host factors ͉ plus-stranded RNA virus ͉ tomato bushy stunt tombusvirus ͉ virus replication ͉ yeast-knockout strains T he success of viruses as pathogens of humans, animals, and plants depends on the viruses' ability to reprogram the host-cell metabolism to support the infection. The virus-host interaction is more complex than the term ''reprogramming'' suggests because host cells have antiviral defense mechanisms. Identifying host genes that can affect virus replication and the infection process is central to understanding the complex role of the host in viral infections. The largest group of viruses, the positive-strand RNA viruses, which include the severe acute respiratory syndrome (SARS) coronavirus and hepatitis C and West Nile viruses, has virion RNAs that are directly translated in the infected cell. Synthesized viral proteins and recruited host proteins mediate processes that lead to efficient multiplication of the viral RNA (1, 2). RNA viruses are important not only as infectious agents but also as tools in biotechnology and gene therapy for expressing selected proteins in cells (3)(4)(5).Yeast is a model eukaryotic cell that has been used extensively to study the roles of individual genes in cellular processes based on genome-wide screens (6-9). Many screens have been based on the yeast single-gene-knockout (YKO) library, because the role of each nonessential yeast gene can be tested for selected functions (10). We and others have developed systems for inducing yeast cells to support the replication of certain positive-strand RNA viruses or their surrogates (11)(12)(13)(14). Here, we apply our previously developed system for robust replication of a small RNA replicon of the tomato bushy stunt virus (TBSV) in yeast (13,15) to screen the entire YKO library for genes influencing the efficiency of viral replication. A total of 96 YKO strains were identified. The identified host genes are either involved in many cellular processes, including nucleic acid, protein, and lipid metabolism, protein targeting͞transport, and general and stress metabolism or have unknown functions. Our results show that the replication of positive-strand RNA viruses of different supergroups is influenced by distinct groups of genes and that TBSV replication is associated with sets of genes that also have been associated with certain human disease states. Materials and MethodsYeast Strains and Expression Plasmids. Yeast strain 4741 and the YKO deletion series (10) were obtained from Open Biosystems (Huntsville, AL). Yeast transformation was modified from the standard lithium acetate (LiOAc)͞polyethylene glycol (PEG) protocol (16) to facilitate a 96-well plate format. Briefly, yeast strains were grown overnight in yeast extract͞peptone͞dextrose medium supplemented with 200 mg͞liter geneticin G418. Cultures were then diluted to Ϸ0.3 OD 600 in fresh medium (0.25 ml per well) and cultured for an additional 4 h at 30°C. The cells were pelleted, washed with sterile water, and resuspended in 0.1 M LiOAc. This procedure was followed by rep...