5880 Nucleic Acids Research, 2011, Vol. 39, No. 14 Several studies have suggested that heat shock could acti- vate the LTR-driven transcription in cells (20,21); however, the mechanism of activation has not been clearly understood (22). During our efforts to understand the mechanism of Nef-dependent upregulation of HSP40 in HIV-1 infection, we have identiﬁed the importance of HSF1 in HIV-1 gene expression and replication in the present study. Our results clearly show that HSF1 positively regulates HIV-1 gene expression and replication by two distinct pathways. First, it induces HSP40 expression in association with viral protein Nef, both of which has been earlier shown to be required for increased viral gene expression (15). Second, activated HSF1 directly interacts with LTR to induce viral gene expression and replication. MATERIALS AND METHODS Cell lines, plasmids and reagents HIV-1 NL4-3 Nef expression vector pcDNA-Nef was ob- tained from Dr M. Federico (23). NL4-3 Nef and its point mutants tagged with HA (HA-Nef) plasmids were obtained from Dr W.C. Greene (24). HSF1 encoding plasmid pCMV-HSF1-Flag was a gift of Dr J. Goldman. This HSF1 was further subcloned in pET-28a(+) vector (Novagen, USA). HIV-1 NL4-3 molecular clone (pNL4-3) was obtained from the NIH AIDS repository (25). The nef-deleted NL4-3 molecular clone (pNL4– 3 Nef) was obtained from Dr J.C. Guatelli (26). The HIV-1 LTR reporter vector, pLTR luc was cloned in our laboratory as reported earlier (27). HIV-1 LTR and its deletion mutant constructs of CD series were obtained from Dr T. Okamoto (28). HSP40 promoter construct was obtained from Dr K. Ohtsuka (29), which was used to subclone HSP40 promoter in pGL3 luciferase vector. HEK-293T and Jurkat cells were obtained from the NCCS Cell Repository, India. CEM-GFP, a CD4+ human T-cell line, was obtained from the NIH AIDS re- pository, USA (30). A polyclonal anti-Nef serum was obtained from Dr S. Jameel (31). Monoclonal and poly- clonal Nef antibodies were obtained from Chemicon, USA and NIH AIDS Repository, respectively. Antibodies against HSP40, HA-tag and phospho-HSF1 were obtained from Santa Cruz Biotechnology, USA. Monoclonal and polyclonal HSF1 antibodies were obtained from Chemicon and Cell Signaling, USA, re- spectively. HSF1 Ab-4 cocktail (Clones 4B4+10H4+ 10H8 Rat monoclonal antibody) was obtained from Thermo-Fisher Scientiﬁc, USA. Mouse monoclonal Flag-tag and Tubulin antibodies were obtained from Sigma, USA. Control and Sp1 siRNA pools were from Santa Cruz Biotechnology, USA and HSF1 siRNA pool was obtained from Dharmacon, USA. Transient transfection and luciferase assay HEK-293T cells were transfected with reporter constructs along with other expression vectors using calcium phos- phate precipitation method and cells were harvested 36 h post-transfection for luciferase assay. For siRNA experi- ments, cells were ﬁrst transfected with siRNA using Lipofectamine 2000, followed by second transfection with other vectors after 24 h. The cells were then lysed and analyzed for luciferase activity using Luclite substrate (PerkinElmer Life Sciences, USA). Luciferase assays were analyzed using TopCount microplate reader (PerkinElmer Life Sciences, USA). The results were normalized with EGFP expression using Fluoroskan Ascent microplate reader (Thermo Labsystems, USA). HIV-1 infection and virus quantitation CEM-GFP or Jurkat cells (5 106) were infected with HIV-1 NL4-3 virus at 0.1 multiplicity of infection (MOI) in the presence of Polybrene (1 mg/ml) as described earlier (15). Human peripheral blood was collected from normal seronegative donors and PBMCs were isolated using Ficoll-Hypaque (Amersham Bioscience, USA). The cells were activated with PHA and later infected at 0.5 MOI of NL4-3 virus as described in detail earlier (15). The culture supernatants from NL4-3 infected and transfected cells were used to determine virus production by p24gag antigen capture ELISA (PerkinElmer Life Sciences, USA). Immunoprecipitation, His pull-down and immunoblotting HEK-293T cells overexpressing HA-Nef and HSF1 were lysed in lysis buffer (50 mM Tris–HCl pH 7.4, 5 mM EDTA, 0.12 M NaCl, 0.5% NP40, 0.5 mM NaF, 1 mM DTT, 0.5 mM PMSF) on ice for 45 min. Clariﬁed lysates were incubated with anti-Flag antibody and the antigen– antibody complex was pulled down by an equal mixture of protein A and G agarose beads followed by resolution on 12% SDS–PAGE. Proteins were transferred on to PVDF membrane and probed with HA antibody. The blots were developed by the ECL Plus system (Amersham Biosciences, USA). Similar co-immunoprecipitation ex- periment was performed with HIV-1-infected CEM-GFP cell lysates, which were immunoprecipitated with Nef antibody followed by immunoblotting with HSF1 antibody. Escherichia coli BL21 (DE3) cells expressing His-HSF1 were induced with isopropyl b-D-thiogalactoside followed by puriﬁcation of His-HSF1 protein using Ni-NTA beads (Qiagen, Germany). Transfected 293T cells, over- expressing wild-type and different mutants of Nef were lysed in cold lysis buffer (25 mM HEPES, pH 7.3, 0.1 M NaCl, 5 mM EDTA, 0.5% Triton X-100 and 1 mM DTT) with protease inhibitor cocktail (Roche Applied Bioscience, Germany). The clariﬁed lysates were incubated with His-HSF1 protein in binding buffer pH 8.0 (50 mM NaH2PO4, 300 mM NaCl, 10 mM Imidazole and 0.1% Tween-20) overnight at 4 C followed by pull down with Ni-NTA beads. The complexes were then resolved on 12% SDS–PAGE. Proteins were transferred onto PVDF membrane and were probed with polyclonal HA-Nef antibody. Furthermore, HIV-1-infected CEM-GFP and Jurkat cell lysates were run on SDS–PAGE, followed by immunoblotting for HSP40 and HSF1, respectively. Reverse transcription PCR RNA was prepared from 2 106 HIV-1 NL4-3-infected CEM-GFP cells using TRIzol Reagent (Invitrogen, USA).