-13 cells potentiated transcriptional activation by the glucocorticoid receptor, which is known to require SWI-SNF function. BRG1 also specifically repressed transcription from a transfected c-fos promoter and correspondingly blocked transcriptional activation of the endogenous c-fos gene. Mutation of lysine residue 798 in the DNA-dependent ATPase domain of BRG1 significantly reduced its ability to repress c-fos transcription. Repression by BRG1 required the cyclic AMP response element of the c-fos promoter but not nearby binding sites for Sp1, YY1, or TFII-I. Using human C33A cervical carcinoma cells, which lack BRG1 and also express a nonfunctional Rb protein, transcriptional repression by BRG1 was weak unless wild-type Rb was also supplied. Interestingly, Rb-dependent repression by BRG1 was found to take place through a pathway that is independent of transcription factor E2F.The modification of chromatin structure is increasingly recognized to be an important facet of transcriptional regulation. Such alterations likely occur in concert with the actions of the general transcription factors and promoter-specific activators and repressors in order to allow regulatory changes to take place. Several multiprotein complexes with roles in this kind of regulation have been identified, including the yeast SAGA (23,72) and RSC (9) complexes, the Drosophila ISWI-containing complexes (16,29,67,69), and the yeast and human SWI-SNF complexes (ySWI-SNF and hSWI-SNF) (8,14,28,32,35,37,50,55,73,74). The regulation of chromatin structure is likely to influence specific cellular processes that rely heavily on transcriptional events, including the control of cellular proliferation.Several studies have suggested a role for hSWI-SNF components BRG1 (32), hBRM (50), and hSNF5-INI1 (49) in the control of cellular proliferation. BRG1 and hBRM, human homologues of yeast SNF2, bind to members of the retinoblastoma (Rb) tumor suppressor protein family and can trigger cellular growth arrest (15,32,61,62). Recently, the hSNF5-INI1-encoding gene was found to be mutated in multiple malignant rhabdoid tumors, strongly suggesting that hSWI-SNF has a tumor suppressor function (71). In addition, we have reported that the N-terminal domain of the adenovirus E1A oncoprotein specifically blocks SWI-SNF-dependent transcription in budding yeast, suggesting that disruption of human and mouse SWI-SNF function may be important in oncogenic transformation by E1A (46).The ySWI-SNF and hSWI-SNF complexes have been studied extensively in vitro and have been demonstrated to trigger chromatin remodeling and facilitation of sequence-specific DNA binding (reviewed in references 7, 31, and 35). In addition, genetic approaches have resulted in the identification of several yeast cellular genes whose transcriptional regulation requires a functional SWI-SNF complex (10,38,39,41,56). In higher eukaryotic systems, transfected promoters under the control of the glucocorticoid receptor (GR) (17, 50, 61) or transcription factor E2F (66) have been shown to be regulated b...

E2F is a cellular, sequence-specific DNA-binding factor that binds to pairs of sites that occur upstream of the E1A and E2 early mRNA cap sites. During adenovirus infection, there is induction of a form of E2F that binds cooperatively to the pair of sites in the E2 control region. Production of the infection-specific E2F activity is dependent on early region 4 (E4), as extracts of cells infected with a mutant that lacks E4 did not contain this activity. Instead, two new forms of E2F were seen with the E4 mutant. Infection with mutant viruses unable to make E1A gene products produced the wild-type infection-specific E2F activity after a delay. Mutations in the E1B-55 kD-, E1B-21 kD-, E2-72 kD-, and E3-coding regions had no effect on production of infection-specific E2F. Analysis of cell lines confirmed the results obtained with mutant viruses. Cells that expressed E1A but not E4 genes (e.g., 293 cells) did not contain infection-specific E2F. Cell lines that expressed the E4 gene contained the activity. These observations demonstrate that E4 participates in the infection-induced change in E2F-binding activity. The data are consistent with E1A playing an indirect role in the process by mediating the efficient expression of E4 gene products which, in turn, induce the alteration in E2F activity.

The innate immune system guards against virus infection through a variety of mechanisms including mobilization of the host interferon system, which attacks viral products mainly at a posttranscriptional level. The influenza virus NS1 protein is a multifunctional facilitator of virus replication, one of whose actions is to antagonize the interferon response. Since NS1 is required for efficient virus replication, it was reasoned that chemical inhibitors of this protein could be used to further understand virus-host interactions and also serve as potential new antiviral agents. A yeast-based assay was developed to identify compounds that phenotypically suppress NS1 function. Several such compounds exhibited significant activity specifically against influenza A virus in cell culture but had no effect on the replication of another RNA virus, respiratory syncytial virus. Interestingly, cells lacking an interferon response were drug resistant, suggesting that the compounds block interactions between NS1 and the interferon system. Accordingly, the compounds reversed the inhibition of beta interferon mRNA induction during infection, which is known to be caused by NS1. In addition, the compounds blocked the ability of NS1 protein to inhibit double-stranded RNA-dependent activation of a transfected beta interferon promoter construct. The effects of the compounds were specific to NS1, because they had no effect on the ability of the severe acute respiratory syndrome coronavirus papainlike protease protein to block beta interferon promoter activation. These data demonstrate that the function of NS1 can be modulated by chemical inhibitors and that such inhibitors will be useful as probes of biological function and as starting points for clinical drug development.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, three highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,8,33,42,(52)(53)(54)61). Protein VII is the major protein component of the core, with an estimated 1,070 copies present per virion (20). Along with , it is bound noncovalently to the DNA in a sequence-independent manner (2, 6, 36, 55). Protein V contacts the DNA as well and also acts as a bridge between protein VII and the outer capsid (19,55). Protein IVa2 makes sequence-specific contacts with the viral DNA packaging sequence and is thought to play a role in DNA packaging (64). Salt-extracted preparations of the core contain only DNA and protein VII, suggesting that this protein is the most tightly DNA bound of all core proteins (60). Similarly, Sarkosyl preparations of the core contain predominantly DNA and protein VII (8).Structural features of the DNA-protein complex within the adenovirus capsid and during infection remain largely unknown. DNA within the capsid is in a highly compact configuration, and electron microscopy studies of purified viral core reveal structures reminiscent of beads on a string, or higherorder chromatin compaction, depending on the method of preparation (8,18,45,49,50,60,63). Nuclease digestion of core preparations results in discrete populations of prote...

Adenovirus protein VII is the major component of the viral nucleoprotein core. It is a highly basic nonspecific DNA-binding protein that condenses viral DNA inside the capsid. We have investigated the fate and function of protein VII during infection. "Input" protein VII persisted in the nucleus throughout early phase and the beginning of DNA replication. Chromatin immunoprecipitation revealed that input protein VII remained associated with viral DNA during this period. Two cellular proteins, SET and pp32, also associated with viral DNA during early phase. They are components of two multiprotein complexes, the SET and INHAT complexes, implicated in chromatin-related activities. Protein VII associated with SET and pp32 in vitro and distinct domains of protein VII were responsible for binding to the two proteins. Interestingly, protein VII was found in novel nuclear dot structures as visualized by immunofluorescence. The dots likely represent individual infectious genomes in association with protein VII. They appeared within 30 min after infection and localized in the nucleus with a peak of intensity between 4 and 10 h postinfection. After this, their intensity decreased and they disappeared between 16 and 24 h postinfection. Interestingly, disappearance of the dots required ongoing RNA synthesis but not DNA synthesis. Taken together these data indicate that protein VII has an ongoing role during early phase and the beginning of DNA replication.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, the highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,5,19,25,31,33,34,42). Protein VII is the major protein component of the core with an estimated 1,070 copies present per virion (9) and is primarily responsible for establishing viral chromatin structure. It can potently condense DNA in vitro and in vivo and also repress transcription (3,6,21,35). This is consistent with the highly condensed configuration of viral chromatin found within the virion. When delivered to the nucleus, the chromatin is silent prior to stimulation by the viral transcriptional activator E1A (13).We and others have reported that protein VII from infectious viral particles enters the nucleus along with viral DNA and remains associated with it, suggesting that the protein VII-DNA complex is the substrate for transcriptional activation by E1A during early phase (8,15,18,21). Moreover we found that protein VII can associate with E1A protein in vitro (21).In this report we have studied the fate and function of protein VII during the early phase of infection. We demonstrate that virus-derived "input" protein VII persists in the nucleus throughout early phase and the beginning of viral DNA replication, suggesting that it has an ongoing role in gene regulation and perhaps DNA replication. During this period protein VII is found in discrete nuclear dot structures and viral DNA continues to associate with protein VII.We have also investigated the association of protein VII with cel...

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.

The transcriptional control regions of several E1A-inducible adenovirus early genes contain sequences similar to the cAMP response element of several cellular cAMP-inducible genes. The cAMP-responsive cell line $49 was infected with wild-type adenovirus and found to contain elevated levels of mRNAs encoded by all early genes tested (E4, E1A, and E1B), following treatment with dibutyryl cAMP. This effect was at the level of transcriptional activation. The effect of cAMP on E4 and E1A transcription was greater in cells infected with wild-type virus than in cells infected with virus that lacked functional E1A proteins, cAMP in combination with E1A generated a greater induction than the product of the increases achieved by each inducer alone. Therefore, cAMP acted in synergy with E1A to induce maximally transcription of the E4 and E1A genes. These data suggest that E1A or E1A-stimulated events can interact functionally with targets of cAMP signaling in the cell to induce transcription of the adenovirus early genes.

Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is a nonspecific DNA-binding protein that condenses viral DNA inside the capsid. Protein VII remains associated with viral chromatin throughout early phase, indicating its continuing role during infection. Here we characterize the release of protein VII from infectious genomes during a time period that corresponds to the late phase of infection. Interestingly, the early viral transactivator E1A, but not other early gene products, is responsible for releasing protein VII by a mechanism that requires ongoing transcription but not viral DNA replication. Moreover transcription per se, in the absence of E1A, is also sufficient to trigger release. Accordingly, a recombinant genome containing only non-coding "stuffer" DNA is unable to support release of protein VII. Our data support a model in which early gene transcription results in a change in the structure of the viral chromatin.