No abstract

p300 and the closely related CREB binding protein (CBP) are transcriptional adaptors that are present in intracellular complexes with TATA binding protein (TBP) and bind to upstream activators including p53 and nuclear hormone receptors. They have intrinsic and associated histone acetyltransferase activity, suggesting that chromatin modification is an essential part of their role in regulating transcription. Detailed characterization of a panel of antibodies raised against p300/CBP has revealed the existence of a 270-kDa cellular protein, p270, distinct from p300 and CBP but sharing at least two independent epitopes with p300. The subset of p300/CBP-derived antibodies that cross-reacts with p270 consistently coprecipitates a series a cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Purification and analysis of various proteins in this group reveals that they are components of the human SWI/SNF complex and that p270 is an integral member of this complex.The cellular protein p300 is a direct target of the transforming functions of the adenovirus E1A gene (19, 51) and as such is implicated in the regulation of both cell cycle-specific and tissue-specific gene expression (18,27,38,43,46,52,54). p300 is highly homologous (about 64% identical) to the cyclic AMP response element binding protein (CREB) coactivator, CBP (CREB binding protein) (5,11,17,30,34). Both p300 and CBP are present in intracellular complexes with the TATA binding protein (TBP) (1, 13). Both act as cofactors for p53 (6,21,33,44) and nuclear hormone receptors (9,24,28). Both also contain intrinsic and associated histone acetyltransferase (HAT) activity (39, 53), suggesting that chromatin modification is an essential part of their role in regulating transcription.A recent detailed characterization of a panel of antibodies raised against a mixture of native p300 and CBP revealed the existence of a 270-kDa cellular protein, distinct from p300 and CBP but sharing at least two independent antigenic determinants with p300 (13). Four of the eleven antibodies in the panel recognize p270. The subset of p300/CBP-derived antibodies that recognizes p270 consistently coprecipitates a series of cellular proteins with relative molecular masses ranging from 44 to 190 kDa. Typical of these is the antibody designated NM1, whose immunoprecipitation pattern is shown in Fig. 1. TBPspecific antibodies coprecipitate a subset of these proteins including p300, CBP, and the phosphoprotein species indicated in Fig. 1 as p64 and p59 (1, 13). Because the TBP-specific antibodies do not coprecipitate all of the p300 family-associated proteins, it is likely that the array of proteins seen in Fig. 1 represents more than one intracellular complex.We have now identified four of the remaining p300/CBP/ p270-associated proteins as members of another important cellular complex: the mammalian SWI/SNF complex. The 190-kDa protein visible in the p300-related complex is BRG1, the human homolog of the yeast transcriptional activator, SWI2/ SNF2. The 170-and 155-k...

A direct comparison of the inhibitory effects of alpha, beta, and gamma interferons (IFNs) on replication of a hepatitis C virus subgenomic replicon in a hepatoma cell line revealed similarities in antiviral potency. However, alternate IFN-induced antiviral mechanisms were suggested following observations of striking differences between IFN-␥ and IFN-␣/␤ with respect to strength and durability of the antiviral response and the magnitude and pattern of IFN-mediated gene expression.Interferons (IFNs) are a family of cytokines that share the property of inducing proteins that inhibit virus replication (23). IFNs are not constitutively synthesized by cells but respond to external stimuli, such as virus infections, that trigger their transient synthesis and secretion. Binding of IFN to its receptor elicits signals that induce the transcription of a specific set of genes. These genes encode proteins that carry out the diverse functions of the IFN system including mediating antiviral activity. IFNs are classified as either alpha/beta IFN (IFN-␣/␤) or IFN-␥. IFN-␣ and -␤ are readily induced by double-stranded viral RNA, primarily in leukocytes and fibroblasts. These IFNs then induce some of the known IFN-responsive antiviral effector mechanisms, including (i) 2Ј,5Јoligoadenylate synthetase (2Ј,5Ј-OAS) (which then activates the constitutively expressed RNase L to degrade viral RNAs), (ii) double-stranded RNAactivated protein kinase (which inhibits viral protein synthesis), and (iii) MxA (a GTPase that blocks transport of viral ribonucleoproteins to the nucleus) (23). The IFN-␣/␤-regulated genes induce direct antiviral activity as part of the early or innate host immune response to limit viral infection. The host adaptive antiviral response mediated by CD8 ϩ cytotoxic T lymphocytes and natural killer cells and by virus-specific neutralizing antibodies then eradicates virus and virus-infected cells. IFN-␥ is induced as part of the adaptive immune response by a wide variety of stimuli and is restricted to T cells and natural killer cells (4). Little is known about the direct antiviral activity of IFN-␥, particularly with respect to the mechanism, magnitude, and kinetics of its antiviral activity in comparison with IFN-␣ and -␤.Hepatitis C virus (HCV), a member of the Flaviviridae family, has a single-stranded positive-sense RNA genome encoding a polyprotein of at least 10 distinct gene products (5). Infections caused by HCV are a major medical concern since as much as 3% of the worldwide population is estimated to be HCV seropositive (1). At present, the most widely prescribed HCV antiviral therapy is the combination of polyethylene glycol-modified IFN-␣2b (PEG-Intron) and ribavirin (18), a treatment capable of inducing sustained virologic responses in 54 to 56% of chronically infected patients (11a, 18). Although this is a significant improvement from the sustained response rate of IFN-␣ monotherapy (6 to 16%) (7, 21, 24) and IFN-␣ plus ribavirin (40%) (8, 19), improvement in sustained response rates still remains the majo...

Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3D pol ) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3D pol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3D po1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.Rhinovirus infections are the leading cause of the common cold (35). There are over 100 serotypes of human rhinovirus (HRV), the majority of which utilize intercellular adhesion molecule 1 as their host-cell receptor. HRV serotype 16 (HRV16) from this major receptor group is a suitable virus for clinical study (26) and can serve as a model for studying the transmission of colds and virus-induced asthma (6). Rhinoviruses are positivestrand, nonenveloped RNA viruses and are members of the Picornaviridae family, which comprise one of the largest families of viral pathogens responsible for many serious human and animal diseases including the common cold, hepatitis, poliomyelitis, and foot-and-mouth disease.HRV16 contains a genome comprised of 7,124 nucleotides and includes a 5Ј nontranslated region, a single open reading frame, a 3Ј nontranslated region, and a poly(A) tail of approximately 40 nucleotides. The open reading frame encodes a single polyprotein of 2,152 residues (

Pyridine derivatives R 0380Identification and Structure-Activity Relationships of Substituted Pyridones as Inhibitors of Pim-1 Kinase. -Chemical library hits along with synthesized derivatives of these compounds act as potent inhibitors of Pim-1 kinase. A complex crystal structure and the mechanism of action are defined for pyridone (IIIa), the most potent Pim-1 inhibitor. Preliminary data further indicate that (IIIa) lacks in vitro activity towards related serine/threonine kinases Pim-2 and MEK1/2. Small molecules similar to (III) may serve as starting scaffolds for the development. -(CHENEY*, I. W.; YAN, S.; APPLEBY, T.; WALKER, H.; VO, T.; YAO, N.; HAMATAKE, R.; HONG, Z.; WU,