus to be isolated [1, 2]. It causes an aggressive malignancy known as adult T-cell leukemia/lymphoma, as well as inflammatory diseases such as HTLV-1-associated myelopathy/tropical spastic paraparesis, after a very prolonged period of latency, often lasting between 20 and 50 years [3, 4]. The tax gene product encoded by the pX region of HTLV-1 appears to be a key element in the development of HTLV-1-associated diseases [5–7]. Tax enhances productive virus replication by driving gene transcription via the cAMP-responsive element located in the viral long terminal repeat (LTR) [8, 9]. Tax also activates the expression of many cellular genes, including genes that encode cytokines, cytokine receptors, and immediate early transcription factors, via the activation of several cellular signal transduction pathways, such as the nuclear factor kappaB (NF-κB) and serum response factor (SRF) pathways [10–12].
These constructed clones were used to transfect the human epithelial 293T cell line. RT-PCR of the transfected 293T cells with Δ3' LTR and Δ3' LTR Bst EII demonstrated that the cells expressed mRNA sequences corresponding to the tax gene. However, the cells transfected with other Δ3' LTR clones, including the complete provirus, did not express the tax gene (Figure 2B). The expression of other HTLV-1 genes (gag, pol, and env) was the same as that of the tax gene (data not shown). The Δreverse TATA mutant did not produce the viral antigen. Recently, the expression of the HTLV-1 basic leucine zipper factor (HBZ), an antisense mRNA transcribed from the 3' LTR, has been shown to be consistently expressed in adult T-cell leukemia cells. Thus, HBZ may have a functional role in cellular transformation and leukemogenesis . HBZ was first found to inhibit the Tax-mediated transactivation of viral transcription from the 5' LTR by heterodimerizing with Jun and CREB2 . None of the constructed Δ3' LTR clones contained the promoter for the HBZ gene located in U5 and only the part R region of the 3' LTR . No HBZ gene transcript was detected by RT-PCR in cells transfected with any clone, including those transfected with the complete provirus clone.
These results suggest that the sequence between 40 nucleotides (nt) and 113 nt at the Aat II site downstream from the beginning of the 3' LTR, which constitutes the C-terminal portion of the tax downstream sequence, might be involved in the inhibition of the replication of HTLV-1 genes in infectious molecular clones. It is suggested that the complete infectious molecular clone could not produce the viral antigen because the expression of Tax was low, and there might exist a binding site for cellular factors that inhibit the expression of the tax gene between 40 nt and 113 nt at the Aat II site downstream from the beginning of the 3' LTR. Recently, Fryrear et al. reported that a Tax mutant (353 amino acids), in which the C-terminal portion (amino acids 323-353) was deleted, displayed higher transcriptional activity than that of the wild-type protein . There is a PDZ-protein-binding motif at this site, which interacts with several PDZ proteins, such as DLG1, the precursor of interleukin 16, and MAGI3 [20–25]. Ishioka et al. reported that inactivation of DLG1 augments the Tax-mediated transformation of cells. This finding suggests that DLG1 regulates the function of Tax through its PDZ-binding motif . HTLV-1-infected cells in the peripheral blood rarely express viral genes in HTLV-1-infected individuals [16, 27]. This might be caused by cellular factors, such as PDZ proteins, inhibiting Tax expression by binding to the PDZ-protein-binding motif in the C-terminal portion of the tax gene.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, the Japan Leukaemia Research Fund, and Kumamoto University's Centers of Excellence (COE) Program.
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