To identify novel inhibitors of transcriptional activation by the HIV Tat protein, we used a combination of in vitro and in vivo Tat-dependent transcription assays to screen >100,000 compounds. All compounds identified blocked Tat-dependent stimulation of transcriptional elongation. Analysis of a panel of structurally diverse inhibitors indicated that their target is the human homolog of Drosophila positive transcription elongation factor b (P-TEFb). Loss of Tat transactivation in extracts depleted of the kinase subunit of human P-TEFb, PITALRE, was reversed by addition of partially purified human P-TEFb. Transfection experiments with wild-type or kinase knockout PITALRE demonstrated that P-TEFb is required for Tat function. Our results suggest that P-TEFb represents an attractive target for the development of novel HIV therapeutics.

The urgent need for efficacious drugs to treat chronic hepatitis C virus (HCV) infection requires a concerted effort to develop inhibitors specific for virally encoded enzymes. We demonstrate that 2-C-methyl ribonucleosides are efficient chain-terminating inhibitors of HCV genome replication. Characterization of drug-resistant HCV replicons defined a single S282T mutation within the active site of the viral polymerase that conferred loss of sensitivity to structurally related compounds in both replicon and isolated polymerase assays. Biochemical analyses demonstrated that resistance at the level of the enzyme results from a combination of reduced affinity of the mutant polymerase for the drug and an increased ability to extend the incorporated nucleoside analog. Importantly, the combination of these agents with interferon-␣ results in synergistic inhibition of HCV genome replication in cell culture. Furthermore, 2-C-methyl-substituted ribonucleosides also inhibited replication of genetically related viruses such as bovine diarrhea virus, yellow fever, and West African Nile viruses. These observations, together with the finding that 2-C-methyl-guanosine in particular has a favorable pharmacological profile, suggest that this class of compounds may have broad utility in the treatment of HCV and other flavivirus infections. Hepatitis C virus (HCV)1 is the most common blood-borne infection and a major cause of chronic liver disease and liver transplantation in industrialized countries. The prevalence of HCV infection is estimated to be ϳ5-fold greater than HIV infection and ranges from 1-5% in most developed countries (1). Current therapy is both poorly tolerated and has limited efficacy, with less than 50% response rates among patients infected with the most prevalent virus genotype (1b) (1). Currently approved drugs for the treatment of hepatitis C are interferon-␣ and ribavirin, neither of which appears to act directly on the virus, and their antiviral effects appear to be mediated by multiple, indirect mechanisms. Therefore, there is a need for more efficient and better tolerated anti-HCV agents.The success of antiviral therapies based on chemotherapeutic agents targeting viral polymerases has prompted intense efforts to develop inhibitors of HCV NS5B, the virally encoded RNA-dependent RNA polymerase (RdRp). Studies with HIV reverse transcriptase validate the clinical utility of two distinct classes of viral polymerase inhibitors, nucleoside and non-nucleoside inhibitors. Nucleoside inhibitors function as competitive substrate analogs that prevent RNA chain elongation when incorporated by the viral enzyme, resulting in premature chain termination (2, 3). HIV reverse transcriptase non-nucleoside inhibitors bind to a site residing outside the enzyme active site and inhibit catalysis by an allosteric mechanism (4, 5). Several putative allosteric binding sites on the surface of HCV NS5B have been suggested based on recent structural studies (6 -8), and several chemical classes of NS5B non-nucleoside inhibitors have ...

The mammalian transcription factor E2F plays a critical role in the expression of genes required for cellular proliferation. To understand how E2F is regulated, we have developed a reconstituted in vitro transcription assay. Using this E2F-responsive assay, we can demonstrate that E2F-mediated transcription can be directly repressed by the tumor suppressor protein pRB. This inhibition is abolished by phosphorylation of pRB with either cyclin A/cdk2 or cyclin E/cdk2. However, these cyclin/kinase complexes exhibit differences in the ability to phosphorylate E2F. Only cyclin A/cdk2 can phosphorylate E2F effectively, and this phosphorylation abolishes its ability to bind DNA and mediate trans-activation. Thus, this in vitro transcriptional assay allows activation and inactivation of E2F transcription, and our findings demonstrate how transcriptional regulation of E2F can be linked to cell cycle-dependent activation of kinases.

Acidic transcriptional activation domains function well in both yeast and mammalian cells, and some have been shown to bind the general transcription factors TFIID and TFIIB. We now show that two acidic transactivators, herpes simplex virus VP16 and human p53, directly interact with the multisubunit human general transcription factor TFIHH and its Saccharomyces cerevisuze counterpart, factor b. The VP16-and p53-binding domains in these factors lie in the p62 subunit of TFIIH and in the homologous subunit, TFB1, of factor b. Point mutations in VP16 that reduce its transactivation activity in both yeast and mammalian cells weaken its binding to both yeast and human TFIIH. This suggests that binding of activation domains to TFIIH is an important aspect of transcriptional activation.Accurate transcription in vitro by human RNA polymerase II involves the general transcription factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF (RAP30 and RAP74), TFIIH (also known as BTF2), and TFIIJ (reviewed in references 13 and 121). Beginning with TFIID, which recognizes the TATA boxes present in many promoters for RNA polymerase II, these factors and RNA polymerase II can be assembled in a defined order onto a promoter (5). TFIIH and TFIIJ are the last of these factors to bind to an assembling initiation complex (12,14,26), and TFIIH, also known as BTF2, is the only factor known to possess associated enzymatic activities. These include an ATP-dependent DNA helicase activity (95, 96) and a protein kinase activity that can phosphorylate the carboxyterminal heptapeptide repeat domain (CTD) of the largest subunit of RNA polymerase 11 (12, 21,68 herpes simplex virus transactivator VP16 (107) and in the N-terminal 73 amino acids of the mammalian tumor suppressor protein p53 (23). The p53 protein has a site-specific DNA-binding domain in its carboxy-terminal portion (101). VP16 does not, by itself, bind specifically to DNA, but instead its amino-terminal portion binds DNA in association with mammalian factors, including the POU homeodomain protein Oct-1 that recognizes octamer sequences in DNA (28,60,102). When fused to the DNA-binding domain of GAL4, the VP16 and p53 activation domains stimulate transcription in yeast and human cells of a gene bearing GAL4-binding sites (9,16,23,74,87,92). These observations imply that common mechanisms for transcriptional activation by acidic activators may exist in fungi and mammals.Many activation domains have been shown to interact with the TATA-box-binding protein (TBP) subunit of TFIID. These include the highly acidic activation domains in VP16 (50,103), p53 (10,67,72,84,97,108),118), and E2F-1 (37), as well as other kinds of activation domains found in the adenovirus activator ElA (48, 62), the Epstein-Barr virus proteins Zta (64) and R (70), the human T-cell leukemia virus type 1 (HTLV-1) activator Taxl (8), the transactivator Tat of human immunodeficiency virus type 1 (HIV-1) (53), and human c-Fos and c-Jun (85), PU-1 (38), and Spl (20). In certain cases, reduced binding of TBP by activation domains wit...

The two forms of RNA polymerase H that exist in vivo, phosphorylated (HO) and nonphosphorylated (HA), were purified to apparent homogeneity from HeLa cells. The nonphosphorylated form preferentially binds to the preinitiation complex. RNA polymerase H in the complex was converted by a cellular protein kinase to the phosphorylated form.Purified RNA polymerase II cannot accurately initiate transcription from class II promoters in vitro unless it is supplemented with general transcription initiation factors (1-3). Seven human general transcription factors (TFIIA, -IIB, -IID, -lIE, -IIF, -IIG, and -IIH) that, together with RNA polymerase II are sufficient for specific transcription, have been identified (O.F. and D.R., unpublished results).Despite progress in the purification of the general transcription factors, determination of their individual activities and contributions to the formation of a transcriptioncompetent complex remains obscure. Complicating these analyses is the existence in eukaryotic cells of two different forms of RNA polymerase II, IIA and IIO, which differ in the level of phosphorylation of a highly conserved heptapeptide repeat present at the carboxyl terminus of the largest subunit (carboxyl-terminal domain; CTD) (4-6). The heptapeptide repeat is essential for viability (7-10); nevertheless, a third species ofRNA polymerase II lacking the CTD (IIB) has been observed in vitro (11)(12)(13)(14). Photoaffinity labeling experiments demonstrated that nascent RNA transcripts crosslink almost exclusively to the phosphorylated IIO form in vivo and in vitro (5,15,16), suggesting that it is the IIO polymerase that elongates RNA chains. Monoclonal antibodies against the nonphosphorylated IIA form inhibited specific transcription initiation (17, 18), suggesting that RNA polymerase IIA was more active than IIO during specific transcription initiation in vitro. Therefore, the CTD phosphorylation state may regulate the transition from initiation to elongation (19).We have purified human RNA polymerases IIO and IIA to apparent homogeneity and analyzed their roles in transcription. We demonstrate that the IIA form associates preferentially with the preinitiation complex where it is then converted by a cellular protein kinase to the phosphorylated 11O form. MATERIALS AND METHODSTranscription Factors, Transcription Reaction Mixtures, and DNA Binding Assays. Transcription reactions and DNA binding assays were performed as described (20). Transcription factor IIA (TFIIA) (P. Cortes and D.R., unpublished data), TFIIB (22), TFIIE (23), and TFIIF (24) were purified from HeLa cell nuclear extracts as described. TFIID was purified to homogeneity as described (20) Purification of the Phosphorylated (HO) and Nonphosphorylated (HA) Forms of RNA Polymerase H. RNA polymerase II was purified from HeLa cell nuclear pellets (7.5 x 1010 cells). Enzyme solubilization and chromatography on DE-52 were as described (26). Active fractions ofthe DE-52 column, between 0.2 and 0.3 M salt, were pooled (109 mg of protein, 170 ml...