5886 Nucleic Acids Research, 2011, Vol. 39, No. 14 Figure 5. HSF1 induces HIV-1 LTR-driven gene expression in Nef independent manner. (A) Schematic representation of HIV-1 LTR and the LTR-luc mutants used in the present study. (B) HSF1 enhances HIV-1 LTR-driven luciferase expression in 293T cells co-transfected with Tat and Nef. (C) HSF1 induces LTR-driven luciferase expression independent of Nef. 293T cells were transfected with pNL4-3 or Nef deleted pNL4-3 along with LTR-luc and HSF1 vectors and were analyzed for luciferase activity. (D) HSF1 induces HIV-1 LTR activity through its enhancer region. 293T cells were transfected with different LTR-luc mutants along with HSF1 and pNL4-3 and luciferase activity was analyzed 36 h post-transfection. The error bars represent the mean ± SEM of three independent experiments. Statistical analysis was performed using Student’s t-test, with the levels of signiﬁcance deﬁned as *P < 0.05. We then wanted to identify the region of LTR which was involved in HSF1-mediated induction of LTR activity. We used different LTR deletion mutants (schematically pre- sented in Figure 5A) for co-transfection with HSF1 and pNL4-3 in 293T cells followed by reporter assay. The results indicate that, region encompassing NF-kB and Sp1 elements ( 117 to 65) is involved in HSF1- mediated induction of LTR activity (Figure 5D). Deletion of this region, as present in CD52 luc ( 65 luc) did not show any induction with HSF1, suggesting the role of NF-kB-Sp1 enhancer region of LTR in HSF1-mediated activation. HSF1 induces HIV-1 gene expression by interacting with newly identiﬁed HSF1 binding sequence in the LTR We have shown above that HSF1-mediated induction of LTR is dependent upon the enhancer region. In order to identify the HSF1 binding sequence in HIV-1 LTR, we then analyzed the LTR nucleotide sequence using TFSEARCH program version 1.3 (36), which revealed putative HSF1 binding site in LTR enhancer region ( 69 to 91, data not shown). Based on this prediction, we made four overlapping oligonucleotide probes P1, P2, P3 and P4 encompassing the enhancer region as shown in Figure 6A. Recombinant His-HSF1 protein was used in electrophoretic mobility shift assay (EMSA) with these four probes along with consensus HSE probe as a positive control. Interestingly, probes P1, P3 and P4 failed to form any complex whereas P2 formed nucleoprotein complex with His-HSF1 (Figure 6B). P2 comprises of nucleotide sequence from 69 to 91 of LTR promoter. To further conﬁrm the speciﬁcity of HSF1 binding, we analyzed P2 binding with increasing amount of His-HSF1 protein, which resulted in a dose dependent increase in complex formation (Figure 6C). Binding speciﬁcity of P2 for HSF1 was also conﬁrmed by competition experiments with speciﬁc and non-speciﬁc oligo (Figure 6D). In order to see the binding with cellular HSF1, we then used nuclear extract from HIV-1-infected Jurkat cells. P2 formed nu- cleoprotein complex with HIV-1-infected nuclear extract (Figure 6E). Loss of P2 binding was observed in pre- incubation with HSF1 antibody; however, post- incubation with anti-HSF1 did not show any effect on binding (Figure 6E). This shows that HSF1 antibody