5888 Nucleic Acids Research, 2011, Vol. 39, No. 14 trimerization of HSF1 by chemical cross-linking using EGS followed by immunoblotting with monoclonal HSF1 antibody (Figure 6I). HSF1 was seen as an inactive monomer in uninfected cells but upon infection it was pre- dominantly present as a trimer (Figure 6I). Furthermore, immunostaining with phospho-HSF1 (S230) antibody showed cytoplasmic localization of pHSF1 in uninfected cells but following infection, it was predominantly local- ized in the nucleus (Figure 6J). Notably, co-localization of pHSF1 with DAPI was distinctly visible in infected Jurkat cells (Figure 6J). This was further conﬁrmed by immuno- blotting of nuclear and cytoplasmic fraction of HIV- 1-infected Jurkat cells using pHSF1(S230) antibody. Phospho-HSF1 was predominantly observed in the cyto- plasm of uninfected cells whereas most of it was present in the nucleus of infected cells (Figure 6K). Put together all these data clearly suggest that HSF1 is functionally activated during HIV-1 infection and in turn regulates HIV-1 gene expression and replication. HSF1 regulates HIV-1 gene expression and replication Our observation of HSF1 induced HSP40 promoter and LTR-driven gene expression led us to further investigate whether expression of HSF1 directly modulates HIV-1 replication or virus production. We thus performed a single cycle replication study in 293T cells by co- transfecting pNL4-3 along with HSF1 and virus produc- tion was analyzed in culture supernatants by p24 antigen capture ELISA. HSF1 overexpression resulted in enhanced virus production as compared to control cells (Figure 7A). Similar result was also obtained with Jurkat cells, trans- fected with HSF1 ﬁrst, followed by infection with NL4-3 virus (Figure 7B). This ﬁnding was further validated by looking at the effect of HSF1 silencing on virus produc- tion. HSF1 silencing was followed by transfection with pNL4-3 in 293T cells and infection in Jurkat cells and virus production was analyzed by p24 ELISA assay. HSF1 silencing in both 293T and Jurkat cells lead to sig- niﬁcant reduction in virus production (Figure 7C and D), Figure 7. HSF1 overexpression increases HIV-1 replication whereas it’s silencing leads to inhibition of HIV-1 replication. (A) HSF1 overexpression increases virus production. Culture supernatants of 293T cells transfected with pNL4-3 and HSF1 vectors were analyzed for virus production using p24 antigen capture ELISA. HSF1 overexpression is shown in the inset (B) HSF1 overexpression leads to increased virus production in HIV-1 NL4-3-infected Jurkat cells. (C) HSF1 downregulation reduces HIV-1 virus production. Culture supernatants from 293T cells co-transfected with pNL4-3 and increasing concentrations of HSF1 siRNA were analyzed for virus production using p24 ELISA. Efﬁciency of gene silencing was checked by RT–PCR (inset). (D) HSF1 silencing leads to inhibition of virus production in HIV-1-infected Jurkat cells. Jurkat cells were ﬁrst transfected with HSF1 siRNA followed by infection with NL4-3 virus. Cells were analyzed for HSF1 silencing by RT–PCR as shown in (inset) and culture supernatant was used for p24 ELISA. The error bars represent the mean ± SEM of two independent experiments. Statistical analysis was performed using Student’s t-test, with the levels of signiﬁcance deﬁned as *P < 0.05.