HIV-1 Tat Induces the Expression of the Interleukin-6 (IL6) Gene by Binding to the IL6 Leader RNA and by Interacting with CAAT Enhancer-binding Protein β (NF-IL6) Transcription Factors

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection is associated with severe psoriasis, B cell lymphoma, and Kaposi's sarcoma. A deregulated production of interleukin-6 (IL6) has been implicated in the pathogenesis of these diseases. The molecular mechanisms underlying the abnormal IL6 secretion of HIV-1-infected cells may include transactivation of the IL6 gene by HIV-1. Here we report the molecular mechanisms of Tat activity on the expression of the IL6 gene. By using 5 deletion mutants of pIL6Pr-CAT and using IL6:HIV-1-LTR hybrid constructs where discrete regions of the IL6 promoter replaced the TAR sequence in HIV-1 LTR, we identified a short sequence of the 5-untranslated region of the IL6 mRNA that is required for Tat to trans-activate the IL6 promoter. This sequence acquires a stemloop structure and includes a UCU sequence that binds to Tat and is necessary for full trans-activation. In addition, we provide the evidence that Tat can function by enhancing the CAAT enhancer-binding protein (C/EBP) DNA binding activity and is able to complex with in vitro translated C/EBP␤, which is a major mediator of IL6 promoter function. By using the yeast two-hybrid system and immunoprecipitation, we observed that the interaction of Tat with C/EBP proteins also occurred in vivo. The data are consistent with the possibility that Tat may function on heterologous genes by interacting with RNA structures possibly present in a large number of cellular and viral genes. In addition, Tat may function by protein-protein interactions, leading to the generation of heterodimers with specific transcription factors.

IκB-α Represses the Transcriptional Activity of the HIV-1 Tat Transactivator by Promoting Its Nuclear Export

“…pSV-␤-gal was purchased from Promega. To generate p3XFLAG-CMV-Tat, p3XFLAG-CMV-Tat C (22,25,27)A, and p3XFLAG-CMV-Tat R(49 -57)A, the sequence of Tat was amplified from the pGEX-2T-Tat expressing vectors (43) and ligated to EcoRI/XbaI-digested p3XFLAG-CMV-7.1 (Sigma). pRc/CMV-HA-I B-␣S32/36A was previously described (44).…”

“…GST Pulldown-GST fusion proteins were produced in Escherichia coli strain BL21 as previously described (43). Bacterial cultures (500 ml) were grown to exponential phase and induced with 0.25 mM isopropyl-␤-D-thiogalactopyranoside (SigmaAldrich) for 3 h to express GST fusion proteins.…”

N-myc oncogene overexpression down-regulates IL-6; evidence that IL-6 inhibits angiogenesis and suppresses neuroblastoma tumor growth

“…Transient transfections were performed, according to the manufacturer's instructions, using either e ectene (Qiagen) or lipofectamine plus reagent (Invitrogen) for CAT and luciferase IL-6 promoter reporter constructs, respectively. A reporter CAT construct containing the 71200 to +15 promoter fragment of the human IL-6 (pIL6-CAT(71200/ +15)) was kindly provided by Dr Scala (University Federico II, Naples, Italy) (Ambrosino et al, 1997). HeLa cells (5610 5 per 60 mm dish) were transfected with 0.4 mg of the pIL6-CAT (71200/+15) construct, alone or in combination with 0.4 mg of wild-type or mutated N-myc expression vectors, for 40 h. In each transfection a constant total DNA concentration was used.…”

Physical and Functional Interaction of HIV-1 Tat with E2F-4, a Transcriptional Regulator of Mammalian Cell Cycle

“…a, Tat was produced and purified in vitro as a GST fusion protein as described previously (29). 5 g of purified GST or GST-Tat proteins were incubated with whole cellular extracts prepared from Jurkat cells.…”

“…The Tat functional interaction with cellular proteins plays a key role in the regulation of Tat transcriptional activity (23,24). Tat-mediated regulation of cellular gene expression is strongly related to its physical and functional interaction with proteins directly involved in the basal transcriptional process including TFIID, TFIIB (25)(26)(27), and eukaryotic transcription factors such as Sp1 (28), and CAAT enhancer-binding protein ( (29). Moreover, Tat binds to cyclin T and recruits CDK9 to increase the processivity of RNA polymerase II (12, 30 -32).…”