NF-kappaB, a key regulator of the cellular inflammatory and immune response, is activated by the HTLV-I transforming and transactivating protein Tax. We show that Tax binds to the amino terminus of the protein kinase MEKK1, a component of an IkappaB kinase complex, and stimulates MEKK1 kinase activity. Tax expression increases the activity of IkappaB kinase beta (IKKbeta) to enhance phosphorylation of serine residues in IkappaB alpha that lead to its degradation. Dominant negative mutants of both IKKbeta and MEKK1 prevent Tax activation of the NF-kappaB pathway. Furthermore, recombinant MEKK1 stimulates IKKbeta phosphorylation of IkappaB alpha. Thus, Tax-mediated increases in NF-kappaB nuclear translocation result from direct interactions of Tax and MEKK1 leading to enhanced IKKbeta phosphorylation of IkappaB alpha.

SPT5 and its binding partner SPT4 regulate transcriptional elongation by RNA polymerase II. SPT4 and SPT5 are involved in both 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole (DRB)-mediated transcriptional inhibition and the activation of transcriptional elongation by the human immunodeficiency virus type 1 (HIV-1) Tat protein. Recent data suggest that P-TEFb, which is composed of CDK9 and cyclin T1, is also critical in regulating transcriptional elongation by SPT4 and SPT5. In this study, we analyze the domains of SPT5 that regulate transcriptional elongation in the presence of either DRB or the HIV-1 Tat protein. We demonstrate that SPT5 domains that bind SPT4 and RNA polymerase II, in addition to a region in the C terminus of SPT5 that contains multiple heptad repeats and is designated CTR1, are critical for in vitro transcriptional repression by DRB and activation by the Tat protein. Furthermore, the SPT5 CTR1 domain is a substrate for P-TEFb phosphorylation. These results suggest that C-terminal repeats in SPT5, like those in the RNA polymerase II C-terminal domain, are sites for P-TEFb phosphorylation and function in modulating its transcriptional elongation properties.Regulation of transcriptional elongation is a critical process in the control of viral and cellular gene expression (reviewed in references 3 and 28). A number of cellular factors that regulate transcriptional elongation have been defined using both biochemical and genetic techniques. These factors include the general transcription factors TFIIF and TFIIS, as well as other factors including the elongin and ELL proteins (20,41,48).In addition, cellular kinases play an important role in the control of transcriptional elongation based on their ability to phosphorylate the RNA polymerase II C-terminal domain (CTD) (27). One of these kinases, CDK-activating kinase (CAK), is composed of the CDK7 kinase in addition to cyclin H and MAT1. CAK is contained in the multiprotein TFIIH complex and is involved in modulating promoter clearance of specific promoters (13,45,47). A second kinase complex, PTEFb, is composed of cyclin T1 and CDK9 and also phosphorylates the RNA polymerase II CTD and stimulates transcriptional elongation (18,32,33,36,64). The Tat protein, which is a potent stimulator of transcriptional elongation, interacts with P-TEFb to stimulate human immunodeficiency virus type 1 (HIV-1) gene expression (4, 7, 17-19, 25, 26, 30, 31, 55, 56, 62, 64).SPT4 and SPT5 are highly conserved proteins which are present in a variety of species from yeast to humans and are involved in the regulation of transcriptional elongation (23,53,58,60,61). Genetic assays in yeast demonstrate that SPT5 conditional mutants can be suppressed by mutations in the genes encoding two largest subunits of RNA polymerase II (23). Furthermore, SPT5 interacts directly with RNA polymerase II via a domain in SPT5 that has homology to the Escherichia coli transcription elongation factor NusG (23, 53, 61). The human homologues of the SPT4 and SPT5 proteins have also been character...

SPT5 and its binding partner SPT4 function in both positively and negatively regulating transcriptional elongation. The demonstration that SPT5 and RNA polymerase II are targets for phosphorylation by CDK9/cyclin T1 indicates that posttranslational modifications of these factors are important in regulating the elongation process. In this study, we utilized a biochemical approach to demonstrate that SPT5 was specifically associated with the protein arginine methyltransferases PRMT1 and PRMT5 and that SPT5 methylation regulated its interaction with RNA polymerase II. Specific arginine residues in SPT5 that are methylated by these enzymes were identified and demonstrated to be important in regulating its promoter association and subsequent effects on transcriptional elongation. These results suggest that methylation of SPT5 is an important posttranslational modification that is involved in regulating its transcriptional elongation properties in response to viral and cellular factors.

Successful antiretroviral pre-exposure prophylaxis (PrEP) for mucosal and intravenous HIV-1 transmission could reduce new infections among targeted high-risk populations including discordant couples, injection drug users, high-risk women and men who have sex with men. Targeted antiretroviral PrEP could be particularly effective at slowing the spread of HIV-1 if a single antiretroviral combination were found to be broadly protective across multiple routes of transmission. Therefore, we designed our in vivo preclinical study to systematically investigate whether rectal and intravenous HIV-1 transmission can be blocked by antiretrovirals administered systemically prior to HIV-1 exposure. We performed these studies using a highly relevant in vivo model of mucosal HIV-1 transmission, humanized Bone marrow/Liver/Thymus mice (BLT). BLT mice are susceptible to HIV-1 infection via three major physiological routes of viral transmission: vaginal, rectal and intravenous. Our results show that BLT mice given systemic antiretroviral PrEP are efficiently protected from HIV-1 infection regardless of the route of exposure. Specifically, systemic antiretroviral PrEP with emtricitabine and tenofovir disoproxil fumarate prevented both rectal (Chi square = 8.6, df = 1, p = 0.003) and intravenous (Chi square = 13, df = 1, p = 0.0003) HIV-1 transmission. Our results indicate that antiretroviral PrEP has the potential to be broadly effective at preventing new rectal or intravenous HIV transmissions in targeted high risk individuals. These in vivo preclinical findings provide strong experimental evidence supporting the potential clinical implementation of antiretroviral based pre-exposure prophylactic measures to prevent the spread of HIV/AIDS.

Recent iPrEx clinical trial results provided evidence that systemic preexposure prophylaxis (PrEP) with emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-B pathway. In this study, we demonstrated that Hsp27 associates with the I 〉 kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor ␣ treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK␤ to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK␤ and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor ␣ stimulation.

No abstract

The NF-B pathway is important in the control of the immune and inflammatory response. One of the critical events in the activation of this pathway is the stimulation of the IB kinases (IKKs) by cytokines such as tumor necrosis factor-␣ and interleukin-1. Although the mechanisms that modulate IKK activation have been studied in detail, much less is known about the processes that down-regulate its activity following cytokine treatment. In this study, we utilized biochemical fractionation and mass spectrometry to demonstrate that protein phosphatase 2C␤ (PP2C␤) can associate with the IKK complex. PP2C␤ association with the IKK complex led to the dephosphorylation of IKK␤ and decreased its kinase activity. The binding of PP2C␤ to IKK␤ was decreased at early times post-tumor necrosis factor-␣ treatment and was restored at later times following treatment with this cytokine. Experiments utilizing siRNA directed against PP2C␤ demonstrated an in vivo role for this phosphatase in decreasing IKK activity at late times following cytokine treatment. These studies are consistent with the ability of PP2C␤ to down-regulate cytokine-induced NF-B activation by altering IKK activity.The NF-B pathway is a critical regulator of the cellular response to a variety of stimuli including cytokines such as TNF␣ 1 and interleukin-1, bacterial and viral infection, and double-stranded RNA (1-7). Cytokines lead to a rapid increase in the activity of the IB kinases, and this is followed by a subsequent decrease in the activity of these kinases, suggesting both positive and negative regulation of the NF-B pathway. A better understanding of the NF-B pathway will be important in defining how these factors modulate the host immune and inflammatory response and prevent apoptosis (1-7).The NF-B transcription factors, p105/50, p100/52, p65, cRel, and RelB, contain a Rel homology domain that mediates their dimerization and DNA binding properties (2). These proteins are sequestered in the cytoplasm of most cells, where they are bound to a family of inhibitory proteins known as IB (1, 3). Treatment of cells with cytokines, including TNF␣ and interleukin-1, stimulates the activity of IB kinases that phosphorylate IB on amino-terminal serine residues, leading to its ubiquitination and degradation by the proteasome (3-7). This process results in the nuclear translocation of the NF-B proteins where they bind to the promoter elements of a variety of genes involved in the control of the immune and inflammatory response (1-7).Activation of the IB kinases is a critical process in regulating the NF-B pathway (7-12). These kinases, designated IKK␣ and IKK␤, are components of a 600 -900-kDa complex (7-12), which also includes a scaffold protein IKK␥/NEMO (13-16) and the chaperone proteins Hsp90 and Cdc37 (17). In addition to binding to IKK␣ and IKK␤, IKK␥/NEMO has been demonstrated to bind to a variety of other proteins that have been reported to be involved in the regulation of the NF-B pathway, including RIP, A20, CIKS, and the HTLV-I Tax protein (18 -22).Althou...