clearly indicating the importance of HSF1 in the viral life cycle. Taken together, all these results indicate that HSF1 positively regulates HIV-1 gene expression and replication by two distinct pathways. Firstly, it induces HSP40 promoter activity along with Nef and secondly it directly interacts with HIV-1 LTR to induce viral gene expression and replication. DISCUSSION Expression of HSP family members is modulated in various disease conditions like cancer (37) and sepsis (38). Acute infection of cells with viruses also induces ex- pression of stress proteins (39,40). In case of acute HIV-1 infection modulation of HSP27 and HSP70, expression in CD4+ T cells have been reported earlier (6); however, the molecular mechanism of this modulation remains obscure. We have shown earlier that HSP40 is upregulated in pres- ence of Nef (15). Here, we ﬁrst conﬁrm this Nef-dependent phenomenon in HIV-1-infected T cells followed by the identiﬁcation of the mechanism. Normally the gene ex- pression of HSPs is regulated by heat shock elements (HSE) present on the promoter (41) and HSF protein, which speciﬁcally bind to these HSE sequences and enhance HSP gene expression (42). In case of HIV-1 in- fection, there is one report showing Vpr-dependent modu- lation of HSP27 expression through HSF1 (43). We report here for the ﬁrst time that Nef induces HSP40 expression by forming a complex with HSF1. Nef, initially reported to be a transcriptional repressor of HIV-1 LTR (9,44) has been later shown to act as a positive regulator of LTR by a variety of mechanisms (12,45). In our effort to understand mechanistic details of Nef-dependent HSP40 upregula- tion, we ﬁnd that positive effect of Nef on HSP40 expres- sion was mediated by its interaction with HSF1. As, HSF1 is known to regulate expression of HSPs by being re- cruited on the promoter of HSPs, we also investigated the recruitment of Nef on HSP40 promoter. Nef seems to be recruited at HSE elements on HSP40 promoter along with HSF1. This ﬁnding was further supported by overexpression and gene silencing studies, where overex- pression of HSF1 enhanced HSP40 expression in Nef-dependent manner whereas its silencing resulted in in- hibition of HSP40 promoter activity in presence of Nef. Our results thus imply that the co-occupancy of Nef and HSF1 on HSP40 promoter activates the promoter to increase HSP40 expression in HIV-1-infected cells, which in turn enhances viral gene expression and production as elucidated earlier (15). In addition to stress response, HSF1 is also reported to be involved in developmental processes by regulating ex- pression of some other genes like inﬂammatory cytokines (46). It can act both as transcriptional activator as well as repressor depending on the presence of HSE elements and cohort of additional factors which it recruits (46). In view of the role that HSF1 plays for regulating the transcrip- tion of non-HSP genes, in second part of our study, we have analyzed direct role of HSF1 in LTR-driven gene expression. This study was also supported by several Nucleic Acids Research, 2011, Vol. 39, No. 14 5889 previous observations where activation of LTR-driven gene expression was reported under hyperthermic condi- tion (20–22). Suppression of HIV-1 LTR by a mutant HSF has been also reported (47); however, the modulation of LTR activity by HSF1 remains to be clearly elucidated. Our transient transfection studies clearly show that HSF1 enhances Tat induced LTR-driven gene expression. Furthermore, our studies with mutant LTR-luc constructs indicate the role of enhancer region of the LTR in positive regulation by HSF1. Computational analysis of HIV-1 LTR enhancer region resulted in identiﬁcation of a putative HSF1 binding site in this region. We then con- ﬁrmed binding of HSF1 with the putative binding site ( 69 to 91) in the enhancer region on HIV-1 LTR both in vitro and in vivo. Furthermore, enhanced expres- sion of HSF1 observed in HIV-1-infected cells may play a role in enhancing the positive effect of HSF1 on LTR-driven gene expression. The activation of HSF1 is associated with transition of the monomeric inactive to trimeric active form and con- comitant post-translational modiﬁcation like phosphoryl- ation and translocation into the nucleus (42). So to further explore the mechanism of HSF1-driven LTR gene expres- sion we have tried to correlate its LTR DNA binding activity with the transcriptionally active state. We observed that following HIV-1 infection, HSF1 not only shows increased trimerization but also shows increased phosphorylarion and nuclear re-localization in infected cells. This functional activation seems to facilitate the binding of HSF1 on both HSP40 promoter and LTR to enhance viral gene expression. However, it is still unclear how this binding activates the promoters. This binding might induce some molecular and physical changes in promoter at the chromatin level that allow binding of some activator molecules to mediate activation as observed in case of IL-6 gene, where the binding of HSF1 on its promoter led to opening of chromatin for binding of activator or repressor molecules (48). Future studies on the molecular details of this phenomenon at chromatin level may unravel novel mechanistic details about HIV-1 gene regulation. Activation of HSF1-mediated stress response in reaction to microbial infection is functionally important for elevated expression of HSPs, which generally act as an alert signal for host to elicit anti-microbial immune response (49). We have attempted to understand the func- tional relevance of increased HSF1 expression and activa- tion during HIV infection and our results clearly show that HSF 1 positively regulates HIV-1 gene expression and replication as HSF1 overexpression enhanced viral gene expression whereas its knockdown reduced virus pro- duction. As regulation of HIV-1 gene expression involves interplay between viral and cellular host factors, our present study adds another complex layer of regulation in HIV-1 gene expression by HSF1. In summary, we conclude that HSF1 regulates viral replication by two distinct pathways. It interacts with Nef and both are re- cruited on the HSP40 promoter to enhance HSP40 expres- sion in infected cells. HSP40 enhances viral replication by binding to Cdk9 and modulating the activity of P-TEFb (15). Second, it also regulates the viral transcription by