The NF-B p50/p50 homodimer is mainly associated with transcriptional repression. Previously, we demonstrated that phosphorylation of NF-B p50 Ser 337 is critical for DNA binding. Here, we report that p50 Ser 337 is constitutively phosphorylated by the protein kinase A catalytic subunit (PKAc) in three different cell types, which may account for the constant binding of p50/p50 to DNA in unstimulated cells. This was demonstrated first by showing that treatment of cells with PKAc-specific inhibitors blocked p50/p50 DNA binding. Second, phosphorylation of p50 by PKAc was prevented by substitution of Ser 337 to alanine. Third, both p50 and PKAc proteins as well as kinase activity that phosphorylates p50 were found to co-fractionate following gel filtration chromatography. Finally, PKAc and p50 were shown to be able to reciprocally co-immunoprecipitate one another, and their physical association was blocked by a PKA catalytic site inhibitory peptide. This indicates that phosphorylation of p50 Ser 337 involves direct contact with the PKAc catalytic center. In contrast to the dramatic elevation of nuclear p50/p65 heterodimers induced by tumor necrosis factor ␣, DNA binding of p50/ p50 homodimers was not greatly altered. Taken together, these findings reveal for the first time that there is a direct interaction between PKAc and p50 that accounts for constitutive phosphorylation of p50 Ser 337 and the existence of DNA bound p50/p50 in the nuclei of most resting cells. This mechanism of DNA binding by p50/p50 following phosphorylation of Ser 337 by PKAc may represent an important means for maintaining stable negative regulation of NF-B gene expression in the absence of extracellular stimulation.The nuclear factor NF-B is a transcription factor identified by Sen and Baltimore nearly 20 years ago (1). NF-B plays a critical role in transcription regulation of genes involved in immune response, inflammation, cell proliferation, differentiation, apoptosis, and oncogenesis (2-6). In vertebrates, five members of NF-B family have been identified. These include RelA (p65), RelB, c-Rel, NF-B1 (p50/p105), and NF-B2 (p52/ p100). All of these proteins share a highly conserved Rel homology domain termed RHD within the N-terminal 300 amino acid region. The RHD is responsible for DNA binding, dimerization, nuclear translocation, and interaction with IB inhibitory proteins. Although all NF-B family members can bind to DNA, only p65, RelB, and c-Rel contain a transactivation domain in their C-terminal regions.The NF-B family members can form various homodimers and heterodimers among which the p50/p65 heterodimer is the most abundant and studied species. In most resting or unstimulated cells, p50/p65 heterodimers are confined in the cytoplasm in an inactive form by forming a complex with IB proteins. Treatment of cells with NF-B stimuli such as cytokines, mitogens, and bacterial lipopolysaccharide leads to phosphorylation of IB by IB kinase and subsequent ubiquitination and degradation of IB by the 26 S proteasome (7,8). This allows NF-B to ...

It has been demonstrated that phosphorylation of the p50 subunit of NF-B is required for efficient DNA binding, yet the specific phospho-residues of p50 have not been determined. In this study, we substituted all of the serine and conserved threonine residues in the p50 Rel homology domain and identified three serine residues, Ser 65 , Ser 337 , and Ser 342 , as critical for DNA binding without affecting dimerization. Although substitution with negatively charged aspartic acid at each of these positions failed to restore DNA binding, substitution with threonine, a potential phospho-acceptor, retained DNA binding for residues 65 and 337. In particular, Ser 337 , in a consensus site for protein kinase A (PKA) and other kinases, was shown to be phosphorylated both in vitro and in vivo. Importantly, phosphorylation of Ser 337 by PKA in vitro dramatically increased DNA binding of p50. This study shows for the first time that the DNA binding ability of NF-B p50 subunit is regulated through phosphorylation of residue Ser 337 , which has implications for both positive and negative control of NF-B transcription.The transcription factor NF-B acts as a central regulator of inflammatory, immune, and stress responses by controlling gene expression of cytokines, chemokines, immunoreceptors, antigen-presenting proteins, growth factors, transcription factors, cell adhesion molecules, stress response proteins, and apoptotic regulators (1, 2). Members of Rel/NF-B transcription factor family include Dorsal, Relish, and Dif in Drosophila and p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100 in vertebrates. All of these proteins have a highly conserved DNAbinding and dimerization domain called the Rel homology domain. The vertebrate Rel family proteins can form homodimers or heterodimers that bind to 10-basepair B sites in the promoters of NF-B target genes (1, 3, 4). The most common and important NF-B transactivating species is the p50/p65 heterodimer. The p65 subunit, which contains a transactivation domain in the C terminus of the Rel homology domain, is responsible for the ability of NF-B to stimulate transcription. By contrast, p50 subunit, which lacks a transactivation domain, functions mainly in NF-B DNA binding (5-9). Another important dimeric species, the p50/p50 homodimer, serves mainly as a negative regulator of NF-B activity through competing with p50/p65 for NF-B response elements on DNA and through its association with co-repressor histone deacetylase (10, 11). The x-ray crystal structures of p50/p65 heterodimer and p50/p50 and p65/p65 homodimer binding to DNA have revealed a conformation often referred to as a "butterfly" (12-16). The DNA recognition loop (L1) in the N-terminal half of NF-B Rel homology domain mediates base-specific DNA contacts, whereas the C-terminal half is responsible for dimerization and nonspecific DNA contacts (17).NF-B activity is regulated by nuclear translocation. In most cell types, p50/p65 heterodimers exist in the cytoplasm as an inactive form associated with the inhibitor protein, I B. A w...

The 3 ends of RNAs associated with turnip crinkle virus (TCV), including subviral satellite (sat)C, terminate with the motif CCUGCCC-3. Transcripts of satC with a deletion of the motif are repaired to wild type (wt) in vivo by RNA-dependent RNA polymerase (RdRp)-mediated extension of abortively synthesized oligoribonucleotide primers complementary to the 3 end of the TCV genomic RNA. Repair of shorter deletions, however, are repaired by other mechanisms. SatC transcripts with the 3 terminal CCC replaced by eight nonviral bases were repaired in plants by homologous recombination between the similar 3 ends of satC and TCV. Transcripts with deletions of four or five 3 terminal bases, in the presence or absence of nonviral bases, generated progeny with a mixture of wt and non-wt 3 ends in vivo. In vitro, RdRp-containing extracts were able to polymerize nucleotides in a template-independent fashion before using these primers to initiate transcription at or near the 3 end of truncated satC templates. The nontemplate additions at the 5 ends of the nascent complementary strands were not random, with a preference for consecutive identical nucleotides. The RdRp was also able to initiate transcription opposite cytidylate, uridylate, guanylate, and possibly adenylate residues without exhibiting an obvious preference, flexibility previously unreported for viral RdRp. The unexpected existence of three different repair mechanisms for TCV suggests that 3 end reconstruction is critical to virus survival.satellite RNA ͉ RNA replication

Smallpox constitutes a major bioterrorism threat, which underscores the need to develop antiviral drugs for rapid response in the event of an attack. Viral processivity factors are attractive drug targets in being both specific and essential for their cognate DNA polymerases to synthesize extended strands of DNA. An in silico model of the vacinnia virus processivity factor, comprised of the A20 and D4 heterocomplex, was constructed and used for lead optimization of an indole-based scaffold identified earlier from a high-throughput screening. On the basis of this model, a new class of potent antivirals against vaccinia virus was designed and synthesized, of which two (24a and 24b) exhibited superior improvement over the parent scaffold (IC50 = 42 and 46 vs 82000 nM, respectively). The ability of 24a to suppress vaccinia DNA synthesis is supported by the inhibition of late viral gene expression, as well as by the diminished incorporation of bromodeoxyuridine into viral replication factories.

Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3'-proximal element (3'-UCCCAAAGUAU) located 11 bases from the 3' terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5'-proximal element, the 3'-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3' minus-strand terminal sequence "3'-OH-CCCUAU," which contains the minus-strand 3'-end sequence "3'-OH-CC(1-2)(A/U)(A/U)(A/U)" found in all carmovirus RNAs. Single mutations in the 3'-terminal sequence, which we have named the carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3'-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3'-terminal 21 bases revealed that all satC species accumulating in plants contained the 3' CCS. In addition, the 3'-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.