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...

Rapid evolution of RNA viruses with mRNA-sense genomes is a major concern to health and economic welfare because of the devastating diseases these viruses inflict on humans, animals, and plants. To test whether host genes can affect the evolution of RNA viruses, we used a Saccharomyces cerevisiae single-gene deletion library, which includes Ϸ80% of yeast genes, in RNA recombination studies based on a small viral replicon RNA derived from tomato bushy stunt virus. The genome-wide screen led to the identification of five host genes whose absence resulted in the rapid generation of new viral RNA recombinants. Thus, these genes normally suppress viral RNA recombination, but in their absence, hosts become viral recombination ''hotbeds.'' Four of the five suppressor genes are likely involved in RNA degradation, suggesting that RNA degradation could play a role in viral RNA recombination. In contrast, deletion of four other host genes inhibited virus recombination, indicating that these genes normally accelerate the RNA recombination process. A comparison of deletion strains with the lowest and the highest recombination rate revealed that host genes could affect recombinant accumulation by up to 80-fold. Overall, our results demonstrate that a set of host genes have a major effect on RNA virus recombination and evolution.host factors ͉ plus-strand RNA virus ͉ tombusvirus ͉ yeast ͉ evolution R apid evolution of RNA viruses with mRNA-sense genomes, which include severe acute respiratory syndrome coronavirus, hepatitis C virus, and West Nile virus, makes controlling RNA viruses a difficult task. The emergence of new pathogenic RNA viruses is frequently due to RNA recombination (1, 2), which can lead to dramatic changes in viral genomes by creating novel combinations of genes, motifs, or regulatory RNA sequences. Thus, RNA recombination can change the infectious properties of RNA viruses and render vaccines and other antiviral methods ineffective (2). RNA recombination likely contributed to outbreaks with denguevirus (3, 4), poliovirus (5), calicivirus (6), astrovirus (7), enterovirus (8, 9), influenzavirus (10), pestivirus (11,12), and severe acute respiratory syndrome coronavirus, a newly emerged viral pathogen of humans (13)(14)(15). RNA recombination also is important in viral RNA repair, which likely increases the fitness of RNA viruses that lack proofreading polymerases (1,(16)(17)(18).Current models of RNA recombination are based on a templateswitching mechanism driven by the viral replicase (1, 16) or RNA breakage and ligation (19). The more common template-switching RNA recombination is thought to occur as an error during the replication process (1, 16). Because viral RNA replication depends not only on viral proteins but also on host factors (20), it is likely that host factors could affect the recombination process, too. However, despite the significance of RNA recombination in viral evolution, the possible roles of host genes in the viral RNA recombination process are currently unknown.Tombusviruses, including ...

Replication of plus-stranded RNA viruses requires many components of the host cells, including host proteins and intracellular membranes, which serve as sites of virus replication in infected cells (1,3,38). Accordingly, the virus-specific replicase complex (RC) consists of virus-and host-coded proteins and the viral RNA, which assemble on intracellular membranes into functional complexes. In addition, the viral replication proteins and host factors likely play roles in template selection for replication and recruitment (intracellular transport/targeting) of the viral RNA into replication (2,20). Host factors could also affect the stability/degradation of viral proteins and the viral RNA (4, 40, 41). Overall, viruses utilize/ depend on many diverse resources of the host cells.To identify the roles and/or effects of host genes on virus replication, systematic genome-wide screens were conducted in yeast, a model host, using the single-gene deletion library (YKO) with two distantly related plus-strand RNA viruses, namely Brome mosaic virus (BMV) and Tomato bushy stunt virus (TBSV) (12,27). These studies led to the identification of ϳ100 host genes for each virus that either stimulated or inhibited virus replication. Interestingly, most of the identified genes had a virus-specific effect, whereas only a small number of genes affected the replication of both BMV and TBSV. These observations suggest that BMV and TBSV, belonging to different supergroups within plus-stranded RNA viruses, could use and/or be affected by mostly different host factors (12, 27). Altogether, the above systematic screens tested only ϳ80% of all the known genes, which are not essential for yeast growth, whereas the effect of essential yeast genes remained untested.Tombusviruses, such as TBSV and Cucumber necrosis virus (CNV), are single-component RNA viruses of ϳ4,800 nucleotides (nt). Among the five virus-coded proteins, only two, termed p33 and p92 pol , are essential for TBSV replication (44). p92 pol is the viral RNA-dependent RNA polymerase (RdRp), whereas p33 replication cofactor (which overlaps with the Nterminal pre-readthrough segment of p92 pol ) is an RNA-binding protein (28,32,33). Earlier work defined that p33 is involved in template selection and recruitment of viral RNA into replication (18,25,32). These proteins interact with each other, the viral RNA, and the host proteins in cells (25,34,35,39), which leads to the assembly of RC on peroxisomal membranes (22, 25). The CNV replication proteins can support the replication of TBSV defective interfering (DI) RNA, a small deletion derivative of the genomic RNA, as efficiently as TBSV replication proteins can (19,24). A recent genome-wide screen of the YKO library for tombusvirus replication led to the identification of 96 host genes, whose separate deletions affected replication of a TBSV replicon RNA (repRNA), which is based on a DI RNA, in yeast (27). Based on the large number of host genes identified, the emerging picture is that the host likely plays a complex role in virus rep...

RNA recombination is a major process in promoting rapid virus evolution in an infected host. A previous genome-wide screen with the yeast single-gene deletion library of 4,848 strains, representing ϳ80% of all genes of yeast, led to the identification of 11 host genes affecting RNA recombination in Tomato bushy stunt virus (TBSV), a small model plant virus (E. Serviene, N. Shapka, C. P. Cheng, T. Panavas, B. Phuangrat, J. Baker, and P. D. Nagy, Proc. Natl. Acad. Sci. USA 102:10545-10550, 2005). To further test the role of host genes in viral RNA recombination, in this paper, we extended the screening to 800 essential yeast genes present in the yeast Tet-promoters Hughes Collection (yTHC). In total, we identified 16 new host genes that either increased or decreased the ratio of TBSV recombinants to the nonrecombined TBSV RNA. The identified essential yeast genes are involved in RNA transcription/metabolism, in protein metabolism/transport, or unknown cellular processes. Detailed analysis of the effect of the identified yeast genes revealed that they might affect RNA recombination by altering (i) the ratio of the two viral replication proteins, (ii) the stability of the viral RNA, and/or (iii) the replicability of the recombinant RNAs. Overall, this and previous works firmly establish that a set of essential and nonessential host genes could affect TBSV recombination and evolution.RNA viruses are successful pathogens because they are capable of rapid evolution that helps them to overcome host resistance and other antiviral strategies (13,14,17,27,54,55,64). RNA recombination, the joining of two noncontiguous RNA segments together, is an especially powerful tool for viruses to create new resistance-breaking or drug-resistant strains and/or viruses (27,64). Accordingly, the generation of novel recombinant RNAs (recRNAs) has been described for many human, animal, and plant viruses as well as RNA bacteriophages (1,4,5,11,16,21,23,24,27,32,42,59,64,65).Progress in our understanding of viral RNA recombination has been slowed down by the difficulty of detection of new recRNAs, the adverse selection pressure on some recRNAs, and the poor predictability of recombination events. Development of powerful model RNA recombination systems, however, has revealed many unique features of viral RNA recombination. For example, sequencing of numerous recRNAs in Brome mosaic virus (BMV) (3,33,36,53), Turnip crinkle virus (TCV) (6,7,37,38,40), and tombusviruses (61, 62) established that recombination does not occur randomly within the viral RNA genome but rather, there are recombination "hot spots". These include AU-rich sequences (31, 34, 58), inter-or intramolecular secondary structures (19,35,62), and cis-acting RNA elements with high affinity toward the viral replicase (8,10,40). Mutagenesis of the replicase proteins has led to altered recombination frequencies or altered the sites of recombination (15,30,47), suggesting that many recombination events are due to template switching (replicase jumping) by the viral replicase (22,27,39...