5882 Nucleic Acids Research, 2011, Vol. 39, No. 14 As shown in Figure 1A and B, HSP40 was induced in HIV-1 NL4-3-infected cells in a time-dependent manner. However, this increase was not observed in Nef deleted NL4-3-infected cells (Figure 1C and D) conﬁrming that HSP40 upregulation during HIV-1 infection is dependent on the Nef protein. In order to understand the mechanism of HSP40 induc- tion, we ﬁrst looked at the role of HIV-1 and Nef on the HSP40 promoter activation, if any. HEK-293T cells were co-transfected with HSP40 promoter-luciferase construct along with wild-type or nef deleted NL4-3 molecular clone followed by analysis of luciferase activity. The result clearly shows that HSP40 promoter activity was signiﬁ- cantly induced by wild-type NL4-3 but not with the nef deleted clone (Figure 1E) suggesting thereby that Nef was responsible for induction of HSP40 promoter activity. In order to conﬁrm this observation, we then co-transfected some individual HIV-1 protein expressing vectors along with HSP40-luciferase construct in 293T cells. The results clearly show that Nef speciﬁcally induces HSP40 promoter activity whereas Tat and Rev did not show any signiﬁcant effect (Figure 1F). We then wanted to identify the region of HSP40 promoter (29) involved in Nef- mediated induction of its activity, for which we created different deletion and point mutations in the promoter as represented in Figure 1G. These mutant constructs were then used in co-transfection assay along with Nef in 293T cells followed by luciferase activity analysis. As there was no signiﬁcant change in the activity of 246 promoter mutant with Nef as compared to the wild-type HSP40 ( 277) promoter-luc construct, the point mutants were made in the 246 HSP40 promoter sequence. The activity analysis of mutants show that HSE elements in the HSP40 promoter were necessary for Nef-mediated upregulation, whereas CAAT box did not seem to play any role (Figure 1H). Mutation at Sp1 sites also seems to moderately inhibit Nef-induced HSP40 promoter activity (Figure 1H). Thus above results indicate the pos- sible involvement of HSF1 and Sp1 transcription factors in Nef-mediated upregulation of HSP40 promoter activity. HSF1 is required for Nef-mediated upregulation of HSP40 in HIV-1-infected cells In order to establish the requirement of HSF1 and Sp1 for Nef-mediated induction of HSP40 promoter, endogenous HSF1 and Sp1 expression was then silenced by gene spe- ciﬁc siRNA transfection in 293T cells (Figure 2A, inset). These cells were then co-transfected with Nef and HSP40 Figure 1. Heat shock elements are required for Nef-dependent upregulation of HSP40 promoter-driven gene expression. (A and B) Expression proﬁle of HSP40 in HIV-1 NL4-3-infected CEM-GFP cells as analyzed by RT–PCR and immunoblotting. (C and D) Expression proﬁle of HSP40 in HIV-1 NL4-3 Nef-infected CEM-GFP cells as analyzed by RT–PCR and immunoblotting. (E) Nef is required for HSP40 promoter activation by NL4-3 virus in transfected 293T cells. (F) HSP40 promoter is speciﬁcally activated by HIV-1 Nef but not Tat and Rev in 293T cells. (G) Schematic representation of HSP40 promoter and its mutants. Star marks indicate the mutated site. (H) Nef induces HSP40 promoter through HSE elements. 293T cells co-transfected with various HSP40 promoter-luc construct along with Nef expression vector were analyzed for luciferase activity (top) and Nef expression (bottom). 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.