rich motif of Nef seems to play an important role in this interaction. HSF1 has been reported to be localized in the nucleus after heat shock or stress (16,33), whereas Nef was reported to be a predominantly cytoplasmic protein but was also shown to be present in the nucleus of HIV- 1-infected cells (34,35). As these proteins were found to physically interact, we then performed immunoﬂuores- cence staining for both HSF1 and Nef in transfected 293T and HIV-1-infected Jurkat cells. As shown in Figure 3E, confocal microscopic analysis clearly indicate that both proteins co-localize in the nucleus of Nef and HSF1 cotransfected, pNL4-3 transfected and HIV-1-infected cells (Figure 3E). All these results further conﬁrm that Nef and HSF1 not only interact with each other but they also co-localize in nucleus and get recruited on HSP40 promoter during HIV-1 infection as a complex. HSF1 is induced in HIV-1-infected cells As HSF1 expression status has not been studied during HIV-1 infection, we then assessed HSF1 expression in HIV-1-infected Jurkat cells by both immunoblotting and immunoﬂuorescence. The results show that HSF1 was up- regulated in HIV-1-infected Jurkat cells (Figure 4A and B). Nucleic Acids Research, 2011, Vol. 39, No. 14 5885 We further conﬁrmed this ﬁnding by quantitative real- time PCR using RNA prepared from uninfected and HIV-1-infected (Day 5) Jurkat cells and human PBMCs. As shown in Figure 4C and D, HSF1 expression was induced in HIV-1-infected Jurkat and PBMCs. All these data clearly indicate upregulation of HSF1 during HIV-1 infection. HSF1 enhances HIV-1 LTR-driven gene expression As HSF1 expression is induced during HIV-1 infection and it has been implicated in LTR-mediated gene expres- sion, we then analyzed the effect of HSF1 on LTR-driven gene expression (Figure 5A). HSF1 overexpression along with Tat or Nef signiﬁcantly induced LTR-driven gene expression, as compared to Tat or Nef alone or together (Figure 5B). This result indicates that HSF1 activates LTR-driven gene expression. As HSP40 gene expression was induced in Nef- dependent manner during HIV-1 infection, we then invest- igated role of Nef in HSF1-mediated induction of LTR-driven gene expression. As shown in Figure 5C, HSF1 induced LTR-driven gene expression in both wild- type and nef-deleted NL4-3 transfected cells, indicating that activity of HSF1 on LTR was Nef independent. Figure 4. HSF1 is upregulated during HIV-1 infection. (A) HSF1 expression in HIV-1 NL4-3-infected Jurkat cells on Day 5 post-infection as analyzed by immunoblotting. (B) Immunoﬂuorescence analysis of HSF1 expression in HIV-1-infected Jurkat cells. (C) qRT–PCR analysis of HSF1 mRNA expression in HIV-1-infected Jurkat cells on Day 5 post-infection. (D) qRT–PCR analysis of HSF1 mRNA expression in HIV-1-infected PBMCs on Day 5 post-infection. GAPDH was used as internal control for normalization. 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.