Nucleic Acids Research, 2011, Vol. 39, No. 14 5887 Figure 6. HSF1 interacts with novel HSF1 binding site on LTR and gets functionally activated during infection. (A) Schematic representation of HIV-1 LTR showing the positions of oligonucleotide probes (P1 to P4) and primers (F1–F3 and R) used in EMSA and ChIP analysis, respectively. (B) His-HSF1 speciﬁcally binds to probe P2 in EMSA analysis. HSE consensus sequence was used as positive control (lane 2). (C) His-HSF1 binds to probe P2 in dose-dependent manner. (D) His-HSF1 binding to P2 is inhibited by cold P2 oligo but not by non-speciﬁc P1 oligo. (E) Probe P2 binds to Jurkat nuclear extract which is inhibited by HSF1 antibody pre-incubation. P2 binding remains unaffected by post-incubation with HSF1 antibody. (F) P2 probe binds with nuclear extract in dose-dependent manner. (G) P2 binding to nuclear extract is competitively inhibited by cold P2 oligo but not by non-speciﬁc P1 oligo. (H) HSF1 is recruited on the putative HSF1 binding sequence in HIV-1 LTR during HIV-1 infection of Jurkat cells. ChiP analysis was performed with Nef antibody followed by PCR ampliﬁcation using F1 to F3 and R primers. (I) HSF1 trimerization increases during HIV-1 infection. HIV-1-infected CEM-GFP cell lysates were used for chemical cross-linking by EGS followed by gel electrophoresis as described in the text. (J) HSF1 is phosphorylated and translocated in to the nucleus of HIV-1-infected cells. HIV-1-infected Jurkat cells were used for immunostaining with polyclonal phospho-HSF1 (Ser230) antibody. The extreme right panel in each row is a magniﬁed image of one cell from the merged image panel. Arrows indicate localization of phospho-HSF1 in the cell. (K) HSF1 is phosphorylated in HIV-1-infected Jurkat cells as analyzed by immunoblotting of nuclear and cytoplasmic fractions. speciﬁcally blocks the binding between P2 and HSF1. Furthermore, increased P2 binding was observed with infected nuclear extract (Figure 6F), which could be explained by increased HSF1 expression during HIV-1 infection. P2 binding speciﬁcity was further checked by competition experiment that showed loss of binding with excess cold oligo but not with non-speciﬁc oligo (Figure 6G). We also analyzed the in vivo HSF1 recruit- ment on HIV-1 promoter by ChIP assay. Cross-linked chromatin from HIV-1-infected Jurkat cells was pulled down with HSF1 antibody and recruitment on LTR was checked by PCR ampliﬁcation using forward primers (F1, F2, F3) and reverse primer (R) spanning the enhancer region as shown in Figure 6A. Strong signal in PCR with primers F1/R and F2/R was obtained, which includes a region downstream to NF-AT1 from 144 to +12 and 93 to +12, respectively. However in the same experiment no PCR signal was obtained with F3/R primer set encompassing a region downstream to third Sp1 site between 75 to +12 (Figure 6H). Thus our in vitro and cellular studies have shown for the ﬁrst time binding and recruitment of HSF1 on a newly identiﬁed HSF1 binding site in HIV-1 LTR ( 69 to 91, GGGACTTTCCAGGG AGGTGTGGC). HSF1 is functionally activated during HIV-1 viral infection Earlier reports have shown induction of HSF1 transacti- vation activity by its phosphorylation, trimerization and nuclear translocation (17,18). We then looked at the