Brome mosaic virus (BMV), a member of the alphavirus-like superfamily of human-, animal-, and plant-infecting (ϩ)RNA viruses, has been studied as a model for viral RNA replication, encapsidation, recombination, and other processes (3). BMV has three genomic RNAs. RNAs 1 and 2 encode the interacting, multifunctional 1a helicase-like and 2a polymerase RNA replication factors (4, 5), which form endoplasmic reticulum (ER) membrane-associated RNA replication complexes with functional similarities to the replicative cores of retrovirus and double-strand (ds)RNA virus virions (6). RNA3 encodes protein 3a that enables infection spread between cells in natural hosts. The negative-strand [(Ϫ) RNA]3 replication intermediate also serves as a template for synthesis of a subgenomic (sg) mRNA, RNA4, which encodes the viral coat protein (Fig. 1A).The yeast Saccharomyces cerevisiae has proven a valuable model for normal and disease processes in human and other cells. The unusual ability of BMV to direct its genomic RNA replication, gene expression, encapsidation, and other processes in this yeast (7,8) has allowed traditional yeast mutagenic analyses that have identified host genes involved in multiple steps of BMV RNA replication and gene expression. Such host genes encode a wide variety of functions and contribute to diverse replication steps, including supporting and regulating viral translation, selecting and recruiting viral RNAs as replication templates, activating the RNA replication complex through chaperones, and providing a lipid profile compatible with membrane-associated viral RNA replication (9-14; reviewed in refs. 2 and 15).Here, we sought to develop a more rapid, global method to systematically identify yeast host factors with effects on BMV RNA replication by using an ordered array of yeast deletion strains (16) to assay virus replication in the absence of each of Ϸ4,500 yeast factors, which is Ϸ80% of the yeast genome. We describe screening this deletion array by using a whole-cell assay based on BMV-directed Renilla luciferase (Rluc) expression by pathways dependent on viral RNA replication and viral RNAdirected sg mRNA synthesis. The assay identified nearly 100 host genes whose absence repressed or enhanced BMV-directed Rluc expression by 3-to 25-fold. The results provide a significantly expanded view of virus-host interactions and should advance understanding of virus and cell pathways. Materials and MethodsYeast. YMI04 and ded1i yeast were described (11). Strains BY4743 (WT; ref. 17) and the homozygous diploid deletion series (BY4743 strain background; ref. 16) were from Research Genetics (Huntsville, AL). Standard yeast techniques were used (18), except for 96-well transformations, which were based on a one-step procedure (19). Briefly, yeast were grown to saturation overnight at 30°C in 96-well plates (1.2 ml per well), pelleted, suspended in 100 l of transformation mix (0.18 M LiAc, pH 5.5, 36% polyethylene glycol-3350, 90 mM DTT, 0.5 mg͞ml sheared salmon sperm DNA, and 20 g͞ml of each plasmid), incubate...
Reduced cell surface levels of major histocompatibility complex class I antigens enable adenovirus type 12 (Ad12)-transformed cells to escape immunosurveillance by cytotoxic T lymphocytes (CTL), contributing to their tumorigenic potential. In contrast, nontumorigenic Ad5-transformed cells harbor significant cell surface levels of class I antigens and are susceptible to CTL lysis. Ad12 E1A mediates down-regulation of class I transcription by increasing COUP-TF repressor binding and decreasing NF-B activator binding to the class I enhancer. The mechanism underlying the decreased binding of nuclear NF-B in Ad12-transformed cells was investigated. Electrophoretic mobility shift assay analysis of hybrid NF-B dimers reconstituted from denatured and renatured p50 and p65 subunits from Ad12-and Ad5-transformed cell nuclear extracts demonstrated that p50, and not p65, is responsible for the decreased ability of NF-B to bind to DNA in Ad12-transformed cells. Hypophosphorylation of p50 was found to correlate with restricted binding of NF-B to DNA in Ad12-transformed cells. The importance of phosphorylation of p50 for NF-B binding was further demonstrated by showing that an NF-B dimer composed of p65 and alkaline phosphatase-treated p50 from Ad5-transformed cell nuclear extracts could not bind to DNA. These results suggest that phosphorylation of p50 is a key step in the nuclear regulation of NF-B in adenovirus-transformed cells.All human adenoviruses are able to transform nonpermissive rodent cells in vitro. The viral E1A and E1B transforming genes are responsible for disruption of the cell cycle and prevention of apoptosis (reviewed in reference 56). Interestingly, only a subset of adenovirus serotypes, including adenovirus type 12 (Ad12), can induce the formation of tumors in immunocompetent rodents following inoculation of virus or transformed cells. The highly tumorigenic phenotype of Ad12 correlates with a sharp decrease in cell surface levels of the major histocompatibility complex (MHC) class I antigens (11,17,59). The diminished class I antigen expression on Ad12-transformed cells enables them to escape detection by cytotoxic T lymphocytes (CTL) and contributes to their tumorigenic potential (11,63,70). In contrast, significant cell surface expression of class I antigens on nontumorigenic Ad5-transformed cells allows for CTL recognition and lysis.E1A is the only gene of Ad12 required for down-regulated synthesis of class I antigens (67). The block in the expression of class I antigens is at the level of transcription (1, 20), and the 47-bp class I enhancer is the target of this transcriptional down-regulation (21, 32) (Fig. 1). In Ad12-transformed cells, the orphan nuclear hormone receptor COUP-TF is strongly bound to the R2 site of the enhancer (39). Additionally, the transcriptional activator NF-B is weakly bound to the R1 site of the enhancer in Ad12-transformed cells (2,38,43,46). The increased binding of COUP-TF and the decreased binding of NF-B to the enhancer are mediated by the first exon (residues 1 to 144...
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