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 can be facilitated by recombination signals present in viral RNAs. Among such signals are short sequences with high AU contents that constitute recombination hot spots in Brome mosaic virus (BMV) and retroviruses. In this paper, we demonstrate that a defective interfering (DI) RNA, a model template associated with Tomato bushy stunt virus (TBSV), a tombusvirus, undergoes frequent recombination in plants and protoplast cells when it carries the AU-rich hot spot sequence from BMV. Similar to the situation with BMV, most of the recombination junction sites in the DI RNA recombinants were found within the AU-rich region. However, unlike BMV or retroviruses, where recombination usually occurred with precision between duplicated AU-rich sequences, the majority of TBSV DI RNA recombinants were imprecise. In addition, only one copy of the AU-rich sequence was essential to promote recombination in the DI RNA. The selection of junction sites was also influenced by a putative cis-acting element present in the DI RNA. We found that this RNA sequence bound to the TBSV replicase proteins more efficiently than did control nonviral sequences, suggesting that it might be involved in replicase "landing" during the template switching events. In summary, evidence is presented that a tombusvirus can use the recombination signal of BMV. This supports the idea that common AU-rich recombination signals might promote interviral recombination between unrelated viruses.

Replication of the nonsegmented, plus-stranded RNA genome of Cucumber necrosis tombusvirus (CNV) requires two essential overlapping viral-coded replication proteins, the p33 replication co-factor and the p92 RNA-dependent RNA polymerase. In this paper, we demonstrate that p33 is phosphorylated in vivo and in vitro by a membrane-bound plant kinase. Phosphorylation of p33 was also demonstrated in vitro by using purified protein kinase C. The related p28 replication protein of Turnip crinkle virus was also found to be phosphorylated in vivo, suggesting that posttranslational modification of replication proteins is a general feature among members of the large Tombusviridae family. Based on in vitro studies with purified recombinant p33, we show evidence for phosphorylation of threonine and serine residues adjacent to the essential RNA-binding site in p33. Phosphorylation-mimicking aspartic acid mutations rendered p33 nonfunctional in plant protoplasts and in yeast, a model host. Comparable mutations within the prereadthrough portion of p92 did not abolish replication. The nonphosphorylation-mimicking alanine mutants of CNV were able to replicate in plant protoplasts and in yeast, albeit with reduced efficiency when compared to the wild type. These alanine mutants also showed altered subgenomic RNA synthesis and a reduction in the ratio between plus- and minus-strand RNAs produced during CNV infection. These findings suggest that phosphorylation of threonine/serine residues adjacent to the essential RNA-binding site in the auxiliary p33 protein likely plays a role in viral RNA replication and subgenomic RNA synthesis during tombusvirus infections.

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by di¡erent experimental approaches are given, including (i) double infections with movement-de¢cient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-de¢cient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.