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 ...

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...

RNA viruses of humans, animals, and plants evolve rapidly due to mutations and RNA recombination. A previous genome-wide screen in Saccharomyces cerevisiae, a model host, identified five host genes, including XRN1, encoding a 5-3 exoribonuclease, whose absence led to an ϳ10-to 50-fold enhancement of RNA recombination in Tomato bushy stunt 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). In this study, we found abundant 5-truncated viral RNAs in xrn1⌬ mutant strains but not in the parental yeast strains, suggesting that these RNAs might serve as recombination substrates promoting RNA recombination in xrn1⌬ mutant yeast. This model is supported by data showing that an enhanced level of viral recombinant accumulation occurred when two different 5-truncated viral RNAs were expressed in the parental and xrn1⌬ mutant yeast strains or electroporated into plant protoplasts. Moreover, we demonstrate that purified Xrn1p can degrade the 5-truncated viral RNAs in vitro. Based on these findings, we propose that Xrn1p can suppress viral RNA recombination by rapidly removing the 5-truncated RNAs, the substrates of recombination, and thus reducing the chance for recombination to occur in the parental yeast strain. In addition, we show that the 5-truncated viral RNAs are generated by host endoribonucleases. Accordingly, overexpression of the Ngl2p endoribonuclease led to an increased accumulation of cleaved viral RNAs in vivo and in vitro. Altogether, this paper establishes that host ribonucleases and host-mediated viral RNA turnover play major roles in RNA virus recombination and evolution.Human-, animal-, and plant-pathogenic RNA viruses are frequently subjected to RNA recombination (1, 3, 20, 51), a process that joins noncontiguous RNA segments together. The resulting novel combinations of genes, sequence motifs, and/or regulatory RNA sequences could cause dramatic changes in the infectious properties of RNA viruses that can potentially lead to the emergence of new viruses or strains. Therefore, RNA recombination can help viruses to "jump species," escape natural resistance mechanisms, and/or render antiviral methods ineffective. Indeed, there is a growing number of examples where RNA recombination likely contributed to viral outbreaks, including outbreaks of caliciviruses (15), astroviruses (48), poliovirus (12, 22), dengue virus (14, 52), enteroviruses (21, 29), influenza virus (17), bovine viral diarrhea virus (12), and the recombinant severe acute respiratory syndrome (SARS) coronavirus, a newly emerged viral pathogen of humans (4,42,46). RNA recombination can also increase virus fitness by facilitating virus genome repair, a process that leads to the correction of mistakes introduced during viral RNA replication by the error-prone viral RNA replicases or due to damage caused by host ribonucleases (1,3,20,27,51). Viral RNA recombination is thought to occur when the viral replicase accidentally switches templates during comple...

RNA recombination occurs frequently during replication of tombusviruses and carmoviruses, which are related small plus-sense RNA viruses of plants. The most common recombinants generated by these viruses are either defective interfering (DI) RNAs or chimeric satellite RNAs, which are thought to be generated by template switching of the viral RNA-dependent RNA polymerase (RdRp) during the viral replication process. To test if RNA recombination is mediated by the viral RdRp, we used either a purified recombinant RdRp of Turnip crinkle carmovirus or a partially purified RdRp preparation of Cucumber necrosis tombusvirus. We demonstrated that these RdRp preparations generated RNA recombinants in vitro. The RdRp-driven template switching events occurred between either identical templates or two different RNA templates. The template containing a replication enhancer recombined more efficiently than templates containing artificial sequences. We also observed that AU-rich sequences promote recombination more efficiently than GC-rich sequences. Cloning and sequencing of the generated recombinants revealed that the junction sites were located frequently at the ends of the templates (end-to-end template switching). We also found several recombinants that were generated by template switching involving internal positions in the RNA templates. In contrast, RNA ligationbased RNA recombination was not detected in vitro. Demonstration of the ability of carmo-and tombusvirus RdRps to switch RNA templates in vitro supports the copy-choice models of RNA recombination and DI RNA formation for these viruses.Viral RNA recombination, a process that joins together two noncontiguous RNA segments, is an especially powerful tool in virus evolution, since it can rapidly lead to dramatic changes in virus genomes by recombining or rearranging "battle-tested" (i.e., evolutionarily successful) sequences. Accordingly, the significant role of RNA recombination in emergence of new viruses or virus strains is well documented for numerous human, animal, plant, insect, fungal, and bacterial viruses (2, 4, 12, 17, 18, 20, 21, 27-31, 51, 57, 61, 62, 64, 65, 71). In addition to increasing sequence variability, RNA recombination can be an efficient tool for viruses to repair viral genomes, thus contributing to virus fitness (6,14,35,36,53,66). In spite of its significance, our understanding of RNA recombination is incomplete. This is due to the complex nature of RNA recombination and the lack of tractable systems for mechanistic studies.RNA recombination may also play a role in the formation of subviral RNAs that include defective interfering (DI) RNAs associated with many animal and plant viruses. DI RNAs are mainly derived from the parent (helper) virus via sequence deletion(s). The DI RNAs are deficient in replication and/or other functions, which makes them dependent on the helper virus for their survival and spread (50, 70). The best-known DI RNAs among plant viruses are those associated with tombusvirus infections. The tombusvirus DI RNAs are mosai...

Rapid RNA virus evolution is a major problem due to the devastating diseases caused by human, animal and plant-pathogenic RNA viruses. A previous genome-wide screen for host factors affecting recombination in Tomato bushy stunt tombusvirus (TBSV), a small monopartite plant virus, identified Xrn1p 5'-3' exoribonuclease of yeast, a model host, whose absence led to increased appearance of recombinants [Serviene, E., Shapka, N., Cheng, C.P., Panavas, T., Phuangrat, B., Baker, J., Nagy, P.D., (2005). Genome-wide screen identifies host genes affecting viral RNA recombination. Proc. Natl. Acad. Sci. U. S. A. 102 (30), 10545-10550]. In this paper, we tested if over-expression of Xrn1p in yeast or expression of the analogous Xrn4p cytoplasmic 5'-3' exoribonuclease, which has similar function in RNA degradation in Arabidopsis as Xrn1p in yeast, in Nicotiana benthamiana could affect the accumulation of tombusvirus RNA. We show that over-expression of Xrn1p led to almost complete degradation of TBSV RNA replicons in yeast, suggesting that Xrn1p is involved in TBSV degradation. Infection of N. benthamiana expressing AtXrn4p with Cucumber necrosis tombusvirus (CNV) led to enhanced viral RNA degradation, suggesting that the yeast and the plant cytoplasmic 5'-3' exoribonuclease play similar roles. We also observed rapid emergence of novel CNV genomic RNA variants formed via deletions of 5' terminal sequences in N. benthamiana expressing AtXrn4p. Three of the newly emerging 5' truncated CNV variants were infectious in N. benthamiana protoplasts, whereas one CNV variant caused novel symptoms and moved systemically in N. benthamiana plants. Altogether, this paper establishes that a single plant gene can contribute to the emergence of novel viral variants.