Abstract
Human T-lymphotropic Virus type 1 (HTLV-1) infection is characterized by viral latency in the majority of infected cells and by the absence of viremia. These features are thought to be due to the repression of viral sense transcription in vivo. Here, our in silico analysis of the HTLV-1 Long Terminal Repeat (LTR) promoter nucleotide sequence revealed, in addition to the four Sp1 binding sites previously identified, the presence of two additional potential Sp1 sites within the R region. We demonstrated that the Sp1 and Sp3 transcription factors bound in vitro to these two sites and compared the binding affinity for Sp1 of all six different HTLV-1 Sp1 sites. By chromatin immunoprecipitation experiments, we showed Sp1 recruitment in vivo to the newly identified Sp1 sites. We demonstrated in the nucleosomal context of an episomal reporter vector that the Sp1 sites interfered with both the sense and antisense LTR promoter activities. Interestingly, the Sp1 sites exhibited together a repressor effect on the LTR sense transcriptional activity but had no effect on the LTR antisense activity. Thus, our results demonstrate the presence of two new functional Sp1 binding sites in the HTLV-1 LTR, which act as negative cis-regulatory elements of sense viral transcription.
Highlights
The complex retrovirus Human T-lymphotropic Virus type 1 (HTLV-1) (Human T-lymphotropic virus type 1) infects about 15–20 million people worldwide and is endemic in Japan, Africa, South America and the Caribbean islands[1,2]
In order to determine the relative contribution of each Sp1 binding site to the basal and Tax-mediated HTLV-1 Long Terminal Repeat (LTR) activity, the point mutations identified in EMSAs (Fig. 3) were introduced individually or in combinations in the context of the promoter cloned in an episomal vector (pREP)-luc episomal luciferase reporter vector in both the sense and antisense orientations with respect to the luciferase transcriptional unit, thereby generating a serie of plasmids described in Supplementary Table S2
After integration into the host cellular genome, the HTLV-1 provirus is transcriptionally regulated by cellular transcription factors such as the ubiquitously expressed Sp1 factor
Summary
The complex retrovirus HTLV-1 (Human T-lymphotropic virus type 1) infects about 15–20 million people worldwide and is endemic in Japan, Africa, South America and the Caribbean islands[1,2]. The U3 region contains the TRE-2 (Tax responsive element 2, nt −163/−117) which is critical for Tax-mediated transactivation of the HTLV-1 LTR and interact with cellular transcription factors Ets and Sp1. 12) and binds CREB/ATF (cAMP-responsive element-binding protein/activating transcription factor) family members, and the TRE-2 which has been demonstrated to collaborate with the promoter proximal TRE-1 via recruitment of Ets, Elk-1 and Sp113–15. Both the TRE-1 and TRE-2 motifs are required in cis to allow Tax transactivation of the HTLV-1 promoter[11]. Sp (Specificity protein) factors are member of the zing-finger Specificity protein/Krüppel-like Factor (SP/KLF) transcription factor family which is characterized by a highly conserved DNA-binding domain in ref
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